An overview on plant cuticle biomechanics

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PlantScience

journalhomepage:www.elsevier.com/locate/plantsci

Review

Anoverviewonplantcuticlebiomechanics

EvaDomíngueza,JesúsCuarteroa,AntonioHerediab,∗

ab

EstaciónExperimentalLaMayora,CSIC,E-29750Algarrobo-Costa,Málaga,Spain

DepartamentodeBiologíaMolecularyBioquímica,UniversidaddeMálaga,E-29071Málaga,Spain

article

info

abstract

Articlehistory:

Received3February2011

Receivedinrevisedform11April2011Accepted26April2011

Availableonline4May2011

Keywords:PlantcuticleBiomechanicsTomatofruitCutin

Fruitcracking

Plantbiomechanicscombinestheprinciplesofphysics,chemistryandengineeringtoanswerquestionsaboutplantgrowth,developmentandinteractionwiththeenvironment.Theepidermal-growth-controltheory,postulatedin1867andverifiedin2007,statesthatepidermalcellsdeterminetherateoforganelongationsincetheyareundertension,whileinnertissuesareundercompression.Thelipidcuticlelayerisdepositedonthesurfaceofouterepidermalcellwallsandmodifiesthechemicalandmechanicalnatureofthesecellwalls.Thus,theplantcuticleplaysakeyroleinplantinteractionwiththeenvi-ronmentandincontrollingorganexpansion.Rheologicalanalysesindicatethatthecuticleisamostlyviscoelasticandstrain-hardeningmaterialthatstiffensthecomparativelymoreelasticepidermalcellwalls.Cuticlestiffnesscanbeattributedtopolysaccharidesandflavonoidspresentinthecuticlewhereasacutinmatrixismainlyresponsibleforitsextensibility.Environmentalconditionssuchastemperatureandrelativehumidityhaveaplasticizingeffectonthemechanicalpropertiesofcuticlesincetheylowercuticlestiffnessandstrength.

Theexternalappearanceofagriculturalcommodities,especiallyfruits,isofgreateconomicvalue.Mechanicalpropertiesofthecuticlecanhaveapositiveornegativeeffectondisorderslikefruitcracking,fungalpathogenpenetrationandpestinfestation.Cuticlerheologyhassignificantvariabilitywithinaspeciesandthuscanbesubjectedtoselectioninordertobreedcultivarsresistanttopests,infestationanddisorders.

©2011ElsevierIrelandLtd.Allrightsreserved.

Contents1.2.3.4.

5.

6.7.8.

Introduction:theplantcuticle.......................................................................................................................Generalaspectsonplantbiomechanics..............................................................................................................Cuticlebiomechanics:ageneralview................................................................................................................Thecompositionofcuticleanditsroleinmechanicalproperties...................................................................................4.1.Cutinandpolysaccharides....................................................................................................................4.2.Waxesandflavonoids.........................................................................................................................Environmentalconditions............................................................................................................................5.1.Hydration.....................................................................................................................................5.2.Temperature..................................................................................................................................Cuticlemechanicsduringgrowth....................................................................................................................Theroleofcuticleinfruitquality:apracticalapproach.............................................................................................Futureandperspectives..............................................................................................................................Acknowledgements..................................................................................................................................References............................................................................................................................................

7878798080808080818181828383

∗Correspondingauthor.Tel.:+34952131940;fax:+34952132000.E-mailaddress:heredia@uma.es(A.Heredia).

0168-9452/$–seefrontmatter©2011ElsevierIrelandLtd.Allrightsreserved.doi:10.1016/j.plantsci.2011.04.016

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“Therearesolidsinrheology,evenifthey...creep”

MarkusReiner[1]

1.Introduction:theplantcuticle

Plantsprotectthemselvesfromthesurroundingenvironmentwiththeextracellularlipidcuticle.Thismembranecoversaerialpartsofhigherplantssuchasleaves,fruits,flowers,seedsandnon-woodystems[2].Suchamembranenotonlyconstitutestheinterfacebetweentheplantanditsenvironmentbutalsofunc-tionsasapermanentbiologicalbarriercontrollingthediffusionofmolecules.Thus,themainfunctionsascribedtothecuticleareprotectionfromwaterlosswhileatthesametimeregulatinggasexchange,protectingagainstmechanicalinjurycausedbymicroor-ganisms,pestsortheenvironment,attenuatingUVlightsorption,andgeneratingamicroenvironmentsuitableforthegrowthofepiphyticorganismssuchassomebacteria,fungiandarthropods[3].Thecuticleisattachedtoandinteractswiththesubtendingouterepidermalcellwallwhichcanberegardedasacutinizedcellwall.Itsstructure,composition,thickness(submicronstoover10microns)andquantity(fewhundredstooverathousandmicro-gramspersquarecentimeter)varywidelyamongplants,organsandgrowthstages[2,4–6].Leavesareaclearexampleofanasym-metricalpatternofcuticledepositionwithinanorgan;withthickeradaxialthanabaxialcuticles[6].Thesedifferencesplayasignificantroleinthemodificationandadaptationofcuticlepropertiestospe-cificenvironmentalandgrowthconditionsthatcanbeobservedbetweenandwithinorgansanddevelopmentalstages.

