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Plant
Science
j our na l h o me p ag e :w w w . e l s e v i e r . c o m / l o c a t e / p l a n t s c i
Mineral
stress
affects
the
cell
wall
composition
of
grapevine
(Vitis
vinifera
L.)
callus
João
C.
Fernandes
a,
Penélope
García-Angulo
b,
Luis
F.
Goulao
c,
José
L.
Acebes
b,
Sara
Amâncio
a,∗aDRAT/CBAA,InstitutoSuperiordeAgronomia,UniversidadeTécnicadeLisboa,TapadadaAjuda,1349-017Lisbon,Portugal bDepartamentodeBiologíaVegetal,ÁreadeFisiologíaVegetal,UniversidaddeLeón,E-24071León,Spain
cBioTrop–InstitutodeInvestigac¸ãoCientíficaTropical(IICT,IP),Av.daRepública,QuintadoMarquês,2784-505Oeiras,Portugal
a
r
t
i
c
l
e
i
n
f
o
Articlehistory: Received8October2012
Receivedinrevisedform15January2013 Accepted19January2013
Available online 6 February 2013 Keywords: Cellulose FT-IR Lignin Mineralstress Pectin
a
b
s
t
r
a
c
t
Grapevine(VitisviniferaL.)isoneofthemosteconomicallyimportantfruitcropsintheworld.Deficitin nitrogen,phosphorusandsulfurnutritionimpairsessentialmetabolicpathways.Theinfluenceofmineral stressinthecompositionoftheplantcellwall(CW)hasreceivedresidualattention.Usinggrapevine callusasamodelsystem,6weeksdeficiencyofthoseelementscausedasignificantdecreaseingrowth. CallusCWswereanalyzedbyFouriertransforminfraredspectroscopy(FT-IR),byquantificationofCW componentsandbyimmunolocalizationofCWepitopeswithmonoclonalantibodies.PCAanalysisof FT-IRdatasuggestedchangesinthemaincomponentsoftheCWinresponsetoindividualmineralstress. Decreasedcellulose,modificationsinpectinmethylesterificationandincreaseofstructuralproteinswere amongtheeventsdisclosedbyFT-IRanalysis.Chemicalanalysessupportedsomeoftheassumptionsand furtherdisclosedanincreaseinlignincontentundernitrogendeficiency,suggestingacompensationof cellulosebylignin.Moreover,polysaccharidesofcallusundermineraldeficiencyshowedtobemore tightlybondedtotheCW,probablyduetoamoreextensivecross-linkingofthecellulose–hemicellulose network.OurworkshowedthatmineralstressimpactstheCWatdifferentextentsaccordingtothe withdrawnmineralelement,andthatthemodificationsinagivenCWcomponentarecompensatedby thesynthesisand/oralternativelinkingbetweenpolymers.Theoverallresultsheredescribedforthefirst timepinpointtheCWofVitiscallusdifferentstrategiestoovercomemineralstress,dependingonhow essentialtheyaretocellgrowthandplantdevelopment.
© 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Thestructuralandmechanicalsupportofplantsisprovidedby cellwalls(CWs),which areload-bearing,extensibleviscoelastic structuresthatsurroundthecells,actingasan“exoskeleton”.The CWplaysavitalroleintheregulationoftherateanddirectionof growthandthemorphologyofplantcellsandorgans[1].Theplant CWisadynamiccomplexwithfurtherfunctionssuchascontrol ofthediffusionthroughtheapoplast,signaling,regulationof cell-to-cellinteractions,storageofcarbohydrates,orprotectionagainst biotic[2]andabioticstressagents[3].
Abbreviations: 2.4-D,2,4-dichlorophenoxy-acetic acid; CDTA, cyclohexane-trans-1.2-diamine-N,N,N,N-tetraacetic acidsodium salt; CW, cell wall; FT-IR, Fourier-transforminfrared;GC,gaschromatography;HRP,horseradishperoxidase; MS,MurashigeandSkoogMedium;-N,nitrogendeficientcallus;-P,phosphorus deficientcallus;PBS,phosphatebufferedsaline;PCA,principalcomponent analy-sis;PVP-40T,polyvinylpyrrolidone;RG-I,rhamnogalacturonan-I;-S,sulfurdeficient callus;SD,standarddeviation;TFA,trifluoroaceticacid.
∗ Correspondingauthor.Tel.:+351213653418;fax:+35133653383. E-mailaddress:samport@isa.utl.pt(S.Amâncio).
