TECHNIQUESFORMOLECULARANALYSISPracticalapproachestoplantvolatileanalys - PDF document

TECHNIQUESFORMOLECULARANALYSISPracticalapproachestoplantvolatileanalysisDorotheaTholl,WilhelmBoland,ArminHansel,FrancescoLoreto,UrsulaS.R.Ro¨seandJo¨rg-PeterSchnitzler emissionofVOCsincludingCgreen-leafvolatiles(e.g.(hexenaland()-3-hexenylacetate),methylsalicylate,methyljasmonate,indole,terpenesandothers.Thesevolatilescanactasdirectdefensecompounds(Andersenetal.,1994;DeMoraesetal.,2001)orplayaroleinindirectdefensebyattractingnaturalenemiespreyinguponorparasitizingherbivores(Dickeetal.,2003;KesslerandBaldwin,2001;etal.,2005;Turlingsetal.,1995).Finally,chemicalvolatilesignalsreleasedfrominjuredplantsnotonlyaffectherbivoresorpathogensbutmayalsosignalalarmtoneighboringplantsbytriggeringdefenseresponses(Arim-etal.,2000;Engelberthetal.,2004).Independentoftissuedamagebyotherorganisms,numerousplantsemitVOCsinresponsetolightandtemperaturechangesorotherabioticstresseslikeßoodingordrought(Ebeletal.,1995;Holzingeretal.,2000;Kreuzwie-etal.,2000).Low-molecular-weightterpenessuchasisoprene(C),monoterpenes(C)andsesquiterpenes(Carereleasedinsubstantialamountsfromwoodyplants.TheyhavesigniÞcantimpactonatmosphericchemistrysincetheycontributetotheformationofozoneandsecondaryorganicaerosolsinthepresenceofanthropogenicpollutants(Atkin-son,2000;Hoffmannetal.,1997;KesselmeierandStaudt,1999;SharkeyandYeh,2001).Furthermore,isoprenoidemissionsinßuencelevelsofatmospherichydroxylradicalsOH)andtheatmosphericresidencetimeofmethane,animportantgreenhousegas(Thompson,1992).Thephysiolo-gicalroleofsuchterpeneemissionsisstillnotcompletelyunderstood.Volatileterpenesarebelievedtoimprovethethermotoleranceofphotosynthetictissuessincetheyarelikelytointercalateintothylakoidmembranesandmaystabilizethemathightemperatures(Loretoetal.,1998;etal.,2001).Thereisincreasingevidencetoshowthatterpenevolatilesexhibitantioxidantactivitiesinplantabyquenchingreactiveoxygenspecies(LoretoandVelikova,2001;Loretoetal.,2001b).Moreover,terpenevolatileemis-sionsarethoughttoactasametabolicsafetyvalvetoavoidtheunduesequestrationofphosphates(Rosenstieletal.TheincreasingscientiÞcinterestinthebiochemistry,physiology,ecologyandatmosphericchemistryofplantVOCshasledtothedevelopmentofavarietyofsystemsforthecollectionandanalysisofvolatiles(LinskensandJack-son,1997;MillarandSims,1998).Inthepastdecadeinparticular,volatileanalysishasimprovedbythedesignofrelativelyinexpensivebutsensitivebench-topinstrumentsforgaschromatographyÐmassspectrometry(GC-MS).ThedevelopedheadspaceanalysistechniquesprovideamorerepresentativevolatileproÞleoflivingplantsthantraditionalmethodsofsolventextractionorsteamdistillation.Besidesmanuallyoperatedheadspacesamplingmethods,automa-tedVOCanalysissystemswithhightimeresolutionandon-linecapabilityhavebecomeindispensableformonitoringfastchangesofvolatileproÞlesduringplantdevelopmentorunderstressconditions.Theneedforreal-timemeasure-mentshasledtoconsiderableinterestinnon-chromato-graphicmethods,mostofteninvolvingchemiluminescenceorinfraredphotoacoustic(PA)spectroscopy,ormassspectr-ometry[e.g.protontransferreaction(PTR)-MS].Suchsys-temscanbecombinedwithadditionalequipmentforparallelmeasurementsofotherstress-sensitivephysiologicalparameterssuchasphotosyntheticactivity.VOCanalysissystemsthataresensitive,fastandfullyautomatedarealsoofincreasingimportancetoelucidatethebiosynthesisofplantVOCs.Inaddition,functionalgenomicsapproachesfordissectingthemetabolicpathwaysofplantVOCsdemandtime-efÞcienttechniquesforvolatileproductanalysisofrecombinantenzymesorhigh-throughputproÞlingofvola-tilemetabolitesofmutantandtransgenicplantlines.InthispaperwewilldescribetheprincipalmethodsandlatesttechnologiesusedintheanalysisofplantVOCsindifferentareasofresearch.Theadvantagesanddisadvantagesassociatedwitheachmethodwillbepoin-tedouttofacilitatethechoiceofthemostappropriatemethodandequipmentrequiredforaparticularresearchSamplingandanalyzingplantvolatiles–whichmethodtoAllmethodsfortheanalysisofplantvolatilesattempttoidentifytheauthenticproÞleofvolatileblendsemittedbyaplant.However,thechoiceofwhichsystemtouseinaparticularexperimentforcollectionandanalysisofplantvolatilesusuallydependsonthebiologicalproblemandplantmaterialbeinginvestigated(Figure1).First,itneedstobedecidedwhethervolatilesshouldbeanalyzedfromplantsgrownunderlaboratoryconditionsorintheirnaturalhab-itat.Toaddressecologicallyorphysiologicallyrelevantfunctionsofvolatilesinnaturalpopulations,ÞeldcollectionsofVOCsrequireportable,robustandoftensimpliÞedequipment.Incomparison,systemset-upsinthelaboratorycanincludecomputer-assistedsamplecollectionandaddi-tionaldevicestoreducecontaminationandtopreciselycontroltemperature,lightintensity,photoperiodandrelat-ivehumidity.StandardizedconditionsallowthemonitoringofVOCemissionsinresponsetochangesofasinglevariablesuchasherbivoredamage.Volatilesaremostconvenientlycollectedinsituwholeplants.However,itisoftenrequiredtosampleVOCsfromplantpartsororgans,forexampletodistinguishthevolatilesofreproductiveandvegetativetissues,todeter-minestress-inducedVOCemissionsaslocalorsystemicresponsesortocorrelateVOCemissionswithtissue-speciÞcenzymeactivities.Inthiscase,VOCsaresampledeitherfromdetachedplantpartsorpreferablyinsitufromenclosedplantorganstoavoidadditionalemissionofVOCsduetowoundingeffects.Plantvolatileanalysis2006TheAuthorsJournalcompilation2006BlackwellPublishingLtd,ThePlantJournal,(2006),,540Ð560 