Theplantcuticleischaracterizedbyaheterogeneouschemi-calnature.Itsmaincomponentisthebiopolyestercutin,amatrixofpolyhydroxylatedC16and/orC18fattyacidscross-linkedbyesterbonds[5,7].Significantamountsofglyceroloccurinafewcutinmatrixes[8],althoughitsimportanceasamajorcompo-nentofthecutinmatrixneedstobeexplored.Cutanisanotherlipidbiopolymerthatispresentinsomeplantcuticles,eitherasanalternativetoorincombinationwithcutin[9–13].Itiscom-posedofpolyunsaturatedfattyacidderivatives,mostlylinkedbyetherbonds[11],whichrenderamatrixhighlyresistanttochemicaldegradation.Thepotentialadvantagesforplantgrowthofthismorechemically-resistantcuticlematrixdeservefurtherstudy.Waxes,mostlymixturesofC20–C40n-alcohols,n-aldehydes,verylongchainfattyacidsandn-alkanes,canbedepositedonthesurface(epicuticularwaxes)orembeddedinthecutinmatrix(intracutic-ularwaxes).Phenolics,principallycinnamicacidsandflavonoids[14],arealsopresentinthecutinmatrixasminorcompounds.Thisphenolicfractioningymnospermsseemstobemoresignif-icantandispresentintheformoflignin[15].Ontheinnersideofthecuticle,cutinismixedwithpolysaccharidesfromtheepi-dermalcellwall.Thispolysaccharidematerialisphysicallyand/orchemicallyinterconnectedwithcutininanasyetunknownfash-ion.Knowledgeoftheseinterconnections,theirextension,whetherchemicallyreversibleorirreversible,wouldbeofimportancetounderstandcuticledepositionandattachmenttothecellwallandwouldalsohaveanimpactonthethermal,mechanicalandhydricpropertiesoftheplantcuticle.Aschemeofacuticlecross-sectionindicatingitsmaincomponentsispresentedinFig.1.

2.Generalaspectsonplantbiomechanics

Anexternalforceappliedtoamaterialproducesinternalforces(stresses)thatresultindeformation(strain).Strainreflectstheratioofthemagnitudeofadeformeddimensiontothatoftheunde-formeddimension.Stressisdefinedastheamountofforceperareauponwhichitacts.Maximumstrainandbreakingstressareparam-etersthatreflectthestressandstrainobtainedatthebreaking

Fig.1.Schemeofatransversesectionofthecuticleofalandplantrepresentingthelocationofthedifferentcomponents.OEW=outerepidermalwall.

Source[38],withpermissions.

point.Foranygivenmaterialandovercertainrangesofforces,thestressesandstrainsarerelatedtooneanotherbyamodulusthatcanbeusedtodistinguishamongmaterialsundercertainconditionsofloading.ThemodulusthatreflectstheratioofnormalstresstonormalstrainiscalledelasticmodulusorYoung’smodulus.Ahighelasticmodulusisdesirableinstiffstructuresthatresiststresses,whilematerialswithlowelasticmodulusallowlargedeformations[16].

Planttissueshaveacomplexmechanicalbehavior;theyarenei-therelasticsolidsnoridealfluids.Besides,plantscanchangetheirmechanicalpropertiesastheygroworinresponsetoanappliedforce.Thewayamaterialdeformsdependsonthenatureofitsinter-andintra-molecularbonds.Mostplantmaterialsaremainlyviscoelastic,butshowanelasticcomponentatsmalldeformations.Viscoelasticmaterialshaveelasticandviscouscomponents.Thesematerialsundergogreatchangesinshapeandinternalstructurethatincreaseovertime(i.e.,therelationshipbetweenstressandstrainistimedependent)andcanbepartiallyortotallyrecoveredoncetheexternalforcediminishesordisappears.Elasticmaterialshaveaproportionalrelationshipbetweentheappliedstressesandtheresultingstrains(Hooke’slaw),anddeformationandrecoveryafterremovaloftheexternalforceareinstant,nottimedependent.Chemically,theforcesappliedareaddedtothebondingforcesthatholdthemoleculesandmacromoleculestogetherandthisstoredenergyreturnsinstantaneouslytotheoriginalshapewhentheforceisremoved[16,17].Rheologyisthescienceconcernedwiththestudyoftheflowanddeformationofmaterialsthathaveacomplexmolecularstructure(e.g.polymersandsomebiologicalmaterials)andcannotwithstandastressinequilibriumwithoutchangingtheirviscosity[18].

Stress–straindiagramisthemostcommonrepresentationincuticleliteratureofatensilemechanicaltest.Fig.2showsanexam-pleofastress–straincurve.Asitcanbeobserved,deformationofacuticlesubjectedtoarapidandconstantextensionrateproducesagraphwithtwophaseswithdifferentslopes.Thefirstslopecorre-spondstothelinearelasticphase,whichcanbeabsentorhighly

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5040)aPM30( ssertS20100010203040Strain (%)

Fig.2.Exampleofastress–straincurveshowingtwophases(elasticandviscoelas-tic)withdifferentslopes.Pointsshowpercentageofcuticledeformationafterincreasingloads.

reduceddependingonthesampleandconditions,andthesec-ondtothenon-linearviscoelasticorplasticphase[19,20].Whereasdeformationduringtheelasticphaseisfullyreversible,thesecondphasecorrespondstoamostlyirreversibledeformation.Duringtheviscoelasticorplasticdeformation,thestressneededtostrainthematerialcanincreaseordecrease,showingeitherstrainhardeningorsoftening.Strainhardeningisanincreaseinthestrengthofamaterialduetoorientationofmolecularchainsparalleltotheaxisofstress[16].

3.Cuticlebiomechanics:ageneralview

Thecuticleprotectsplantsbyfulfillingadoublemechanicalrole:itamelioratestheimpactofexternalstressesonplantorgansand,inconjunctionwiththeepidermis,preventsdamagesuchastis-suebreaking.Inaddition,itcontrolsorgangrowthbyimpartingamechanicalrestrainttoinnertissueexpansion[21–23].Epidermalcellsareorganizedinacontinuous(withoutairspacesbetweencells)anduniformmonolayerofstronglyattachedcellswithanasymmetricalcellwalldeposition[24].Thisasymmetryleadstoamuchthickeroutercellwallincomparisonwiththeinnerepi-dermalwallorparenchymacellwalls,althoughhypodermalcellscanalsohavewallthickeningsthatcontributetothemechanicalsupport[21,25].Inaddition,thisoutside/insideasymmetryoftheepidermisisreinforcedbythesecretionofcuticleintothethickenedexternalcellwall.