IntheprimaryCW,celluloseisthemainload-bearing polysac-charide which interlinks with cross-linking matrix glycans, predominantlyxyloglucanindicots[4],toformanextensive frame-workthatprovidesmostofthetensilestrengthtotheCWmatrix. Thisnetworkisembeddedinasurroundingphaseconstitutedby pecticpolysaccharides,forminghydrophilicgelsthatdeterminethe regulationofthehydrationstatusandiontransport,thedefinition oftheporosity,stiffnessandcontrolofthewallpermeability[5]. Thesefeaturesare,inturn,definedbythechemicalstructureof pec-ticpolysaccharides,particularlythebranchingdegreeandpattern, thedecorationwithneutralsugarsandthedegreeandpatternof acetyl-andmethyl-esterification,whichcanleadtoeitherstiffening orlooseningoftheCW[6].Theoccurrenceofmicro-domainsinside thepecticpolysaccharidesmeansthelocalizationofpreciseareas withdistinctproperties,providingahighlyfine-tunedregulation ofthewallpropertiestocopewiththecellfunctioning.Inaddition topolysaccharides,athirdnetworkcomposedbystructural glyco-proteinscontributestothebiophysicalpropertiesoftheprimary CWandcelladhesion[7,8].Insometissues,aftercellgrowthhas ceased,asecondaryCWisformedwithhighercellulosecontentand adifferentorganizationofitsdeposition.Aftercellulose,ligninis 0168-9452/$–seefrontmatter © 2013 Elsevier Ireland Ltd. All rights reserved.
thesecondmostabundantplantpolymerinvascularplants[9].In secondaryCWs,ligninisdepositedwithin,aroundoramongthe cel-lulosemicrofibrilsestablishingcovalentbondswithcarbohydrates, providingadditional strengthand rigiditythat, along evolution, allowedplantstogrowupward[10].
Themostconsensual dicotprimary CWmodel hasbeenthe “tethered network”, a representation in which the hemicellu-losepolymerslinkcellulosethroughhydrogenbondstocreatea loadbearingtether,insertedinanamorphouscement-likepectin matrix [11]. However,recent results disclosed thepresence of covalentlinkagesbetweenrhamnogalacturonan-I(RG-I)-arabinan side-chainsandcellulosemicrofibrils[12]andcovalentlinkages betweenxyloglucanandpectinsinmuro,[13,14]providing struc-turallinksbetweentwomajorcellwalldomains.Moreover,since notallofthecellulosemicrofibrilsurfacesarecoveredwith xyloglu-cansandnotallxyloglucansareadsorbedtocellulose[15–17],the existenceofsuchotherlinkageswithintheCWisexpectedto main-tainitsstructure.
Duringdevelopment,thefinestructureoftheplantCWmatrix isextensivelymodified.Theamountandcompositionofspecific moleculesandtheirarrangementsdifferamongplants,organs,cell typesandevenindifferentmicro-domainsofthewallofagiven individualcell[18].
LocalizedchangesinCWcompositionandstructurealsoprovide thecellwithanotableabilitytotolerateabioticstresses,suchas osmotic[19]andchemical[20,21].
Deficiencies in mineral nutrition, particularly nitrogen (N), phosphorus(P),potassium(K)andsulfur(S),whicharerequired inrelativelylargeamountsbytheplant,stronglyaffecttheplant metabolismwithsubsequentimpact ontheplantgrowth,crop yieldandinbothnutritionalandorganolepticqualityofthe agro-nomicproduct[22–25].Essentialnutrientsaremajorregulatorsof plantgrowthanddevelopmentduetotheirinvolvementinprimary metabolicpathways,e.g.aminoacidandnucleotidebiosynthesis, proteinphosphorylationordisulfidebondsbetweencysteine.
Plant development and anatomy are impacted by abiotic stressesandacommon“stress-induced”setofresponseshavebeen reported:promptingoflocalizedcelldivision,arrestmentofcell elongation,andmodificationsincelldifferentiationstatus[26].
Limitedmineralnutrientavailabilityhasbeenreportedtoaffect organgrowthrates,throughinhibitionoftheproductionofnew cells and/or cell expansion [27] via reduction of CW plasticity [28,29].Ithasbeenproposedthatnutrient-inducedstressactby modifying xylem tension which then signals the onset of CW rearrangementsingrowingtissues[30,31].Thesecomponentsare determinedbythedynamicregulatorypropertiesoftheCW. Nev-ertheless,and eventhough theimportanceof mineralnutrition inplantdevelopmenthasbeenwidelyrecognized,onlyresidual attentionhasbeengiventoitsinfluenceontheCWdynamics.More recently,globaltranscriptomicstudiesinvolvingnutrientdepleted plants revealeddifferentialregulation of CW-relatedgenes and proteinsin variousspecies[32,33],emphasizingtheCWrole in survivalresponsemechanisms.
Despitethegrapevine(VitisviniferaL.)economicvalueand sci-entificrelevanceasamodelspecies,thereislittleinformationabout theCWstructureandpolysaccharidecompositioninthisspecies. Investigationhasbeenmainlyfocusedtotheeconomicimportant organ,thefruit,bothberrypulpandskin,reviewedin[34].