Dependentontheplantspeciesinvestigated,VOCemis-sionratesmayvarysigniÞcantlyandhencedeterminewhichinstrumentationisrequiredforadequatecollectionefÞciencyandanalysissensitivity.WhiletraceamountsoftrappedvolatilesmaybesufÞcientforanalyticalpurposes,largerquantitiesarenecessaryforNMRanalysisorbioassays.Inaddition,theresearcherhastodecidewhethertotakeamorequalitativeÔsnap-shotÕofvolatilesreleasedataparticulartimepointortomeasurequantitative,developmentalorstress-relatedchangesinVOCemissionswithappropriatetimeresolution.Finally,specializedequipmentallowingon-lineorhigh-throughputanalysishastobeconsideredforstudiesofplantvolatilemetabolismormutantscreenings.ToguidetheinexperiencedresearcherinthechoiceofappropriateVOCanalysisprotocols,themostimportantmethodsusedtoinvestigatethebiologicalrolesofplantvolatilesandtheirmetabolismarepresentedinmoredetailinthefollowingsections.Samplingvolatilesintheheadspaceofwholeplantsandplantorgans–practicalaspectsandapplicationsVolatileshavebeeninvestigatedmostextensivelyintheairspace(headspace)surroundingabove-groundplantparts.MajorÞeldsofstudyincludeanalysesofßoralvola-tilesinrelationtopollinationbiology,measurementsofvolatilessuchasisoprenereleasedfromphotosynthetictissuesinresponsetochangesinlightandtemperature,andvolatileemissionsinducedbyherbivoredamage.Inmostcases,theemittedvolatileshavetobesampledandconcentratedpriortosubsequentanalysis.Headspacesamplingisanon-destructivemethodforcollectingvola-tiles.Comparedwithsolventextractionsofvolatilesfromplanttissues,headspaceanalysisgivesamorerealisticpictureofthevolatileproÞleemittedbyplantsanddetectedbyinsectsthatrespondtoplantvolatiles,makingthismethodmostsuitableformanyecologicallyrelevantappli-cations.Ingeneral,devicesusedforheadspacecollectionsshouldbefreeofmaterialsthatretainvolatilesorcausebleedingofcompoundsthatmaycontaminatethesystemandinterferewithvolatileanalysis.Commonlyusedmate-rialsthatdonotshowbleedingincludeglass,metalandspecialplasticssuchasTeßon,althougheventhismaterialmaynotbecompletelyinert.DetailsofmaterialssuitablefortheconstructionofheadspacecollectionchamberscanbefoundinMillarandSims(1998).SamplingvolatileorganiccompoundsinstaticheadspaceForstaticheadspaceanalysis,theplantoritspartsareenclosedinacontainerandtheemittedvolatilesaretrappedontoanadsorbent.TheairsurroundingtheplantremainsÔstaticÕ,whichmeansitisnotcirculatedinthechamber.Volatilesareenrichedontheadsorbingmatrixwithoutsamplingimpuritiesofacontinuousairstreamthatmayobscurethedetectionoflow-abundantVOCs.Thus,this LaboratoryMethod requirementsSimplicityPortabilitySensitivityQualitative/quantitativeTime resolution Plant organs– in situ GC supported methods:Static headspace samplingDynamic headspace samplingFast, GC-independent analysis(e.g. PTR-MS) Light and temperature dependent tissues; e.g. isoprene andmonoterpenes (b) Figure1.Strategiesforplantvolatileanalysis.(a)TypicalsourcesofplantVOCemissions.(b)ConsiderationsforplanningVOCanalysisexperiments.DorotheaTholletal.2006TheAuthorsJournalcompilation2006BlackwellPublishingLtd,ThePlantJournal,(2006),,540Ð560 methodisadvantageousforsamplingVOCsfromlow-emittingplants.Animportantadvanceinstaticheadspaceanalysiswasthedevelopmentofsolidphasemicroextraction(SPME)whichisafastandsimplemethodforcollectingvolatilesatdetectionlimitsintheppbv(partsperbillionbyvolume)range.Solidphasemicroextractionisbasedonad/absorp-tionanddesorptionofvolatilesfromaninertÞbercoatedwithdifferenttypesofad/absorbents.TheÞberisattachedwithintheneedleofamodiÞedsyringeandvolatilescanbesampledbyinsertingtheneedlethroughaseptumofaheadspacecollectioncontainerandpushingtheplungertoexposetheÞber(Figure2).FollowingequilibrationbetweentheÞberandthevolatilesample(afewminutestohalfanhour),theÞberisretractedintotheneedleandcanbetransferredtoagaschromatographfordirectthermaldesorption.SolidphasemicroextractionÞberscanbereusedapproximately100times.ThermaldesorptionofVOCsfromtheÞbereliminatestheneedforsolventsthatmaycontainimpuritieswhichwillinterferewithsampleanalysis.However,bydesorbingtheentiresampleintotheinjector,norepeatedinjectionsofthesamplearepossible.BycarefullyselectingthepolarityandthicknessoftheÞbercoating,compoundsofdifferentpolarityandvolatilityrangingfromhigh-boilingorsemivolatiletovolatilecom-poundscanbesampled(TableS1).Whilethincoatingsensureafastdiffusionandreleaseofsemivolatilecom-pounds,thickercoatingsmaybetterretainhighlyvolatilecompoundsuntilthermaldesorption.Theamountofcom-poundadsorbedbytheSPMEÞberdependsnotonlyonthethicknessoftheÞbercoatingbutalsoonthedistributionconstantoftheanalyte,whichgenerallyincreaseswithitsmolecularweightandboilingpoint.Solidphasemicro-extractionwillnotallowtrappingofsufÞcientamountsofvolatilesforstructureelucidationofunknowncompounds.LargeramountsofVOCscanbesampledonironstirbars,whicharecoatedwiththesamesorbentsasSPMEÞbersandwereoriginallydevelopedtoextractorganiccompoundsfromaqueoussamples(Bicchietal.,2000).ThebarisplacedintheheadspaceoftheplantsampleandtrappedvolatilesarereleasedbythermaldesorptionaftertransferofthebarintotheGCinjectorliner.QuantiÞcationofvolatilesbySPMEisgenerallypossiblebytheapplicationofinternalorexternalcalibration.Toobtainreproduciblequantitativeresults,theÞberandsampleshouldreachequilibrium,atwhichtheamountofanalyteremovedfromtheÞberisproportionaltotheconcentrationofthecompoundinthesample.Equilibra-tiontimeisdependentonthevolatilityandpolarityoftheanalyteandthepropertiesoftheadsorbent.Carefulcontrolofsamplingparameters(samplevolume,tempera-ture,time)andtheuseofappropriatestandardmixturesforcalibrationarecrucialforquantitativeanalysis.How-ever,quantiÞcationbySPMEmaystillbedifÞcultorimpracticalwhendealingwithawiderangeofcompoundswithdifferentdistributionconstants.TheSPMEdeviceandfurtherdetailedinformationonthetheory,optimizationanddifferenttypesofÞberadsorbentsareavailablefromSupelco(Bellefonte,PA,USA).PortableÞeldsamplersarealsoavailablethathaveasealingmech-anismtoallowstorageofsamplesforlateranalysisinthelaboratory.SeveralcompaniesofferGCautosamplerswithaSPMEoptionforrapidprocessingofmultiplevials.How-ever,thesizeandshapeofvialsinthesesamplersareÞxedandoftenimpracticalforsamplingfromplants.Forrapidscreeningofmultiplesamplesinvariabletypesofcontain-ers,anautomatedadjustableroboticarmhasbeendevel-oped(Pham-Tuanetal.,2001).AutomatedSPME-GCallowshigh-throughputanalysisofvolatileproÞlesemittedfromplantpartssuchasßowersorleavesofnaturalvariants,mutantortransgeniclinesasshownbyAharonietal.ArabidopsisthalianaNumerousstudieshaveusedSPMEextractionfortheanalysisofabroadrangeofvolatilecompoundsinfood,air,soilandwatersamples.BiologicalapplicationsincludeSPMEanalysesofVOCsfromwholeplantsorßowerandbarktissues(e.g.Flaminietal.,2002;RohloffandBones,2005;Shaetal.,2004)andessentialoils(e.g.Tomovaetal.,2005).AsanalternativetotrappingVOCsonadsorbents,directheadspacesamplingispossiblebyremovinganaliquotoftheheadspacewithagas-tightsyringeandinjectingitdirectlyintothegaschromatograph(seealsosectionon Fiber holder Plant sample coated fibe r Container seal Figure2.Staticheadspacesamplingwithasolidphasemicroextraction(SPME)device.ForvolatileextractionbySPME,theplantsampleisenclosedinaglasscontainerwithabroadopeningforeasyremovaloftheplant.Theadsorbent-coatedÞberismountedonaSPMEÞberholder,similartoamodiÞedsyringethatisinjectedthroughtheseptumofthesamplecontainer.BypushingtheplungeroftheSPMEÞberholder,theÞberisextendedfromtheneedleandexposedtovolatiles.Aftercollection,theÞberisretractedintotheneedleandtheSPMEdevicecanberemovedfromthecontainerforGCanalysis.Plantvolatileanalysis2006TheAuthorsJournalcompilation2006BlackwellPublishingLtd,ThePlantJournal,(2006),,540Ð560 VOCanalysistechniquesforstudyingthebiosynthesisofplantvolatilesandtheirroleinstressphysiology).Theprocesscanbeautomatedwithcommercialheadspaceautosamplers.However,directheadspacesamplingre-quiresasufÞcientlyhighconcentrationofVOCsintheheadspacetoprovideatleastnanogramquantitiesinthesampletakenforGCanalysis.Thus,themethodislimitedbytheneedforsatisfactorysensitivity.DespitethelatestadvancesinSPMEtechnology,staticheadspacecollectionhassomecleardisadvantages.Thestaticairspaceaccumulateshumidityaswellasheat,especiallywhensamplesarecollectedunderillumination,andtheseconditionsmayinterferewithnormalphysiologi-calprocessesandaffecttheemissionofvolatiles.Becausenotalloftheemittedvolatilesareremovedduringonesamplingtime,changesinemissionovertimewillbedifÞculttodetermine.Inconclusion,staticheadspacesamplingissuitedforqualitativeanalysesofVOCsandsurveysofVOCproÞlesofdifferentplantspeciesorcultivarsatasingletimepointratherthanforquantitativemeasure-mentsofchangingVOCemissions.DynamicheadspacesamplingtechniquesDynamicheadspacesamplingrepresentsthemostfre-quentlyusedtechniqueinallareasofplantvolatileanalysis.Inthissamplingmethod,acontinuousairstreamßowsthroughthesamplecontainerasacarriergas,whichincreasestheheadspacesamplesize.Whiletheanalytesaretrappedonadsorbents,thecarriergasiscirculatedthroughorpurgedoutofthecontainer,allowingforthecollectionofamountsofvolatilessufÞcientfordetectionandevenstructureelucidation.Inopendynamicheadspacesystemsinparticular,someoftheproblemsrelatedtoastatichead-spacesuchasincreasesintemperatureandhumidityoranaccumulationofdeleteriousvolatilesintheheadspaceareeliminatedbyaconstantairstream.However,careneedstobetakentoprovidecleanincomingairthatisÞltered,forexamplethroughactivatedcharcoal,toavoidinterferenceofimpuritieswithheadspacevolatilecompoundsintheensu-inganalyticalsteps.Inmostcasesofdynamicheadspaceanalysis,volatilesaretrappedandenrichedonanadsorbingmatrixpriortotheiranalysisbyGC.Alargenumberofdifferentadsorb-entmaterialsareavailableandseveralreviewshaveprovidedexcellentinformationregardingtheirchoiceandapplications(DettmerandEngewald,2002;LinskensandJackson,1997;MillarandSims,1998;RagusoandPellmyr,1998).TableS1givesanoverviewofthemostcommonmatricesincludingcarbon-basedadsorbentsandorganicpolymers,withinformationontheirthermalstability,bindingafÞnitiesandartifactscausedbyreac-tionsoftheadsorbingmaterialitselfortheadsorbedVOCsonthematrixsurface.Theadsorbentmaterialisusuallypackedinsidenarrowglassormetaltubesinbedsofapproximately2Ð50mmbetweenglasswoolplugsormetalgrids.Theaircontain-ingtheVOCsispassedthroughtheadsorbentbedataparticularßowrateduringthesamplingprocess.Trappedvolatilescanbeelutedfromtheadsorbingmatrixintoglassvialswithpuresolventsormixturesoflow-boiling-pointorganicsolvents.ThesolventshouldcontainadeÞnedamountofoneortwostandardcompounds(e.g.1--octane)forsemiquantitativeanalysis.AdsorbentmaterialswithhighthermalstabilitysuchasTenax,carbonmolecularsievesandgraphitizedcarbonblackscanbeemployedinthermaldesorptionofVOCsasanalternativetosolventextraction.Inthermaldesorption,VOCsaredirectlyreleasedfromtheadsorbentunderhightemperaturesandareusuallyconcentratedbycryofocusingpriortoseparationbyGC(seebelow).Comparedwithsolventextraction,thermaldesorptionprovidesincreaseddesorptionefÞcienciesandpreventssampledilution,lead-ingtoincreasedanalyticalsensitivity.Otheradvantagesarereducedmanualsamplepreparationtimesandtheavoid-anceofimpuritiesintheorganicsolventinterferingwithGCanalysis.However,thermaldesorptionisnotfreeoflimitations,whichincludethelackofrepeatedsampleinjectionsandtheoccurrenceofartifactsduetothedegradationofthermallyunstablecompoundsorreactionsofthetrappingmedia(TableS1).AmajorproblemwithalltrappingmaterialsistheirincompleteadsorptionofVOCs.Forexamplecarbon-basedmatriceshaveveryspeciÞcafÞnitiesforVOCs.ThosematerialsthateffectivelytrapVOCsoflowpolarityandlowmolecularweightareinefÞcientforadsorbingVOCswithoppositecharacteristics.Therefore,whencollectingcom-plexmixturesofVOCscaremustbetaken,andeventuallymorethanoneadsorbentmayberequiredtotrapablendthatisqualitativelyandquantitativelyrepresentative.Thisisespeciallycriticalwhenthemixtureisusedforsubsequentbioassayssuchasinsectattractionexperiments.Thisprob-lemcanbesolvedbyÔmultiplelayeradsorptionÕwherecarbonmatriceswithdifferentretentioncharacteristicsareplacedinserieswithairßowingÞrstintothematrixadsorbingveryvolatileVOCs(e.g.CarbographandCarbo-trapC).ThetheoryandpracticeofVOCsamplingwithmulti-bedtubesgobeyondthescopeofthisreportandhasbeenextensivelydetailedelsewhere(Cicciolietal.,2002).Multi-bedtubesarecommerciallyavailable(e.g.Carbotrap/CarbosieveSIIIbeds,MarkesInternational,Pontyclun,UK)orcanbeself-made(Schnitzleretal.,2004b).Closed-loopstripping.Closed-loopstrippingsystemshavebroadutilityforthecollectionofherbivore-inducedvolatiles,asshownforinducedVOCemissionsfromlimabean,aswellasfortrappingvolatilesfromdetachedßowers(Dudarevaetal.,2005;Kochetal.,1999;Tholletal.,2005).IntheseDorotheaTholletal.2006TheAuthorsJournalcompilation2006BlackwellPublishingLtd,ThePlantJournal,(2006),,540Ð560 systems,volatilesarecollectedduringcontinuouscircula-tionofheadspaceairinsideclosedchambers.Asimpleclosed-loopstrippingsystemdevelopedbyBolandetal.(1984)andDonathandBoland(1995)consistsofsmall1-or3-lglassdesiccatorstowhichaircirculationpumpsareat-tached(Figure3).Plantsorplantpartsareplacedinsidetheglasschambersandheadspaceairiscontinuouslycirculatedthroughavolatiletrapplacedinastainlesssteelhousing,allowingquantitativetrappingoftheemittedVOCs(DonathandBoland,1995).Sincethecirculationofairinaclosedsystemminimizestrappingofaircontaminantsascomparedwiththeairßowinanopensystem(seebelow),closed-loopstrippingisapplicabletoVOCanalysesofplantswithlowvolatileemissions.Forexample,thesmallßowersofthemodelplantA.thalianashowasigniÞcantlyreducedemis-sionrateofvolatilescomparedwithßowersofhighlyscentedplants(Chenetal.,2003)makingitvirtuallyimpossibletocollectvolatilesfromasingleßower.ForadetailedanalysisofArabidopsisßoralterpenevolatileproÞles,70Ð120detachedinßorescenceswereplacedinsmall,water-containing,glassbeakersinsideasealed1-ldesiccator,andtheclosed-loopstrippingprocedureaccordingtoDonathandBoland(1995)wasapplied.Followingthecollectionofvola-tileson1.5mgcharcoalor25mgSuperQtrapsfor8h,compoundswereextractedfromthetrapswith40Ð100dichloromethaneandanalyzedbyGC-MS.Forcomparativeanalysisofvolatilesfromundetachedßowers,ßoralvolatilesweresampledbysemi-opendynamicheadspacevolatiletrapping(seebelow)forasimilarlengthoftimefromwholeßoweringplantswithanequivalentnumberofinßores-cences.ProÞlesandcompositionsofßoralvolatilesobtainedbybothmethodswerehighlycomparable.However,volatileproÞlesobtainedwiththeclosed-loopstrippingmethodshowedasigniÞcantlyhighersignaltonoiseratioduetothetrappingoffeweraircontaminants.Inthisway,minorcom-poundsofthecomplexßoralterpenevolatilemixturecouldbeanalyzed(Chenetal.,2003;Tholletal.,2005).SignaltonoiseratiosmaybeimprovedevenfurtherbysamplingVOCsfromalargernumberofinßorescences.Otheradvantagesofclosed-loopstrippingsystemsarethattheyareeasytosetupincontrolledclimatechambersanditispossibletocollectVOCsfromseveralindividualplantsatthesametime,whichmakesthismethodsuitableforscreeningpurposes.However,resultsfromclosed-loopstrippingsamplingshouldalwaysbecomparedwiththoseobtainedbyopenheadspacetrappingtoexcludeartifactsduetoeffectsontheenclosedplantcausedbychangesintheatmosphereofthechamberintheabsenceofairexchangewiththeoutsideofthechamber.Inaddition,volatilessuchasethylenethatarenottrappedontheadsorbingmaterialmayaccumulateinthechamber,andrelativehumiditymightincreasewithoutoccasionalventingofthesystembetweentrappingcycles.‘Pull’and‘push–pull’systems.Incontrasttoclosed-loopstrippingdevices,pullorpushÐpullsystemsoperatewithaconstantßowofairwhichistakenupfromtheoutsideandleavesthesystemwiththevolatilesemittedbytheplant.Inapullsystem,airispulledovertheplantsamplethroughanadsorbenttrapthatisconnectedtoavacuumpump.Asimpleformofapullsystemisanadsorbenttrapthatisdirectlypositionednexttoaplantoraplantorganwithoutanyfurtherenclosureoftheplant(Burgeretal.,1988).Inopen-topchambers,theplantoritspartsareplacedinacontainerthatallowstheßowofambientairwithoutpriorcleaning(Figure4a,b).Halitschkeetal.