Theepidermis-growth-controltheorypostulatedin1867andcorroboratedin2007[22]statesthattheepidermisisresponsi-blefortherestrictionoforgangrowth.However,notmuchworkhasbeendonecomparingthemechanicalpropertiesofanintactorgananditsisolatedskin(epidermispluscuticle).Inmostcases,theseexperimentshavebeendonewithcoleoptilesandhypocotylssincetheyrepresenttissuesunderrapidexpansion,withoutsec-ondarygrowth,andcanbeeasilyemployedinmechanicaltests.Resultsindicatedthatthepeelisstretchedorundertension(lon-gitudinalandtransverse)whiletheinnertissuesaremaintainedundercompression.Theskincontractsaround18%uponisola-tionwhiletheinnertissuesexpandabout5%[26].Thisalsoholdstruefortheisolatedcuticle[Heredia,unpublisheddata].Thus,mechanicalcomparisonofanintactcoleoptileandapeeledone(withouttheskin)revealedthattheinnertissuesarecapableofasignificantplasticdeformation,whilenosignificantdifferencesinelasticdeformationwereobservedbetweenintactandpeeledtissue[21,26].Theseresultsindicatethatthepeelisamorerigid

structurethantheinnertissuesandthattheskinismainlyresponsi-bleforthemechanicalpropertiesoftheintactorgan.Thigmonastic(plantnasticresponsetotouch)movementofthistlestaminalfilamentshasbeensuggestedtodependstronglyontheelasticmechanicalpropertiesofthecuticlethatprovidestheforceforfilamentcontraction[27].Strongviscoelasticbehaviorwasalsoobservedinfilamentnasticmovementthatcouldbecorrelatedtoahighratioofpectintocelluloseinthecellwalls[28].

Accordingtoengineeringtheory,stressesarehighestonthesur-faceofabody;therefore,thecuticledepositedontheoutersurfaceoforganswouldplayanimportantroleasastructuralelementaddingmechanicalsupporttotheepidermaltissue[16].How-ever,littleresearchhasbeenperformedtocomparethemechanicalpropertiesoftheepidermalpeelanditsisolatedcuticle[29,30].Althoughtheisolationofastripofepidermislackingcuticlewouldbeidealforstudyingtheprobablemechanicalsynergybetweenepidermalcellsandcuticle,noprocedureknowntodateisabletospecificallyeliminatethecuticlewithoutaffectingthesurroundingtissue.Comparisonoftomatofruitskinandisolatedcuticlerevealedthatthebiomechanicalbehavioroftheskinmirrorsthatofitsiso-latedcuticle,bothbeingisotropic,viscoelasticandstrain-hardeningmaterials[29–31].Themechanicalpropertiesoftheisolatedcuti-cleandskinoftomatofruitswerealsocomparedatdifferentstagesofdevelopment[30].Therewasanincreaseintheelas-ticmodulusduringgrowthtogetherwithatendencytodecreasethemaximumstrainandbreakingstressinboththeskinandcuticle.

Theisolatedcuticleofcherrytomatofruitdisplayedahigherelasticmodulus(E)butalowerworkoffracture(theamountofenergyrequiredtopropagateacrack)thanthepeel[29].Thus,whilethecuticlecanbeinterpretedasamembranethatstiffensthecellwalls,epidermalandsubepidermalcellsgivestrengthtothecuti-cle.Epidermalandsubepidermaltissuesarecapableofsignificantelasticdeformationbeforebreaking[16],theycanbetheninter-pretedasenergysinksthatabsorbstrainenergyastheskindeformssuchthatmoreenergyisrequiredtopropagateacrackthroughcellwallsthanthroughisolatedcuticle[32].Contrarily,comparisonoftheskinanditsisolatedcuticleinnormal-sizedtomatoesshowedahigherE,breakingstressandbreakingstrainintheskinthanintheisolatedcuticle[30].Thesemechanicaldifferencesobserved[29,30]couldbeattributedtothetypeoftomatofruit(cherryvs.normal-sizedvarieties),aneffectofthesolutionemployedtokeepthesampleshydrated,orsimplytothefactthatthesubepidermaltissuewasnotconsidered[30]whenmeasuringthecross-sectionalareaoftheskin.Althoughthecuticlemayhaveasignificantroleinsupportingmechanicalstress,onecannotdismissthemechanicalroleoftheepidermalandhypodermalcells.

Thebiomechanicalnatureoftheisolatedplantcuticlehasbeenmostlystudiedintomatofruitwithafewexceptions.Thetomatofruitcuticlehasamostlyviscoelasticnaturewithanelaticcom-ponentatlowstresses[33].Thismeansthat,inthetime-courseofcuticlecreep,twophasescanbeobserved.Inthefirstphase,thecuticlerespondstoeachloadbyinstantaneousextensionwithnofurtherextensionuntilthenextloadisadded;thestraininthisphaseispurelyelastic.Inthesecondphase,thecuticlerespondsbyinstantaneousextension(elasticstrain)togetherwithsomeaddi-tionalextension(viscoelasticstrain)duringthetimethattheloadismaintained.Thetransitionfromelastictoviscoelasticisusuallygradualwithelasticstrainpredominatingoverviscoelasticstrainatlowerloadsandviscoelasticstrainbeingpredominantatgreaterloads[34].Thisbehaviorimpliesthatthecuticleisstiffandresistsdeformationatlowstresses.Athigherones,itsviscoelasticandstrain-hardeningnatureallowsdeformationandmightthusreduceitsriskofmechanicalfailureduetoenergydissipation.Thegen-eralnatureofthisbehaviorhasbeenconfirmedinseveralotherspeciesaswellasinothertissues[35–37].Asix-foldvariabilityin

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theelasticmodulushasbeenreportedamongspeciesandtissues(0.2–1.3GPa)[35–37].