Theaimofthepresentworkwastoinvestigatetheresponse of the CW to mineral depletion of individual major nutrients, nitrogen,phosphorusandsulfur,usingVitiscallusasexperimental model.Here,anintegratedapproachemployingcomplementary methodologieswas followed.Fourier-transform infrared (FT-IR) spectroscopy coupled with chemometrics was used to detect changesinCWpolymersandputativecross-links[35,36]toretrieve themajorcandidateeventsoccurringintheCWinresponsetothe
imposedconditions.Candidateeventswerefurthertestedby chem-ical methods and immunochemical staining using monoclonal antibodies[37] and throughthe determinationof monosaccha-ridecompositionoffractionatedCWs.Thecombineduseofthese methodologiesalloweddraftingamapofCWresponsestospecific changesinthemineralhealthinVitiscallus.
2. Materialandmethods
2.1. Cellcultureandmineralstressimposition
V.viniferacvTourigaNacionalcallustissuewasmaintainedin thedarkat25◦C,asdescribedinJacksonet al.[38].Fourand a halfgramsofcallustissuewasusedasinitialexplantinmedium containingMSbasalsalts[39](DuchefaBiochemie,Haarlem,NL) supplemented with 2.5M 2.4-d (2,4-dichlorophenoxy-acetic acid); 1M kinetin; 5gl−1 PVP-40T; 20gl−1, sucrose; 2gl−1 Gelrite®,pH5.7.Thecallusesweresub-culturedevery3weeks.Four
treatmentswere applied:full nutrients(control),nitrogen defi-ciency(-N),phosphorusdeficiency(-P)andsulfurdeficiency(-S). CommercialMSwasusedtoobtaincontrolsampleswhile mod-ifiedMSmediainwhichnitrates,phosphatesandsulfateswere substitutedforchlorideswereconsidered-N,-Pand-Streatments respectively.Callusesweresub-culturedtotherespectivemedium after3weeksofgrowing.Aftereachculturecycleintherespective treatmentmediumeachsample,correspondingto10Petridishes (9cmØ)containingfourcalluses,wascollectedtomonitorgrowth. Basedontheresultsobtained,6weeksgrowncallus(2×3weeks) sampleswereusedforCWanalyses.
2.2. Cellwallisolation
Twentygramsofcallussampleswerehomogenizedinliquid nitrogenusing a mortar and pestle, washedwith cold 100mM potassium phosphatebufferpH7.0(2×),and treated overnight with2.5Uml−1 ␣-amylaseVIfromhogpancreas(Sigma–Aldrich Co.,St.Louis)at37◦C.Thesuspensionswerecentrifugedandthe pelletwassequentiallywashedwithdistilledwater(3×),acetone (3×),methanol:chloroform(1:1;v/v)(3×),diethylether(2×),and thenair-dried[40].
2.3. FT-IRspectroscopyandmultivariateanalysis
FT-IRanalysiswasperformed accordingtothemethodology describedinAlonso-Simónetal.[36].TabletsforFT-IRspectroscopy werepreparedinaGraseby-SpecacPress,using2mgofCW sam-plesmixed withpotassium bromide(KBr)(1:100,w/w) froma minimumof11biologicalreplicatespertreatment.Spectrawere obtainedon a Perkin-Elmer System 2000FT-IR at a resolution of1cm−1.InordertotackleCWstructuremodifications,a win-dow between 800 and 1800cm−1, which contains information ofpolysaccharidecharacteristiclinkages,wasselectedfor analy-sis.Normalizationandbaseline-correctionweremadeusingthe Perkin-ElmerIRDatamanagersoftwareandthedataexportedto MicrosoftExcelforareanormalization.Principalcomponent anal-ysis(PCA),usingPearsoncoefficientfordistanceestimation,was performedwithamaximumoffourprincipalcomponentsusing theStatistica6.0softwarepackage(StatSoft,Inc.,USA).
2.4. Cellulosequantification
CellulosewasquantifiedbytheUpdegraffmethod[41],using thehydrolyticconditionsdescribedbySaemanetal.[42]and quan-tifyingtheglucosereleasedbytheanthronemethod[43].
2.5. Ligninquantification
Klasonligninwasdeterminedusingthemethoddescribedby Hatfieldetal.[44].Briefly,60mgofCWmaterialwassolubilizedin 2mlof72%H2SO4at30◦Cfor60min.Thesolutionwasdilutedto
2.48%H2SO4priortosecondaryhydrolysisbyautoclavingat115◦C
for60min.Thenon-hydrolyzedresiduewascollectedbyfiltration anddriedat60◦Cuntilconstantweight.Theresultsareexpressed asglignin(mgCW)−1.