(2000)haveusedsuchsystemtoinvestigateherbivore-inducedvolatileemissionsofhydroponicallygrownNicotianaattenuataplantsorleavesofsoil-grownplants,andKaiser(1991)hasappliedsimilardevicesforsamplingofßoralvolatiles.Theset-upisrelativelysimple,inexpensiveandhighlyportablemakingitsuitableforsimultaneouscollectionsfrommanychambersandforsamplingintheÞeldasshownbyKesslerandBaldwin(2001).Thissystemworkswellforplantsthatemithighamountsofvolatiles;however,theriskishighoftrappingimpuritiesfromtheambientairunrelatedtotheinvestigatedVOCblendthatwillobscurethedetectionofminorsamplecompoundsduringGCanalysis.Byenclosingplants,leavesorßowersentirelyinaglasscontainerorapolyacetatecookingbagthatreleasesveryfewvolatiles(Dobson,1991),theamountofimpuritiesfromambientairmaybereduced.AirentersthecollectionchamberthroughapurifyingÞlterandisdrawnfromthechamberbypullinga TDFS Figure3.Volatilecollectionbytheclosed-loopstrippingprocedure.Thephotographshowscollectionofvolatilesfromdetachedsnapdragonßowersduringfeedingwithisotope-labeledprecursors(Dudarevaetal.2005).Airiscontinuouslycirculatedthroughtheglasscontaineratßowratesof2Ð3lmin.P,circulationpump;T,steelhousingcontainingthevolatiletrap;S,Teßonstopperwithholesforpumpadaptors;D,1-ldesiccator;F,detachedsnapdragonßowersinaglassbeakerwiththefeedingsolution.Plantvolatileanalysis2006TheAuthorsJournalcompilation2006BlackwellPublishingLtd,ThePlantJournal,(2006),,540Ð560 deÞnedvolumethroughtheadsorbenttrapthatisextractedforfurtheranalysis.Suchdeviceshavebeendescribedbyetal.(2003)andRagusoandPellmyr(1998)forheadspaceanalysisofvolatilesfromsnapdragonandßowers.Caremustbetakentoachievehomogen-eousairßowovertheplantandasufÞcientßowratemustbesettopreventincreasesintemperatureandhumiditywhenthesystemisexposedtodirectsunlight.Ifaßexiblecookingbagisused,itmaycollapseanddamagetheplant,whichmayaltervolatileemission.InpushÐpullsystems(Figure4c),airispushedintotheheadspacesamplingcontaineratarateregulatedbyaßowmeter.Priortoenteringthechamber,theairiscleanedbyßowingthroughapurifyingÞltermaterialsuchascharcoalthatadsorbsimpuritiesandmaybehumidiÞedatadesiredratebymixingitwithasecondairstreamwithsaturatinghumidity.Insidethecontainer,auniformairßowovertheplantshouldbecreated.Aportionoftheairispulledoutofthechamberthroughanadsorbenttrapconnectedtoavacuumpump.Theßowrateoftheoutgoingairstreamisregulatedbyasecondßowmeter,allowingthecollectionofadeÞnedpercentageofthevolatilesemittedbytheplant.Theremainingairßowescapesthecollectionchamberthroughaventpreventingoverpressure.Comparedwithpullcollectiondevices,thissystemoffersmoreßexibilityinregulatingin-andoutgoingairßowdependentontheemissionratesofVOCsintheinvestigatedplant.Wehaveemployedthissystemtocollectvolatilesfromaphid-infes-tedplantsoverseveraldays(Kunertetal.,2005)andtrapVOCsfromßoweringArabidopsisplantsover8h(Chenetal.,2003).Insomeapplicationsitisdesirabletocollectvolatilesfromonlycertainpartsoftheplant,suchasthosereleasedfromundamagedleavesafterlocalizedherbivoredamage(Figure4d).Whileleavingtheherbivore-damagedleavesoutsidethecollectionsystem,headspacefromupperundamagedleavescanbecollectedforseveraldaysasdemonstratedforVOCemissionsfromGossypiumhirsutumetal.,1996).Suchmeasurementsweremadepossiblebyusingopen-bottomedglasscollectionchamberssetontopofamultiportguillotinebase.Theguillotinebasecontainsconcentricgas-samplingportsandtwoTeßon-coatedremovablebladesthatclosethebottomofthechamberaroundthestemoftheplant,leavinganopeningforthestemwherethebladesÞttogether(Figure4d).Thepositive-pressureventingaroundthestempreventsambientairfromenteringthecollectionchamber.Thissystemallowsforthecollectionofvolatilesfrompartsofintactgrowingplantswhilecompletelyisolatingthelowersectionoftheplantincludingsoilandpots(HeathandManukian,1994;etal.,1996).ThetechniquewasdescribedindetailbyHeathandManukian(1992)andhasbeenautomatedto Vent Ambient airin (a) airin Vacuum Vacuum Air in Volatile Vacuumpump Figure4.Examplesofdynamicheadspacecollectionsystems.(a)InasimpleÔpullÕheadspacecollectionchambertheplantisenclosedinanopen-topcontainer.Anairstream,regulatedbyaßowmeter,ispulledovertheplantandthroughaVOCcollectingadsorbenttrap.(b)AÔpullÕheadspacecollectiondevicewithanopen-topchamberforcollectingVOCsfromasingleleaf.(c)InaÔpushÐpullÕheadspacecollectionsystem,pressurizedairentersthetopofthecollectionchamberregulatedbyaßowmeter.IncomingairispuriÞedbypassingthroughacharcoalÞlterplacedbehindorinfrontoftheßowmeter.Alternatively,high-puritysyntheticairmaybeused.Afterpassingovertheplantsample,theairispulledthroughanadsorbentvolatiletrapatthelowersideofthechamberatadeÞnedratecontrolledbyasecondßowmeter.Excessaircanescapethroughtheventonthelowersideofthechamber.(d)ExampleofamodiÞedÔpushÐpullÕheadspacecollectionchamberforcollectingVOCsfrompartsofaplant.Teßon-coatedguillotine-likebladesclosethebaseofthechamberaroundthestemoftheplantallowingtrappingofVOCsfromtheupperpartoftheplant.DorotheaTholletal.2006TheAuthorsJournalcompilation2006BlackwellPublishingLtd,ThePlantJournal,(2006),,540Ð560 monitortime-dependentchangesinVOCemissionsbyswitchingtrapsfordrawingsamplesatdeÞnedtimeinter-valsoverseveraldays(HeathandManukian,1994;Manu-kianandHeath,1993).Forexample,rhythmicvolatileemissionsfromßoweringMirabilisjalapaweremeasuredwiththissystemoveratimecourseof48h(Effmertetal.2005),anditwasalsosuccessfullyappliedfortime-courseanalysesofherbivore-inducedVOCemissionsfrommaize(DegenhardtandGershenzon,2000).Theentirecollectionchambercanbeinstalledinagreenhouseorinaclimatechambertocontrolenvironmentalparameters.BesidespushÐpullsystems,otherlaboratorieshavedesignedcollectionchambersinwhichpressurizedairenteringthecontainerispushedthroughtheadsorbenttrapwithoutapplyingadditionalvacuum(ÔpushÕsystems,Dickeetal.,1999;Kunertetal.,2002).InthesesystemscontainersmustbesealedwelltopreventleakageoftheaircontainingVOCs,andnewuncloggedvolatiletrapsshouldbeusedtopreventoverpressureandensureefÞcientairßow.InmostofthesystemspresentedaboveenvironmentalparametersaresimplyregulatedbytheconditionsoftheclimatechamberandmanualadjustmentoftherateofairßowandairhumidiÞcationinsidethecollectiondevice.SystemswithimprovedautomatedparametercontrolshavebeendescribedbyJakobsen(1997),forexample,whichcontainelectronichumiditysensorsinsidetheheadspacechamberandtemperaturecontrolbycontinuouscirculationofwaterbetweendoublewallsoftheglasscontainer.Smallersystemsusingcuvetteswithcomparableparametercontrolhavebeendeveloped(foradetaileddescriptionseethesectiononVOCanalysistechniquesforstudyingthebiosynthesisofplantvolatilesandtheirroleinstressphysiology)formeasurementsofVOCsfromleavesorGaschromatographicseparationanddetectionofplantPlantVOCstrappedonadsorbingmatricesareroutinelyanalyzedbythestandardtechniqueofGC.Awealthoflit-eratureisavailabledescribingGCanalysisprotocolsandrecentadvancesinGCanalysistechnology,ofwhichonlyasmallportionwillbecoveredinthispaper(e.g.DewulfandVanLangenhove,2002;HandleyandAdlard,2005;Lock-wood,2001;Marriottetal.,2001;Merfort,2002;Ragunathanetal.,1999).ForGCanalysisofVOCs,samplesareeitherinjectedassolventextractsintotheheatedinjectorinasplitorsplitlessmodeordesorbedfromtheadsorbentbyplacingitdirectlyinathermaldesorptiontube,heatedto250Ð300C.Inatwo-stagethermaldesorber,thethermallyreleasedvolatilesareconcentratedbyacoldtrap(orcryotrap)priortotheirinjectionintotheGCcolumn.Recenttechnologyhasledtothedevelopmentofon-linesystemswhichcombinevolatilesamplingwithautomatedthermaldesorption(seebelow).Furthermore,aninterestingmethodhasbeendescribedfordirectthermaldesorptionofvolatilesfromßoralorgansbyplacingtheminquartzmicrovialsthatareinsertedintoamodiÞedGCinjector(JurgensandDotterl,2004).CaremustbetakentopreventdecompositionofcompoundsathighForanalyticalpurposes,volatilesarecommonlysepar-atedonfusedsilicacapillarycolumnswithdifferentstation-aryphases,suchasthenon-polardimethylpolysiloxanes(e.g.DB-1,DB-5,CPSil5),andthemorepolarpolyethyleneglycolpolymers,includingCarbowax20M,DB-Wax,andHP-20M.Morein-depthguidanceoncolumnselectioncanbeobtainedfromcolumnvendors.FollowingseparationonaGCcolumn,volatilecom-poundscanbeanalyzedbyavarietyofdifferentdetectors.Flameionizationdetectors(FID)arecommonlyusedforquantitativeanalysisbecauseoftheirwidelineardynamicrange,theirverystableresponseandtheirhighsensitivitywithdetectionlimitsoftheorderofpicogramstonanogramspercompound.Anotherdetector,preferredfortheanalysisofvolatileterpenes,isthephotoionizationdetector(PID)whichisconsiderablymoresensitivethantheFIDinthepresenceofreactivedoublebondsbutrequirescarefulcalibrationforquantitativeanalysis.Massspectrometry(MS)detectorsarethemostpopulartypeofdetectorforroutineplantvolatileGCanalysis.InthemassspectrometersofmoststandardGC-MSbench-topinstruments,compoundsexitingtheGCcolumnareionizedbyelectronimpact(EI)andtheresultingpositivelychargedmoleculesandmoleculefragmentsareselectedaccordingtotheirmass-to-charge()ratiobyenteringaquadrupoleiontraporaquadrupolemassÞlter.Totalionchromatogramsareobtained,whichprovideinformationontheretentiontimeofeachcompoundanditsmassspectrumconsistingofacharacteristicionfragmentationpattern.Detectionlimitsofhighlysensitivemassspec-trometersareinthepicogramrangeforthefullscanmode(scanningionsoverawidemolecularrange)andmaybeaslowasinthefemtogramrange(inquadrupolemassÞlters)intheselectedionmonitoring(SIM)modescanningselectedionsthatarerepresentativeofacompound.QuantiÞcationispossibleinfullscanorSIMmode,butrequiresthoroughcalibrationforeachindividualForidentiÞcationofcompoundsinGC-MSanalysis,suggestionscanbeobtainedfrompopularmassspectrallibrariessuchasWileyandNISTMSdatabasesanddatabasesprovidingretentionindexdatasuchastheKovatsindexsystem,relatedto-alkanes(seeTableS2).However,identiÞcationbasedsolelyonretentionindicesorlibrarymassspectraldataisnotreliableandoftenleadstofalseassignments.AcorrectidentiÞcationrequiresatleastthedeterminationofKovatsindicesontwocolumnswithPlantvolatileanalysis2006TheAuthorsJournalcompilation2006BlackwellPublishingLtd,ThePlantJournal,(2006),,540Ð560 differentpolaritiesandamatchofthemassspectrumofthecompoundofinterestwiththatofanauthenticstandard.Asimpleco-injectionofsampleandstandardononlyasinglecolumnisnotsufÞcient.Ion-trap-derivedmassdatamaybelesssuitableforidentifyingunknowncompoundsduetopoormatcheswithavailableMSlibraries.Massspectraobtainedbysofterchemicalionization(CI)insteadofEIshowlimitedmolecularfragmentationbutarehelpfulforobtainingstrongermolecularions,especiallyforaliphaticInadditiontotheidentiÞcationofthebasicstructureofplantVOCs,itisoftendesiredtodeterminetheirchiralitysincetheenantiomericcompositionofvolatilescanbecrucialintheirroleaschemicalsignals.Severalenantiose-lectivecapillarycolumnsareavailable(Schurig,2001)withchiralphasessuchasdifferenthydrophobiccyclodextrinderivatives(Bicchietal.,1999;KonigandHochmuth,2004).Generally,acylatedcyclodextrinderivativesarepreferredforseparatingpolaranalytes,whilenon-polarcompoundsarebetterresolvedonpre-alkylatedcyclodextrinderivatives.TheapplicationofchiralseparationinßavorandfragranceanalysisandenantiomericidentiÞcationofsesquiterpenehydrocarbonshasbeenwelldocumentedbyMaasetal.(1994a,b),Schreieretal.