4.Thecompositionofcuticleanditsroleinmechanicalproperties

Thecuticleisacomplexcompositemembranethatcontainsremnantsofcellwall,acompositestructureitself[38].Althoughmostcuticlesshareasimilarbasiccomposition,differencesinthepercentageofeachcuticlefractionorinthecompositionofafrac-tionhavebeenfoundbetweenspeciesandorgans.Understandingthepossiblecontributionofcuticlecompositiontoitsbiomechani-calandrheologicalperformanceisofgreatinterest.Littleworkhasbeendoneonthistopic,mostlyduetotheinconveniencesderivedfromtheremovalofaspecificcuticlefractionwithoutaffectingtheremainingmaterialandtothelackofcomparablematerial(samespeciesandgenotype)thatdifferonlyinthecomponentofinter-est.Notwithstanding,somestudieshavepostulatedaneffectofthechemicalcompositionofthecuticleonitsbiomechanics[39,40].Thus,cuticleswithahighcontentoftri-hydroxyC18weremoreeasilydeformed[40]andapossibleroleofglucuronicacidincuticledeformabilitywasalsosuggested[27].

4.1.Cutinandpolysaccharides

Importantmechanicalroleshavebeenascribedtocutinandpolysaccharides,thetwomajorcuticlefractions[34].Abiome-chanicalcomparisonbetweencutinandintactcuticleshowedadecreaseoftheelasticmodulusandbreakingstressinthecutinbyafactorof8–13and2–4,respectively[34].Atthesametime,atwo-foldincreaseinthemaximumstraincouldbeobservedinthecutin,whichonlyshowedaviscoelasticphase.Therefore,itcouldbeconcludedthatthepolysaccharidefractionofthecuticleismainlyresponsibleforthelinearelasticbehaviorofthecuti-cleandhighelasticmodulusandbreakingstress,whereasthecutinmatrixischieflyresponsiblefortheviscoelasticbehaviorandhighmaximumstrain[34].Thepresenceofapolysaccha-ridefractioninthecuticle,andthemodificationofthecuticle’smechanicalpropertiesoncethisfractionisremoved,isaclearsignofthesignificantcontributionoftheepidermalcellwalltotheoverallmechanicalbehaviorofthecuticle.Unfortunately,thisepi-dermalmaterialcannotbeisolatedinordertobetested.However,estimationofitselasticmodulususingaVoigt-modelindicatedavaluemuchlowerthantheoneobservedfortheisolatedcuti-cleorskin,andsimilartotheonereportedintheliteratureforhypodermaltissue[29,41].Thefactthatthepolysaccharidefrac-tionofthecuticleischieflyresponsibleofthecuticle’shighelasticmodulus,butthatthisepidermalcellwallfractionisestimatedtohavealowelasticmodulusitself,impliesasignificantsynergyinthemechanicalpropertiesbetweenthecellwallandthecutinmatrix.

4.2.Waxesandflavonoids

Theroleofcuticularwaxesonthemechanicsoftomatofruitcuticlehasalsobeenstudied[33].Waxesactasfillers,sincetheirremovalcausedadecreaseinbreakingstressandanincreaseintheplasticbehavior[33].Unfortunately,onlythemechanicalroleoftotalwaxeshasbeenstudied,althoughmostauthorsagreethattheirroleasfillerscanonlybeattributedtointracuticularwaxes[37],withepicuticularwaxesmakingalimitedcontribution,ifany,totheoverallmechanicalproperties.Nevertheless,itwouldbenec-essarytoverifythisassumptionbycomparingthecontributionofeachwaxfractiontocuticlemechanics.

Finally,thecontributionofphenolics,mostlyflavonoids,hasbeenrecentlyanalyzed.Tomatofruitcuticleaccumulates

flavonoidsduringtheripeningprocess,turningfromcolorlesstoayellow-orangecolor.Thereareseveralknownmutationsintomatowithacolorlesscuticleatmaturityduetothelackofflavonoids.Cuticlebiomechanicalanalysesoftwoofthesemutants,non-ripening(nor)andcolorlessfruitepidermis(y),havebeenperformed[42,43].Thecuticleofnorwassignificantlylessstiffandweakeratmaturitycomparedtothewild-type[42].Thisresultsuggestedapossibleroleofflavonoidsintheincreaseofstiffnessobservedintomatoesduringripening[42].Cuticleoftheymutanthadalowerelasticphaseandconsiderablehigherviscoelasticonecomparedtothewild-typecuticle[43].Examinationofthebiomechanicaldif-ferencesfoundbetweenredripeandmaturegreentomatoshowedthatcuticleswerelessrigidandmoredeformableatmaturegreen,wherenoflavonoidshadaccumulatedyet[34].However,thispro-posedroleofflavonoidsincuticlestiffnesscouldalsobeattributedtootherchanges,suchaspolysaccharidesorchemicalmodificationofthecutinmatrix,observedbetweendevelopmentalstages,orbetweenthewild-typesandbothnorandymutantcuticles.Lately,tomatonaturalvariabilityhasbeenusedtostudythemechanicalroleofflavonoids[44].Genotypeswithflavonoidsinthecuticleatredripeshowedamarkedincreaseinstiffnessduringtheripeningperiod.Bycontrast,tomatoesthatdidnotaccumulateflavonoidsshowedacuticlerheologicalbehavioratredripesimilartomaturegreen,mostlyviscoelasticwithlowstiffness[44].Tosummarize,flavonoidsreinforcetheelasticcontributionofthepolysaccharidefractionand,togetherwithwaxes,playaroleasfillers,sincebothseemtoreducecutinmatrixmobilityandactascompoundsthatincreasecuticlerigidity.

5.Environmentalconditions

Asithasbeendiscussedinarecentandcomprehensivereview,environmentalconditionssuchashydrationandtemperaturearekeyfactorsinthebiophysicalbehaviorofplantcuticlesand,conse-quently,intheirbiomechanicalproperties[38].