2.6. Quantificationofpectinmethyl-esterification
Theextentofesterificationofpectinswasassessedby quan-tificationofthereleasedmethanolusingthemethoddescribedby WoodandSiddiqui[45].Toeach10mgCWsample,0.75ml dis-tilledwaterand0.25ml1.5MNaOHwereadded.Thesampleswere incubatedatroomtemperaturefor30min,chilledoniceandadded with0.25ml4.5MH2SO4.Aftercentrifugation,0.5mlofthe
super-natantwasmixedwith0.5ml0.5MH2SO4,chilledoniceand0.2ml
2%KMnO4(w/v)wereadded.Thesampleswereallowedtostandin
anicebathfor15minandaddedwith0.2ml0.5Msodiumarsenite in0.06MH2SO4.Thesampleswerethoroughlymixedandleftfor
1hatroomtemperature.Finally,2mlacetylacetone–ammonium acetatereagentwasadded,tubeswerevortexed,closedandheated to59◦Cfor15min.Aftercoolingtoroomtemperature,absorbance wasreadat412nmandtheresultswereexpressedasmlCH3OH
(mgCW)−1,usingmethanolasstandard.
2.7. Cellwallfractionation
CWfractionationwasdoneaccordingtoSelvendranandO‘Neill [46] with minor modifications. Dried CW were extracted at room temperature with 50mM cyclohexane-trans-1.2-diamine-N,N,N,N-tetraaceticacidsodiumsalt (CDTA)adjusted topH6.5 withKOH,for8h(2×),collectedbycentrifugation,washedwith distilledwaterandthecombinedsupernatantsreferredtoasthe CDTA-solublefraction. Theresidue wasincubatedfor18hwith 0.1MKOH containing20mM NaBH4 centrifugedand thepellet
washedwithdistilledwater.Thecombinedalkaliandwater super-natantswereadjustedtopH5.0withaceticacid.Thisfractionwas referredtoasthe0.1MKOH-solublefraction.Then,theresiduewas incubatedfor18hin6MKOHcontaining20mMNaBH4,processed
as described for the 0.1M KOH-soluble fraction, to obtainthe 6MKOH-solublefraction.Allfractionsweredialyzedagainst dis-tilledwaterwitha dialysismembraneof12–14kDacut-off and lyophilized.
2.8. Totalsugarandmonosaccharidequantification
Total sugars and uronic acids were determined using the phenol-sulfuricacidassay[47]andthem-hydroxybiphenylassay [48]respectively,usingglucoseandgalacturonicacidasstandards. NeutralsugarswerequantifiedasdescribedbyAlbersheimetal. [49].Lyophilized samplesof eachCW fractionwerehydrolysed with2Ntrifluoroaceticacid(TFA)at121◦Cfor1handthesugars werederivatizedtoalditolacetatesandanalyzedbygas chromatog-raphy (GC) using a Supelco SP-2330 30m×0.25mm×0.20m capillarycolumninaPerkinElmerAutosystemgaschromatograph fittedwithaflame-ionizationdetector.Helium(2mlmin−1)was usedasthecarriergas.Sugarquantificationwascarriedoutafter determinationofeachsugarresponsefactorusingpurerhamnose, fucose,arabinose,xylose,mannose,galactoseandglucoseas stan-dards.Inositolwasusedasinternalstandard.
2.9. Immunodotblotassays
Forimmunodotassays,1laliquotsfromCDTA-,0.1M KOH-and6MKOH-solublefractionsinthreereplicateddilutionseries (1:5dilutions)asdescribedbyGarcía-Anguloetal.[50]were spot-tedontoanitrocellulosemembrane (Scheicher&Schull,Dassel, Germany).Themembraneswereblockedfor2hwith0.1M Phos-phatebufferedsaline(PBS)containing4%fat-freemilkpowderand eachprimaryantibody(2F4,LM1,LM5andLM6)ata1:5dilution. AfterwashingwithPBS,themembraneswereincubatedfor1hwith a1:1000dilutionofananti-ratIgG1secondaryantibodylinkedto horseradishperoxidase(HRP).Forsignaldetection,themembranes wereincubatedwith25mlofdeionizedwater,5mlmethanol con-taining10mgml−1 4-chloro-1-naphtol and30l6%(v/v)H2O2,
andphotographedwithadigitalcamera. 2.10. Statisticalanalysis
Dataispresentedasmeanvalues±standarddeviation(SD).The resultswerestatisticallyevaluatedbyvarianceanalysis(ANOVA) andBonferronitestasposthoctestswithap=0.05significance,to comparethetreatmenteffect.TheSigmaPlot(SystatSoftwareInc.) statisticalpackagewasusedintheanalyses.