(1995)andKonigandco-workers(e.g.KonigandHochmuth,2004;Koetal.,1992).Becauseblendsofplantvolatilescanconsistofmanychemicallydiversecompoundsaswellasisomers,asimpleGC-MSanalysisrunmightnotbesufÞcientfortheidentiÞ-cationofallproducts.Tandemmassspectrometry(MS-MS)hasbeenestablishedallowingseparateanalysesofsinglecompoundsofcomplexGCpeaks(Graneroetal.,2004;etal.,1999).InMS-MS,ionsofinterest(parentions)aremass-selectedandtypicallyfragmentedbycolli-sionwithaneutralgasfollowedbymassanalysisoftheresultingproductions.Furthermore,GC-MSanalysiscanbecomplementedbycapillaryGCÐFouriertransforminfraredspectroscopy(FT-IR).ThisspectroscopicmethodhasbeenemployedtodifferentiatecloselyrelatedisomerswithverysimilarEImassspectra(Ragunathanetal.,1999).Fouriertransforminfraredspectroscopyprovidesinformationontheintactmolecularstructureandallowsuniquespectratobeobtainedevenforsimilarisomers.TheutilityofGC-FT-IRislimitedbydifÞcultiesinquantiÞcationandtime-consu-mingdatainterpretation,althoughagrowingcollectionofhigh-qualitydataisprovidedbytheSadlerdatabase(SadlerDivisionofBio-Rad,Philadelphia,PA,USA).IfitisimpossibletoseparatecomplexvolatilemixturessufÞcientlyonasinglecolumn,two-dimensionalcapillarygaschromatographycanbeused.Here,compoundsareseparatedonaÞrstcolumnfromwhichselectedfractions(heart-cuts)aredirectedtoanothercolumnastheseconddimension.Thisapproachhasbeenused,forexample,todeterminetheenantiomericcompositionofmonoterpenehydrocarbonsintissuesofPiceaabiesbycombiningaconventionalGCcolumnwithachiralcolumn(Borg-Karlsonetal.,1993).Morerecently,so-calledÔcomprehensiveGCsystemsÕhavebeendevelopedthatconsistoftwocolumnswithdifferingpolaritythataredirectlycoupledbyacryogenicmodulatorcompressingregionsoftheefßuentfromtheÞrstcolumnandinjectingtheminthesecondcolumn.Thesimultaneoustwo-columnseparationleadstoasigniÞcantincreaseintotalseparationspace.ThemethodisofparticularinterestintheÞeldofanalysisofessentialoilstoincreasepeakresolutionandimprovequantiÞcationoridentiÞcationofvolatilecomponents(Dietal.,2004;Marriottetal.,2003).TheidentiÞcationofchemicalÞngerprintsviacomprehensiveGCGCmaybeofinterestinthestudyofplantspecieswithverycomplexpatternsofnaturalvariationinvolatileblends.AnotherrecenttrendinGCanalysisofVOCsparticularlyfavoredbyresearchersinvolvedinessentialoilanalysisisthedevelopmentoffastgaschromatography.FastGCanalysisiscarriedoutin3Ð5minwithacomparableefÞciencytoconventionalhigh-resolutionGConthesamematrices(MatisovaandDomotorova,2003).TheapplicationoffastGCcouldbeconsideredforhigh-throughputanalysesinVOCmetaboliteproÞling.However,fastGCrequiresadditionalnon-standardinstrumentationtosupport,forexample,fasttemperaturechangesandhighinletpressures.ForefÞcientdetectionofcompoundsincombinationwithfastGCanalysisandcomprehensiveGCGC,theuseoftime-of-ßight(TOF)-MSdetectorshasbeendiscussed(Ma-tisovaandDomotorova,2003).ThetechniqueofTOF-MS,generatinginstantaneousmassspectra,incombinationwithpeakdeconvolutionalgorithms,resultsinincreasedratesofacquisitionofmassspectraldata(upto5000massspectrasec)comparedwiththoseofquadrupoleMSandallowstheidentiÞcationofoverlappingpeaks.Whethertheresearcherneedshigh-speedGCanalysisorimprovedsampleresolutioncapacity,asdescribedabove,hastobedecidedonacasebycasebasis.TheidentiÞcationofallcompoundsofavolatileblendisnotalwaysrequiredandrelativelysimpleVOCproÞlesmaystillbeanalyzedbystandardGC-MS.However,ongoingadvancesinGCtechnologywillcertainlyfurtherimproveseparationefÞciencyandanalysistimeeveninroutineFinally,theelucidationofthestructureofunknownvolatilecompoundsmeritssomegeneralremarks,althoughdetailsarenotwithinthescopeofthispaper.Multipleanalyticalstepsusuallyneedtobeconsideredforunequi-vocaldetermination.Mostoftenamountsofasinglecom-poundsufÞcientforone-andtwo-dimensionalnuclearmagneticresonance(NMR)analysistechniquesneedtobeisolatedonapreparativescaleusingpreparativepackedGCcolumnsandthick-Þlmcapillarycolumnswithhighlyselect-ivecyclodextrinmatrices(KonigandHochmuth,2004).ArecentpublicationbyNojimaetal.(2004)describesasimpleDorotheaTholletal.2006TheAuthorsJournalcompilation2006BlackwellPublishingLtd,ThePlantJournal,(2006),,540Ð560 andefÞcienttechniqueforpreparationofNMRsamplesofvolatilechemicalsusingamicropreparativeGCsystem.TodeterminetheabsoluteconÞgurationofanovelcompound,itcanbecomparedwithasyntheticreferencecompoundusingenantioselectiveGC.Ifnosyntheticrefer-encecompoundisavailable,chemicalcorrelationisapplied,meaningthatthenewstructureischemicallymodiÞedtoaknowncompoundoraproductthatcanbecomparedwithaproductderivedfromastructurewithknownabsoluteconÞguration(KonigandHochmuth,2004).MonitoringenvironmentallydependentchangesofVOCManyinvestigationshavedocumentedthatemissionsofplantvolatilesshowtime-dependentchanges.Forexample,emissionofßoralvolatilesoftenfollowsrhythmscontrolledbydiurnalorcircadiancycles.Manyplantsemitlow-molecularterpenevolatilessuchasisopreneandmono-terpenesfromphotosynthetictissuesinalight-andtemperature-dependentmanner.Furthermore,thereleaseofvolatilesasdirectorindirectdefensesignalscanbeinducedbyherbivoredamage.Inordertomonitorchangesinvolatileemissionsinresponsetotheseenvironmentalfactorsandcorrelatethemwithalterationsinphysiologicalparameters,geneexpressionandenzymeactivities,volatileproÞlesandemissionratesneedtobedeterminedatmul-tipletimepoints.AutomateddynamicheadspacecollectionsystemsasdescribedearlierallowsamplingofVOCsatpre-deÞnedtimeintervals.However,samplestrappedinthesesystemsneedtobedesorbedandsubsequentlyanalyzedbyGC.Thisstandardoff-lineGCanalysisexcludesthepossibilityofrecordingfast-changingemissionsofvola-tiles,whichoccurontimescalesofsecondsandminutes.ThisdeÞciencyhasledtothedevelopmentofseveralon-linetechniquescombiningafasttimeresponsewithlowdetectionlimits.Dynamicheadspacesamplingwithon-linethermalAVOCanalysissystemwithmoderatetimeresolutionwasdevelopedbyattachinganautomateddynamicheadspacesamplingsystemtoanon-linethermaldesorptiondevice(GerstelOnline-TDSG,Gerstel,Germany).TheGerstelOn-line-TDSconsistsofathermodesorptionunitconnectedtoacryofocusingdeviceforrefocusingoftheanalytepriortoGCinjection.Byregulatingthemassßow,thesystemcanautomaticallydrawvolatilesamplesandswitchbetweensamplingandthermodesorption.Vercammenetal.connectedtheTDS-Gsystemtotwosamplechambers,theheadspacesofwhichwereanalyzedconsecutivelyusingarotaryvalve.Atimeresolutionof5Ð60mincanbeachieveddependingonthetimenecessarytocollectsufÞcientamountsofVOCsfromtheemittingplantandthetimeforGCseparation.On-linethermaldesorptionwasalsoemployedbyAharonietal.