5.1.Hydration

Studiesoncuticlewatersorptionhavebeenperformedonalimitednumberofplantspecies.Fruitandleafcuticleshaveawatersorptionbetween1and8%oftheinitialcuticledryweight[45–47].Waxremovaldidnotmodifythecuticlewatersorp-tion,butisolatedcutinhadadrasticallyreducedsorptioncapacity(around63%)incomparisontothecuticle[45].Thisindicatesthatpolysaccharidesareprimarilyresponsibleforcuticlewatersorption[45,46].Intomatofruitcuticle,waterclustering(accumulationofwaterinaliquidstate)above60%relativehumiditywaspredictedtogetherwiththeexistence,attheintramolecularlevel,ofwaterbindingsitesofvariablestrength[47].Watersorption–desorptionincuticleshasasignificantdegreeofhysteresis;thatis,theratesofwatersorptionanddesorptionaredifferent[15].Plantcuticlesreadilyabsorbwaterbutstronglyretainitinitsstruc-ture,makingwaterdesorptionaslowerprocessincomparisontosorption[15].

Theeffectofthewaterhydrationstatusoncuticlebiomechanicshasbeenstudiedinseveralleafandfruitcuticles.Watercausedageneraldecrease,between35and50%,intheelasticmodulusandanincreaseintotalextensibility(deformation)[33,34,36].Asimilareffectofwateronthesurfaceelasticmoduluswasobservedinnanomechanicalstudiesoftomatofruitcuticle[48].Hence,wateractsasaplasticizerincreasingtheviscouscomponentofthecuticleandmodifyingitsmacroscopicappearance.Theeffectofwateronthemechanicalpropertiesofeachcuticlecomponenthasrecentlybeenstudied[34].Waterhadahigh(seven-fold)softeningeffectonthecuticlebutalow(1.5-fold)effectonthecutinmatrix,the

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maincuticlecomponent.Nosofteningeffectofwateronflavonoidswasrecorded,despitetheirsignificantcontributiontotheoverallcuticlestiffness[44].Flavonoidsintrinsichydrophobicity[49]andintramolecularlocalizationasisolatedclustersinthecutinmatrixmightwellexplainthisbehavior.

Takentogether,theaboveresultsindicatethatpolysaccharidesplayacrucialroleinthecuticlerheologicalbehavior.Theyareresponsible,alongwithflavonoids,fortheelasticbehavioratlowstressesand,atthesametime,theyarethecuticlecomponentthatmainlyinteractswithwater.Thus,atlowrelativehumidity,cuticleappearsasastiffcompositematerialsincedrycrystallinecelluloseandhemicellulosesareverystiff[50]and,athighrelativehumidityorwetconditions,polysaccharidessorbwater,swellandsoftenduetothewaterplasticizingeffect.

5.2.Temperature

Fruitandleafepidermisesarephysicallyexposedtoawiderangeoftemperaturesthathaveconsequencesonplantorgantranspira-tion,growthandbiochemicalmetabolism.Plantbiopolymersarestronglyinfluencedbytemperaturechanges,butonlyafewstud-iesonthistopiccanbefoundintheliteratureaboutplantcuticles.Atransitiontemperature,between25and30◦Chasbeenobservedintomatofruitcuticlethatcanbeattributedtocutin[20,51].Thistemperature,withinthephysiologicallysignificantrange,repre-sentsasecond-orderphasetransitionorglasstransitionthatmarkstheborderbetweenarigidmacromolecularstage,likeglass,atlowertemperaturesandaviscousstageabovethetransitiontem-perature,i.e.,athighertemperatures.Cuticlemechanicsshowedastrongdependenceofcuticlestrengthandstiffnessontempera-ture[20,36].Hence,anincreaseintemperaturewasaccompaniedbyadecreaseincuticleandcutinstrengthandstiffness.Unfortu-nately,themechanicalroleoftemperaturehasonlybeenanalyzedintomatofruitcuticleand,althoughthecutinmatrixseemstoberesponsibleforthissofteningeffect,theroleofothercuticlecom-ponentsneedstobetested.

Finally,theoverallrheologicalbehaviorofthecuticlecannotbeunderstoodwithouttakingintoaccountthecloserelation-shipbetweenwaterandtemperature:thehighertherelativehumidityandtemperature,thelowerthecuticlestrengthandstiffness[20,34,36].Thus,thesignificanceofadynamicplantouterenvelopethatmodifiesitsmechanicalpropertiesaccord-ingtochangesinenvironmentalconditions,seasonallyorevendaily,seemstobeclear.Asithasbeenthoroughlydiscussed,hydric,thermalandmechanicalpropertiesconstitutethebasisforunderstandingthebiophysicalbehaviorofisolatedplantcuticles[38].Abehavior,variablewithinlimits,whichdeservesfurtherresearch.

6.Cuticlemechanicsduringgrowth

Acorrelationbetweenthemechanicalbehaviorofcuticleandsomeanatomicalpropertiessuchascuticlethicknessandinvagi-nationhasbeenpostulated[29].Nevertheless,theredoesnotseemtobeaclearrelationshipbetweentheseparameterssincemoremechanicallyresistantcuticlesdonotalwayshavegreaterthick-nessesoraremoreinvaginated[44].Thisrelationshipisprobablymorecomplexandinvolvesnotonlytheseanatomicalfeaturesbutdifferencesincuticledensityaswellaschangesinsomecuticlecomponents.

Littleworkhasbeenfocusedontheevolutionofthemechanicalpropertiesofthecuticleandskinduringplantgrowth,andmostlydealswithfruitgrowthandripening,sincethereareseveraldis-ordersofeconomicalsignificancerelatedtofruitsurfaceintegrityduringgrowthand/orripening.Nosimilarresearchonthemodifi-cationofthecuticlemechanicalpropertiesduringgrowthhasbeen

performedonvegetativetissues.Similarly,studiesonthevariationofthechemicalcompositionofcuticleduringgrowthhavebeenperformedinalimitednumberofspecies,andmostlyfocusedinwaxes[52–55].Thesignificantchangesobservedinwaxchemi-calcompositionduringleafandfruitdevelopmentsuggestthisisatopicofgreatinterestthatneedsadeepanalysis,andshouldbeextendedtoothercuticlecomponents.Inaddition,thesechangescouldhaveaneffectofonthecuticlemechanicalpropertiesduringgrowtheitherdirectly(bymodifyingsomemechanicalparameter)orindirectly(byaffectingthehydricorthermalbehaviorofthecuticle).