3. Results
3.1. Effectofmineralstressoncallusgrowth
Ourmainaimwastoanalyzetheeffectofnitrogen, phospho-rusorsulfurnutrientdepletioningrapevineCWcompositionand structure.Theeffectoftheimposedindividualmineralstresseson thefunctioningofthebiologicalexperimentalsystemusedwas firstlyassessedbymeasuringthegrowthofcallusalongtime.The absolutegrowthofVitiscallusinfullMSculturemedium(control) andinmodifiedMSmediawithoutnitrogen(-N),phosphorus(-P) orsulfur(-S)alongtwocyclesof3weekseachisshowedinFig.1. Afterwithdrawingofnutrients,theabsolutegrowthofthe cal-luswassignificantlyaffectedinbothcycleswhencomparedtothe control.Duringthefirst3weeks,phosphorusandsulfurdepleted mediaaffectedgrowthinamorepronouncedwaythanunder nitro-genabsence.Afterasecondgrowingcycleundernutrientdepletion, amoreseverereductioningrowthwasnoticedinallindividual
Fig.1.Absolutegrowth(AG)ofVitiscallusgrowingundernitrogendeficient(-N), phosphorusdeficient(-P),sulfurdeficient(-S)andwithfullnutrients(Control)for threeand6weeks.BarsrepresentmeansoftheAGofVitiscallusfrom10Petridishes (n=10)±SD.Ineachsubgraph,differentlettersindicatesignificantdifferencesat p=0.05.
stresses,withareductioningrowthofca.80%incomparisonwith thecontrol.Thistimescale(2cyclesof3weeks)wasselectedto producetemplatematerialtobeusedinthesubsequentanalyses. 3.2. FT-IRspectroscopydetermination
PutativechangesinCWrelative compositionduringnutrient deprivationweremonitoredbyFT-IRspectroscopybyanalysisof atleast11FT-IRCWspectrapertreatment.
ForaclearanalysisoftheFT-IRspectraamultivariateanalysis (PrincipalComponentAnalysis;PCA)wasperformed(Fig.2). Prin-cipalcomponents1and2(PC1andPC2),whichexplain71.9%of thetotalvariation,wereusefultoseparatethesamples.PC1clearly
differentiatesthecontrolsamplesfromtreatments-Nand-P,while PC2separates thesamplesaccordingtoeach individualmineral stress.Samplesfromthe-Streatmentalsotendtobediscriminated fromthecontrol,consideringtheirdistributionacrossthegradient fromthenegativetothepositiveareasofthePC1andPC2axes (Fig.2a).PC1loadingfactorplot(Fig.2b)showedseveralnegative peaksassociatedwithcellulose,suchas1160cm−1,assignedtothe C OandC Cvibration[51],1120cm−1,associatedwiththe gly-cosidicC O Cvibration[52]or988cm−1,associatedwithbonds sharedbycello-triose,-tetraoseand-pentose[53],indicatingthat sampleslocatedinthenegativepartofPC1(allcontrolandsome -Ssamples)shouldhaveahighercontentofcellulose.Inthesame samples,analterationintheesterificationpatternsofpectinsisalso
Fig.2.Principalcomponentanalysis(PCA)ofallcallusspectra.(a)PlotofthefirstandsecondPCsbasedontheFTIRspectraobtainedfrom6weekscallusgrowninthe absenceofnitrogen(-N),phosphorus(-P),sulfur(-S)andunderfullnutrients(Control)(n>11).(b)LoadingfactorplotforPC1( )andPC2( )explaining PCAclustering.Arrowspointtomeaningfulwavenumbers(1–1650cm−1;2–1630cm−1;3–1430cm−1;4–1160cm−1;5–1120cm−1;6–1041cm−1;7–988cm−1;and
Fig.3.Differencesbetweennormalizedandbaseline-correctedFT-IRspectraofCWobtainedfromcallusintheabsenceofnitrogen(-N)(a),phosphorus(-P)(b),orsulfur (-S)(c)relativetocontrol(n>11).VerticallinesrepresentwavenumbersassignedtoCWpolymers.
suggested,asreveledbythecontributionofthe950cm−1peak[54], indicatingthattheabsenceofnutrientsalterspectinbiochemistry. PC2loadingfactorplot(Fig.2b)showednegativepeaksassociated withxyloglucan (1041cm−1,relatedto-glucan)[52]and with proteins(1650cm−1,related toC OamideIlinkage). This evi-dencepointstodecreasedconcentrationsoftheseCWcomponents upon-Nand-Pconditions.PC2positivepeakswerealsoobserved, mainlyrelatedtophenoliccompounds(1630and1430cm−1)[55], suggestinganincrease ofthesecomponentsinCWunder some mineralstresses.Otherpositivepeakswerealsoobserved,mainly at840,1295,1565and1700cm−1,forfactor1and885,1180,1250 and1390cm−1forfactor2,butnoinformationisavailableabout thenatureofthelinkagesandwallcomponentsassociatedwith thesepeaks.It shouldbenoted that-N and,in aminorextent, -P callussamples tend tocluster more compactlyin the plots, whilecontrolsamplesappearasamoredispersedgroup(Fig.2a) suggestinglowervariabilityinsamplesexposedtohigherstresses. Toacquireadditionalinformationonthesignificanceofspectral differencesbetweenthecontrolandthemineralstressedcallus,a subtractionofthespectrawasalsoperformed(Fig.3).Usingthis approach,thedifferencesin-Nspectraobservedweremorestriking asnegativepeaksinwavenumbers1033cm−1characteristicof cel-lulose,relatedtodeformationsofC OHgroups[52]and1650cm−1,
assignedtoproteins,asstatedabove.Thesenegativepeakspoints toaminoramountofcelluloseandproteinsin-Ncondition.They wereaccompaniedbypositivepeaksat950and1740cm−1,both relatedtoahigheramountofpectins.Thesetrendswerenot main-tainedin-Por-Sconditions.In-S,alterationsincelluloseassigned tothevibrationsassociatedwiththeCH2group,asrevealedbythe
1265cm−1wavenumberpeak[53],werealsoobserved(Fig.3).A positivepeakat1180cm−1 wasalsoobservedinallcasesbutit hasnotbeenassociatedwithanyknownwallcomponentorgroup linkage.Acleardifferenceintheabsorbancepeaksforcellulose betweenthethreetreatmentswasobservedinagreementwiththe PC1separations(Fig.2a).Theoverallresultspinpointcelluloseand pectinsasmajorcandidatestobeaffectedbymineralstress,what promptedustoamoredetailedinvestigation.
3.3. Celluloseandlignincontent
Cellulose is themain constituent of the CW. Fig. 4a shows thevariation in cellulose amountin responsetomineralstress imposition.A significantreduction in cellulosewasobservedat theextentofca.43%intheabsenceofnitrogenand,toalessextent ofca.12%,intheabsenceofphosphorus(Fig.4a),whencompared to the control. Sulfur depletiondid not affect significantly the
Fig.4.(a)Cellulosecontent,(b)lignincontentand(c)totalmethyl-esterification extentof6weeksCWcallusgrowninnitrogendeficient(-N),phosphorusdeficient (-P),sulfurdeficient(-S)andfullnutrientmedia(Control).Barsrepresentmeansof 6Vitiscallus±SD(n=10).Differentlettersindicatesignificantdifferencesatp=0.05 significance.
synthesisordepositionofcellulose.AlthoughligninKlasonlevels weresmallcomparedwithcelluloseorothercellwallcomponents, ligninquantificationin controland stressedcallusesreveledan increaseofca.36%relativetothecontrolwhennitrogenisremoved fromthegrowingmedium(Fig.4b).Theabsenceofphosphorus andsulfurdidnotaffecttheamountofligninintheCWofVitis callusafter6weeksgrowth.
3.4. Degreeofpectinmethyl-esterification
Tofurtherexaminetheputativemodificationsinpectin ester-ificationsuggestedbyFT-IRanalysis,theextentofpectinmethyl
esterification wasdetermined by quantification of thereleased methanol.Thedecreaseinthemethanolreleasedobservedin sam-plesfromcallusgrowingunderabsenceofnitrogenintheculture medium,indicatesalowerdegreeofmethylesterificationin com-parisonwiththecontrol(Fig.4c),confirmingtheFT-IRresultsfor thisstress.Neitherphosphorusnorsulfurabsenceaffectedthetotal degreeofpectinesterification.
3.5. Immunodot-blotofcellwallpolysaccharidespecific antibodies
TheresultsofimmunodotassaysarepresentedinFig.5.The abundanceof pectins witha degreeof esterificationup to40% wastestedbyimmunodetectionusing2F4monoclonalantibody [56].Asexpected,labelingwasonlydetectedintheCDTAand,to alessextent,inthe0.1MKOH-solublefraction.Astrongersignal wasdetectedunder-Nand-Sconditions(Fig.5).TheLM1 anti-body,specifictoanextensinepitope[57],showedahigherlabeling understressconditionsinallsolublefractions,indicatingthat min-eralstressincreasesthedepositionofthisstructuralprotein.This ismorestrikingin-Nand-Sconditions,theformermoretightly attachedto theCW. LM5 and LM6antibodies recognize 1,4- -galactanand1,5-␣-arabinanepitopes,respectively[58,59].Under -N,the amountof1,5-␣-arabinanand 1,4--galactan decreases relativetothecontrolintheCDTA-solublefraction. Areduction of labeling was observed in the 0.1M KOH-soluble fraction in allstressesforbothantibodies.Conversely,anincreasein1,5- ␣-arabinanissuggestedtooccurunder-Sconditions,bothinweakly and tightlyCWattachedpolysaccharides (CDTA-and 6M KOH-solublefractions,respectively).
3.6. SugaranalysisinCWfractions
CWfractionationshowedthatthemajorityofpolysaccharides were extracted from the CW by the strongalkaline treatment (Fig. 6).Allmineralstress treatmentsdecreased theamountof polysaccharidessolubilizedinCDTAand0.1MKOH.Converselyin 6MKOH-solublefractions,theyweremoreabundantlydetectedin the-Nand-Ptreatments(Fig.6).
Asexpected,themajorityofpectins(quantifiedasuronicacids, UA)wereextractedbytheCDTAand0.1MKOH(Fig.7aandb).GC analysisofCWfractionsshowedthatthemoresignificantneutral monosaccharidepresentinallfractionswasarabinosefollowedby galactose,xyloseandglucose(Fig.7a–c).Theamountofuronicacids decreasedinalkalinesolublefractionsuponthethreemineralstress treatmentsimposed(Fig.7bandc).
Arabinose-containing polysaccharides responded to -N by decreasing its amount in weak alkaline-soluble fraction but increasingsignificantlyin6MKOH-solubleone.Adifferenttrend was observed for xylose- and galactose-containing polysaccha-rides,withadecreaseinbothalkaline-solublefractions.Regarding xylose,anincreaseinthe6MKOH-solublefractionwasnoticed in the-Streatment. Finally,glucose-containing polysaccharides showedincreasedextractionbybothalkaline solutionsfrom-P CWs.
Theincreaseinneutralsugarsobservedinthe6MKOH solu-blefraction,undernitrogenstarvation(Fig.7c),isonlyduetothe increaseinarabinose,sinceallothermonosaccharidesdecreased ormaintainthesamelevelsrelativetocontrolwhennitrogenis withdrawnfromtheculturemedia.
4. Discussion
Plantmodelsystemsanalyzedincontrolledexperimental condi-tionsareusefultoolstoassesslimitingnutrientsituations.Usingthe
Fig.5. ImmunodotassaysofCW-solublefractionsfromcallusgrowninnitrogendeficient(-N),phosphorusdeficient(-P),sulfurdeficient(-S)andfullnutrients (Con-trol),probedwithmonoclonalantibodieswithspecificityforhomogalacturonanwithadegreeofmethylesterificationupto40%(2F4),forextensinhydroxyproline-rich glycoproteins(LM1),for1,4--galactan(LM5)and1,5-␣-arabinan(LM6).Ineachrowtheantibodywasusedtoprobesamplesata1:5sequentialdilution.
modeldescribed,weobservedthatduringthefirst3weeksof devel-opment,callusgrowthwasimpairedbytheabsenceofphosphates andsulfates(Fig.1).Callusfromplatesdepletedinnitrateswasless affected,probablyduetopreviousnitrogenaccumulation,sincethe nitrateconcentrationintheMSmediumismuchhigherthanthose ofphosphateorsulfate.Bythesixthweek,inthesecondculture cycle,thecallusgrowthwasdrasticallyreducedtolevels signifi-cantlylowerthanthoseobservedat-Pandsimilarto-S(Fig.1). Thecollectiveresultsconfirmtheessentialroleoftheseelements andvalidatetheexperimentmodelemployedtoaccesstheeffectof mineralnutritioninCWconstituentsanddynamics.Previous stud-iesalsoreporttheeffectofmineraldepletionongrowth[24,60], includingofVitiscallus[23].
OurgoalwastoaddresschangesinCWcompositiontriggered bymineraldeficiency.FT-IRprovedtobeareadilyemployedand efficientmethodforsimultaneouslyidentifyingabroadrangeof structuraldifferencesinCWs[36].TheoverallFT-IRresults sug-gestchangesinallofthemaincomponentsoftheCWascandidate events,suchasmodificationsinthebiosynthesisorrearrangements ofcellulosemicrofibrilsandofmatrixlinkedglycans,inpectin
bio-Fig.6. TotalsugarsquantifiedinCWfractionsobtainedfromcallusgrownfor6 weeksintheabsenceofnitrogen(-N),phosphorus(-P),sulfur(-S)andinfull nutri-entmedia(Control).Unitsaregofglucoseequivalents(mgCW)−1.Barsrepresent meansof6replicates±SD.Ineachsub-graph,differentletterswithineachfourbar blockindicatesignificantdifferencesatp=0.05.
chemistryandintheamountsofstructuralproteins(Figs.2band3). Moreover,PCAanalysis(Fig.2a)showedthatCWisdifferentially affected,accordingtothespecific stressimposed.These results promptedustopursueamoredetailedbiochemicalanalysisofthe stressedmaterial.
The statistically significant reduction in cellulose content observed under -N and -P (Fig.4a) couldcompromise the CW integrityand,probably,theviabilityofthecellstosurvive.Dueto theparamountimportanceoftheCWinsurvivalandenvironment adaptation, plants are equippedwith compensatoryalternative mechanismstoreinforcetheirCWswhenthebiosynthesisor depo-sitionofagivencomponentisimpaired[61,62].Ourresultsagree withsuchmodel. In fact, CWsfrom callusgrowingin medium exhaustedinnitratesrespondtolowlevelsofcellulose(Fig.4a) byslightlyincreasingtheirlignincontent(Fig.4b)andreducing their degree of pectin methyl esterification (Fig. 4c). If occur-ringinlongstretchesofthegalacturonicacidchainsassuggested byimmunodotdetectionwiththe2F4antibody(Fig.5),itcould leadtoreinforcethewallviatheformation ofcalciumbridges, e.g. “egg-box” structures [63] and supramolecular pectic gels, bothimportantincontrollingtheporosityandmechanical prop-erties of CW and maintenance of intercellular adhesion [4,64]. Moreover,anexpectedloosercellulosenetworkprods arabinan-containingpolysaccharides tobecomemore cross-linked tothe cellulose–hemicellulosenetwork(Fig.7bandc),providingthewall withadditionalmechanicalsupportandinstigatestheformation ofextensinnetworks(Fig.5).Likewise,thelowercellulose quanti-fiedin-Pcallusisaccompaniedbyanincreaseinneutralsugars enrichedin arabinosepolysaccharides (Fig.7b)including1,5- ␣-arabinans(Fig.5)detectedinpolysaccharidestightlybondedtothe CW(Fig.6).