Therheologicalpropertiesoftomatofruitcuticleandskinhavebeenstudiedatonlythreestagesofdevelopment(immaturegreen,maturegreenandredripe)[30].Nosignificantchangesinthemechanicalpropertiesduringgrowthwerefoundinthecuticleortheskin[30].Similarly,thenormutationdidnothaveanysignif-icanteffectoncuticlemechanicsduringgrowth,althoughEwasalwayslowerinthemutant[42].However,asignificantincreaseinstiffnessandadecreaseinstrainwasobservedduringripeninginboththecuticleandskin[30,34,42,44].Thisincreaseintheelasticmodulusduringripeningwasnotobservedinnorcuticles,whichhadasimilarEvalueduringgrowthandripening[42].Sweetcherryistheonlyotherspecieswhosecuticlemechanicalpropertieshavebeenanalyzedduringgrowth[56].Atearlystagesofgrowth,thecuticlewasmostlyplastic,withanirreversibledeformationandalowelasticphasethatincreasedduringgrowth.Althoughthemethodologyemployeddidnotallowthedetectionofaviscoelas-ticcomponent,thereprobablywasviscoelasticity,especiallyatearlystagesofdevelopment.Theseresultswithsweetcherryareinagreementwiththoseabovedescribedfortomato:atearlystagesthecuticleseemstobeeasilydeformabletoaccommodatesignif-icantgrowth;atlatestages,whenmostgrowthhasceased,thecuticleincreasesitsstiffness.

7.Theroleofcuticleinfruitquality:apracticalapproach

Anydefectsinthesurfaceappearanceoffruitshaveanegativerepercussiononshelf-life,consumer’sappreciationoftheproductandonitseconomicalvalue.Hence,theinabilityofthecuticletofullyprotectfromexcessivewaterlossoruptake,orpestattackwouldaffectyield,depreciatefruitsorevenrenderthemunmar-ketable.Cuticlemechanicalpropertiesareimportantforfungalpathogenpenetrationbyhighlylocalizedappressorialpressure[57]andalsoforovipositionsubstratechoicebyinsects[58].Recently,ithasbeensuggestedthatcuticlemechanicalpropertiescouldcon-tributetofruitfirmnessandshelf-life[59],atopicthatdeservesfurtherattention.Inaddition,chemicaldifferencesincomposi-tionorquantityofcutincanincreaseepidermalpermeabilityandfacilitateplantinfestationbyflylarvae[60].Cuticlemorphologi-calcharacteristicsalsoplayanimportantroleinfruitpreferenceforindustrialandfreshmarketpurposes.Forexample,thecanningindustrypreferstomatoeswithathickcuticle,whichfacilitatesfruitpeeling,whereasconsumersoffreshtomatoespreferathinnerskin,whichcontributestoincreasedpalatability[61].

Fruitcrackingisadisorderthatcausesanimportantreductionofthecommercialyieldinmanycrops,includingtomatoes[62],sweetpeppers[63],apples[],pears[65],grapes[66],sweetcher-ries[67],watermelons[68],nectarines[69],pomegranates[70],litchis[71]andotherfruits.Itcanoccuratallstagesoffruitgrowth,butmostlywhenfruitsareclosetoripeorduringripening.Cuti-clemechanicalpropertieshavebeenshowntoplayaprominentroleinthisdisorder[61].Thisrelationshipbetweencuticlebiome-chanicsandcrackinghasbeeninvestigatedmostlyintomatoesandsweetcherries.Intomato,acorrelationhasbeenobservedbetweenfruitsizeandcuticleaccumulationduringfruitontogeny

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[72–74],whoserelationshipwithcrackinitiationwouldbeinter-estingtoinvestigate.Cuticlewasslowlyaccumulatedduringfruitgrowthuntilitreacheditsmaximumatripeninginmediumorlargetomatoes[72,73],whileincherrytomatoes,maximumaccu-mulationoccurredinthefirst15daysfollowedbyasteadystatethatmaintainedtheamountofcuticlereached[74].Sweetcherry,theonlyotherspecieswhosecuticleaccumulationduringfruitgrowthhasbeenstudied,showedanotherpatternofcuticlesynthe-sis[75].Therewasafastaccumulationofcuticleinthefirststagesofdevelopmentfollowedbycuticlesynthesiscessationandcuticlethinningduringtherestofthegrowingperiod[75].Interestingly,inmediumorlargetomatoeswherethecuticleisslowlyaccumulated,crackinitiationcantakeplacethroughouttheentireperiodoffruitdevelopmentandripening[76].Onthecontrary,incherrytomatoandsweetcherrywherecuticleissynthesizedatearlystages[76],cracksonlyoccurduringtheripeningprocess[76,77].

Sweetcherrycrackingismainlyduetowateruptakethroughtheskinattheendoftheripeningprocess,whichproducesafastvol-umetricincreasethattheskincannotwithstand[77].Differencesintheratioofcuticledepositionandfruitsurfaceexpansionduringgrowthresultinthedevelopmentofincreasingstressestoacon-tinuouslythinningcuticle,leadingtotheformationofmicrocracksthat,alongwithchangesinwaxconstituentsduringripening,facil-itatewaterpenetrationinthefruitand,subsequently,fruitcracking[77].