AlterationsinthebiosynthesisofindividualCW constituents canaffectthesynthesisand/ordepositionofotheCWpolymers. Infact,changesinthecellulosecontentareknowntobe compen-satedbyanincreaseinligninpolymers.Itwasdemonstratedthat cinnamoyl-CoA reductase (CCR) down-regulation in transgenic tobacco(NicotianatabacumL.)linesleadtoa24.7%reductionof itsKlasonligninwithaconcomitantincreaseof15%incellulose content [65].Similarly, different worksreport that highnitrate availabilityreducestheligninintheCW[66–68],sotheopposite
Fig.7. MonosaccharidecompositionofCWCDTA-solublefraction(a),0.1MKOH-solublefraction(b)and6MKOH-solublefraction(c)obtainedbyGCanalysisanduronic acidslevelsmeasuredbythem-hydroxybiphenylassayofcallusgrowninnitrogendeficient(-N),phosphorusdeficient(-P),sulfurdeficient(-S)andfullnutrients(Control) media.Unitsaregofeachmonosaccharideandgalacturonicacidequivalentsforuronicacids(UA)(mgCW)−1.Barsrepresentmeansof6replicates±SD.Differentletters withineachfourbarblockindicatesignificantdifferencesatp=0.05.
trendisexpectedtohappen.Infact,amutationinCesA3,oneofthe genesencodingcellulosesynthases,wasprovedtoleadtoCW rein-forcementthroughligninsynthesis[69].Ithasalsobeensuggested that,underconditionswherecellulosesynthesisisinhibited, com-pensationwithahigherquantityofhemicellulosicpolysaccharides occurs[26].Severalother linesof evidence have demonstrated alterations in the CW architecture of the kor1-1 Arabidopsis mutant[70,71],inwhichadeficiencyinanendo-1,4--glucanase that is not directlyimplicated in pectin metabolism, induces a 150%increaseofpectinsintheprimaryCW[72].Moreover,other cellulose synthase Arabidopsis mutant, MUR10/CesA7, showed anincreaseinpecticarabinan contentsinresponsetoimpaired cellulosebiosynthesis [73].On theotherhand, augmented ara-binoselevels may indicate a higher substitution by arabinosyl residuesintherhamnogalacturonan-I(RGI)sidechainsofpectic polysaccharides.RGIarabinosylsidechainscanworkas plasticiz-ersinCWsthatundergolargephysicalremodelingunderabiotic stressessuchasextremewaterdeficientconditions[74].Ulvskov etal.[75]analyzedthemechanicalpropertiesoftheCWofpotato
(Solanum tuberosum) tubers from wild-type and transformed plantswithdecreasedcontentsofarabinanandcertifiedthatthe forceneededtoinducefailureoftheCWdecreasedintransgenic tubers.
Absenceof sulfates in the media impairedcallus growth in levelssimilartothoseobservedundernitratedeficiency(Fig.1), but no significant reduction in cellulose wasnoticed (Fig. 4a). Nonetheless,amongtheassaysconductedinourwork,increases inweaklyandtightlybondedextensins(Fig.5)andincreasesin arabinose-andxylose-containingpolysaccharidesinalkali-soluble fractions(Fig.7bandc)weredetected,unveilingCWmodifications. ExtensinsareabundantconstituentsoftheprimaryCW[7],known toincrease inresponsetoseveralstresses[76–78].These struc-turalproteinshavebeenimplicatedinthecontrolofCWextension andstrengtheningbytheformationofperoxidase-mediated inter-molecularcross-links[38].
Insummary,grapevinecallusessubmittedtoindividualmineral stressesareimpairedonspecificCWcomponentsorsuffer reorga-nizationoftheirdeposition.OurresultshighlightthatV.vinifera
callusfolloweddifferentstrategiestoovercometheadverseeffects inducedintheCWbytheimposedmineralstressaccordingtothe severityperceivedanditsprimarybiologicalrole,confirming previ-ousassumptionsthatplantshaveevolvedfine-tunedmechanisms toturnondifferentpathwaysrelatedwithspecificwall compo-nents,inresponsetodifferentstimulus.
Acknowledgements
The research was funded by Fundac¸ão para a Ciência e Tecnologia (FCT) Grant SFRH/BD/64047/2009 to JCF and CBAA (PestOE/AGR/UI0240/2011).
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