Incherrytomato,norelationshipbetweentheearlystagesofcuticleformationandfruitcrackingwasobserved,suggestingthattheeventsleadingtofruitcrackingprobablyhappenduringripen-ing[74].Inthisperiod,thereisadecreaseincuticlepolysaccharidesduetocellwalldegradationthatiscompensatedbyasignificantaccumulationofflavonoids,whichistranslatedtoanetincreaseincuticlestiffness.Cherrytomatoincreasesitsvolumeforatleasttwoweeksduringtheripeningperiod[Cuartero,unpublisheddata].Thus,therigidredripecuticlemaynoteasilyallowthenecessarycuticledeformationforthisfinalvolumetricincrease,especiallyinfastgrowingfruits,andthecuticlethencracks.

Fromtheaboveresults,thereseemstobeapossiblerelationshipbetweencuticlesynthesisandthestageofdevelopmentatwhichafruitismoresusceptibletocrack.Nevertheless,thepatternofcuti-cleaccumulationshouldbeinvestigatedinmorespeciesinordertoascertainthispossibility.Alternatively,putativecuticlevariationsamongfruitswithinaplantduetodifferentmicroenvironmentsorphysiologicalconditions,orevensubtlecuticlevariationswithinafruitshouldbeconsidered.Integrationofcuticlepropertieswiththeenvironment(waterandtemperature)andplantphysiologycouldallowtheidentificationoftraitssuitableforselectioninaplantbreedingprogram.

Theeffectofexogenouslyappliedcompoundsoncuticlebiome-chanicsisaninterestingsubjectwithimportantagriculturalapplications.Surfactantsarewidelyusedtoincreasecuticularper-meability[78,79]andfacilitatethepenetrationoffoliarappliedchemicals,suchaspesticides,intotheplant.Thisincreaseincuti-clepermeability,probablyaresultofchangesinthecutinpolymermatrix,couldalsoaffectcuticlemechanicalproperties.Thereisonlyonestudyintheliteraturereportingtheeffectofasur-factant,TritonX-100,oncuticlerheology[33].AlthoughresultsconcludedthatTritonX-100didnotaffectthemechanicalper-formanceofthecuticle,morestudieswithsurfactantsofdifferenthydrophilic–hydrophobicbalance(HLBindex),inthecaseofnon-ionicsurfactants,andwithionicsurfactantsofdifferentpolarityareneededbeforerulingoutitspotentialroleoncuticlemechan-ics.Theseanalyseswillallowustodiscernifthesorptionofanadjuvantcanmodifytheinteractionsbetweencutinmacromolec-ularchains,responsibleofthecuticleviscoelasticbehavior,and/orspecificinteractionsbetweenthesechainsandintracuticularwaxesandphenolics/flavonoids.

8.Futureandperspectives

Theabilityofaplantcuticletomodifyitsmechanicalbehaviorinresponsetoenvironmentalcuesaswellasinternalpressuresispivotalinunderstandingplantgrowthandadaptationtothesurroundingenvironment.Thus,cuticlemechanicsshouldideallybestudiedwithinthecontextoftissue,organandevenwholeplant.Unfortunately,littlehasbeendonetopursuethisapproachsincethecompositenatureofthecuticleitself,itsyetunknowninteractionwithepidermalcellwalls,aswellasthecomplexrela-tionshipbetweendifferenttissueswithinanorganandorganswithinaplant,hinderthisapproximation.Someanalyseshavebeenmadeonthecontributionofthecuticletoepidermalmechanics[29,30]butnottowholeorganmechanics,aparticularlyinterestingapproachforinvestigatingsomedisordersofagriculturalinterest.Anotherreasonthathashamperedthisapproachisthatcuticlesneedtobeenzymaticallyisolatedinordertostudythem.Althoughitisassumedthattheisolatedcuticleissimilartothecuticlethatisstillattachedtotheplant,thisisnotnecessarilytrue.Itsinti-mateinteractionwiththeepidermismostprobablymodifiescuticlemechanicsandexhibitssynergisticproperties.

Mechanicalanalyseshavebeenprimarilyperformedusingtomatofruitcuticleasamodel;atmostadozenotherplantspecieshavebeenstudied[33,35,36].Thesignificantdifferencesobservedbetweentomatogenotypesimplyanimportantsourceofintraspe-cificvariationthatneedstobeexploredandcorroboratedinotherspecies.Also,changesinthemechanicalpropertiesduringorgangrowthshouldbeanalyzedindepth,notonlyinfruitsbutalsoinleaves,wherealmostnoworkhasbeenperformed.Theeffectofenvironmentalconditionssuchaswaterandtemperatureshouldbeexaminedinconjunctionwithplantgrowthtounderstandhowtheyaffectnotonlythemechanicalbehaviorofanalreadyformedcuticlebutalsogeneexpressionlevelsduringgrowth.Itiswellknownthatwaterandtemperatureaffecttheexpressionofgenesinvolvedinwaxsynthesis[80,81]andthatlightaffectstheamountofflavonoidsaccumulated[82–84].Thus,itwouldbeimportanttoknowwhetherwaterandtemperaturealsoplayasimilarroleongenesinvolvedincutinsynthesisandhowthiscouldultimatelyaffectthemechanicalperformanceofcuticle.

Severalcuticlemutantshavebeencharacterizedinthelastfewdecades[7andreferencestherein]andthegenesresponsi-bleforthemutationshavebeenidentified.Thismaterialshouldprovideanexcellentsourceforunderstandingtheroleofspecificcomponentsormorphologicalarrangementsincuticlemechan-ics.Unfortunately,mostofthesemutantshavebeenidentifiedinArabidopsis,aspecieswithanextremelythincuticlethatisdif-ficulttoisolate.Therefore,rheologicalanalysesofthesecuticlemutantshavenotbeenperformed.AnorthologoftheArabidop-siscuticlegeneCER6hasbeenidentifiedandanalyzedintomato[85].Rheologicalcharacterizationofthemutantcuticles,identi-ficationandstudyofotherorthologsofknownArabidopsiscuticlegeneswouldallowthetransferofknowledgealreadyinArabidopsistospeciesthatcouldextendourcomprehensionoftheplantcuti-cle.Identificationofthemajorgenesinvolvedincuticlesynthesiswouldbebeneficialforbetterunderstandingthemechanicalroleofeachcuticlecomponent.Thankstotheavailabletechnology,genesofinterestcouldbesilencedinmodelplantswithwell-knowncuticlesandtheirmechanicalandbiophysicalrolecouldbestud-ied.Althoughcompletegenesilencingwouldbemostdesirableintheanalysisofthemechanicalroleplayedbyaspecificcuti-clecomponent,somecuticlegeneswouldpossiblyonlybetestedwithknock-downmutants,sincetheirknock-outcouldbelethal.Oncediscernedthemechanicalroleplayedbyaspecificcuticlecomponent,knock-downandover-expressionmutantscouldbeusefultoidentifytherangeofvariationbeneficialforplantperfor-mance.Inthelongrun,thisinformationwillallowbreedingnew

E.Domínguezetal./PlantScience181(2011)77–84

83

cultivarswitharationalcuticledesignabletowithstandcertainenvironmental,pest,ormechanicalpressuresandthusimprovetheiragriculturalvalue.

Themeasurementofcuticlemechanicsisdependentontheequipmentavailable.Mostoftheversatileequipmentofferedbycommercialcompanieshasbeendevelopedtoanalyzeindustrialmaterials,sothesizeofthesample,theattachmentofthesampletotheclampsandthesensitivityoftheequipmentdonotmatchtheneedsofcuticlemechanics.Isolatedcuticlessamplesareofsmallsize,verythinandfragile,thustheirhandlingformechanicalpur-posesiscomplicated.Therefore,researchgroupshaveinvestedincustom-designedequipmentsforstudyingtherheologicalproper-tiesofcuticles.Thisisalsotrueforstudyingmechanicsatthewholeorganorplantlevel.But,arethedataobtainedfromthesedifferentapparatusestotallycomparable?Tensiletestisthemostcommontypeofmechanicaltestperformedonthecuticleandmostcustom-builtequipmenthasbeendesignedtoperformit,butthereareotherteststhatcangiveimportantinformationonbiologicallyrelevantmechanicalpropertiessuchasload-unloadandstressrelaxation(materialisquicklystrainedtoafixedamountandthestressneededtomaintainthisstrainismeasuredasafunctionoftime).Itwouldbemostdesirabletoextendthemechanicalanalysesandincludethesetypesoftestsiftheequipmentallowsthemorcanbemodifiedtoperformthem.

Mostifnotallmechanicaltestsperformedonthecuticleareuni-axialtests:stressesappliedononlyoneaxis.Nevertheless,plantsorplantorgansaresubjectedinvivotostressesappliedonmorethanonedimension,evenwhengrowthpreferentiallyoccursinoneaxisduetodifferentgrowthratesorturgorpressures.Equip-mentallowingbiaxialteststhatapplythesameordifferentialstressesoneachaxisandareabletoworkwithbothcuticleandpeel(cuticleplusepidermalcells)wouldbedesirableforapproxi-matingtheinvivoconditionsofsomefruits,especiallybellpeppers,tomatoes,cherries,etc.,wheresurfacecracksproducesignificanteconomicallosses.Inthissense,two-dimensionalrheologicalprop-ertiesofgrapeberriesinvivohavebeenstudiedwiththeaidofaninjectiontesterthatusedwatertoincreaseinternalpressure,andallowedthecorrelationoffruitskinstrainwithsplittingsus-ceptibility[86].Morerecently,ahydraulic2Dtestingdevicewassetuptoanalyzemechanicalpropertiesofsweetcherryskinsub-jectedtohydraulicpressure[87].Suchapparatuswouldallowustobetterunderstandtheequilibriumofforcesafruitissubjectedto.Thesebiaxialtestingdevices,althoughusefulforspheroidfruits,didnotallowthesimulationofmechanicalstressesthatwouldoccurinnon-spheroidfruitsororgans.Clearly,informationfrombiax-ialmechanicalanalyses,thoughstillinitsinfancy,willbeastepforwardinthecomprehensionofinvivoplantcuticlemechanics.Additionally,thedevelopmentoftheoreticalmodelsthatintegratethemechanicalinformationofcuticleandepidermisintoanidealfruitwillalsoadvanceourknowledgeonthesubject.Littleprogresshasbeenmadeonthistopicandonlyrecentlyamathematicalmodelthatdescribestherheologicalbehaviorofisolatedtomatocuticlesfromexperimentallymeasuredpropertieswasformulated[88].

Thestudyofcuticleandplantmechanicsisnotonlylimitedbythedevelopmentofadequateequipment.Viscoelasticityandplas-ticityaremechanicalphenomenathatplayahighlysignificantroleinbiologicalsamples[].Nevertheless,atheoreticalcorpusthatfullyexplainsthesemechanicalbehaviorshasnotbeendevelopedyet.Thus,ourcomprehensionofthemislimitedtoaphenomeno-logicalapproach.

Tosummarize,thecurrentmechanicalmodelregardingthecuticlerheologicalbehaviorwouldbeaviscoelasticmatrix(cutin)containingfibrillarcomponentscapableofpassiverealignmentwhenplacedintension.Thesefibrilswouldbecellwallderivedsinceithasbeenobservedthatcellwallfibrilscanaligninthe

directionoftheappliedforcesuchthattheeffectiveYoung’smod-ulusincreases[90].Insidethismatrix,fillerssuchaswaxesandflavonoids,presentatlowconcentration,canmodulatethestiffnessandothermechanicalparameters.

Acknowledgements

TheauthorsaregratefulforfinancialsupportreceivedthroughgrantsAGL2006-12494,AGL2009-12134andTRA2009-0375fromthePlanNacionaldeI+D,MinistryofEducationandScience,Spain,andcollaborationswiththeFundaciónCajamarandRijkZwaanIberica(Almería,Spain).

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