(2003)forcollectionsofVOCsFastandtransportableGC(zNoseArecentlydevelopedminiaturizedGCinstrument,the(ElectronicSensorTechnology,NewburyPark,CA,USA;Figure5),combinesfasttrappingofVOCswithfaston-lineGCanalysis.Theinstrumentoperateswithahighlysensitivesurfaceacousticwave(SAW)quartzmicrobalancedetector,inwhichVOCanalytesarecondensedonthesur-faceofanoscillatingcrystal.Thisleadstoanincreaseintheoscillatormassandlowersthevibrationalfrequencypro-portionaltotheamountofcondensate.DuetothehighsensitivityoftheSAWdetector(intheppbvrange)thetimeforvolatilesamplingonaTenaxtrapcanbereducedto20Ð40sec.Followingrapidthermaldesorption,compoundsareseparatedonacapillarycolumn(DB-5)of1or5mlengthandquantitativelyanalyzedbytheSAWdetector.Becauseoftheshortoperationtimeandfullyautomatedsamplinganddataacquisition,airsamplescanbecollectedintimeinter-valsaslowas3minoverlongerperiodswithoutsupervi-ThezNosehasbeenemployedformonitoringrhythmicVOCemissionsfromßowersandinducedVOCemissionsfromherbivore-damagedplants(Kunertetal.,2002).A traps Figure5.MonitoringofinducedplantVOCemissionsbyfastandtransport-ableGC(zNose)accordingtoKunertetal.Detachedplantletsoflimabean(Phaseoluslunatus)wereplacedinaglassvialcontainingasolutionofthefungalelicitoralamethicinandtransferredtoasealed4-lglassvessel.PressurizedpuriÞedairpassesthroughtheglasscontainer(dashedarrows)ataßowrateof120mlminandexitsthechamberthroughacharcoalÞlteradsorbenttrap(ÔpushÕsystem).Volatilesaretrappedonadsorbentcartridgesin4-hsamplingperiods.Simultaneously,airsamplesareanalyzedevery15minbythezNosewithasamplingtimeof20sec.Volatileemissionsweremonitoredfor4days.RecordingsofVOCemissionproÞleswerecomparablebetweenadsorbenttrappingandzNosemeasurements,withasigniÞcantlyhighertimeresolutionobtainedbyPlantvolatileanalysis2006TheAuthorsJournalcompilation2006BlackwellPublishingLtd,ThePlantJournal,(2006),,540Ð560 limitationofthisGCistheshortcolumnwhichreducestheresolutionofVOCswithsimilarretentiontimessuchasmonoterpenesorsesquiterpenes.However,theinstrumenthasbeenausefultoolforfastquantitativeestimationsofknownvolatileproÞles,whichmakesitapplicableformeasuringfastchangesinVOCemissionsorscreeningVOCsonahigh-throughputbasis.Evenmoreimportant,theportabilityoftheinstrumentallowssuchmeasurementsnotonlyinthelaboratorybutalsoinÞeldexperiments.Real-timeanalysisofvolatileterpenesandotherVOCsinplantheadspaceandtheatmosphereByfarthemostadvancedtechnologyemployedfortheanalysisofplantvolatileemissionsinrealtimehasbeendevelopedinrecentyearsintheÞeldofatmosphericchemistry.TheparticularinterestinfastmonitoringofVOCssuchasisopreneandotherlow-molecular-weightisopre-noidsthathavesigniÞcantimpactonozoneandaerosolformationhasstimulatedthedevelopmentofdifferenton-linedetectionsystems.High-frequencysamplingisdesiredformeasurementsofvolatileßuxintreecanopieswhereterpenevolatilesshowhighandrapidreactivitywithactivechemicalspeciesintheatmosphere.HillsandZimmerman(1990),fromNCARinBoulder,Colorado,describedtheFIS(fastisoprenesensor)chemilu-minescencedetectorforon-linedetectionofisopreneinair.Thissensor,incombinationwithacuvettesystem,allowedtheÞrstreal-timeobservationoffastchangesinisopreneemissionsfromleaves(HillsandZimmerman,1990;Monsonetal.,1991).Inthechemiluminescencedetector,isoprenereactswithozone,producingformaldehydeinanexcitedstate.Thisreactionproductsubsequentlyrelaxestothegroundstateemittinglightat450Ð550nm,whichisdetectedbyablue-sensitivephotomultipliertubewithadetectionlimitof400partspertrillionbyvolume(pptv)forisopreneandaresponsetimeof0.1sec.ThedetectionisspeciÞcforisoprenesinceothernaturallyabundantvolatiles,forexam-plemonoterpenes,areweakemittersofozone-inducedchemiluminescence(HillsandZimmerman,1990).ThisinstrumenthasbroughtaboutsigniÞcantprogressintheunderstandingofisopreneemissionbyplants(e.g.BruemannandSchnitzler,2002a;Rosenstieletal.,2003;Sings-aasandSharkey,1998)andthedeterminationofisopreneßuxesfromforestcanopies(e.g.Fuentesetal.,1996)andwillremainasigniÞcantinstrumentforthestudyofisopreneßuxes.However,becauseofitsspeciÞcity,itsuseislimitedtoisopreneresearch.Overthelastdecade,laser-basedinfraredphotoacoustic(PA)spectroscopyhasbeendevelopedasanadditionalversatiletoolforsensitivereal-timedetectionofplantvolatilecompounds.Forexample,CO-basedPAspec-trometerswereusedforthedetectionofthephytohor-moneethylene(Arimuraetal.,2002;Waetal.,1999).Thetechniqueisbasedonthestrongabsorptionoftheinfraredwaveband(3Ð10m)byvolatilehydrocarbons,aldehydesandalcohols.Themeasuringprincipleisthedetectionofsoundwaves:absorptionofdistinctpulsedlaserlightbyvolatilesleadstoheatingandconsequentexpansionofthegassample,resultingintheappearanceofsoundwaveswhichcanbedetectedphotoacoustical-lybyverysensitivemicrophones(HarrenandReuss,RecenttechnicaldevelopmentsshowedthatCO(carbonmonoxide)overtonelaserscanbeusedforon-linedetec-tionofisoprene(Dahnkeetal.,2000;Wolfertzetal.,2003,2004),andoxygenatedcompoundssuchasacetaldehydeetal.,2000;Zuckermanetal.,1997)enlargingtheÞeldofapplicationofPAspectroscopyinplantphys-iology.Forexample,thetechniqueallowsthedetectionofisoprenedowntoafewppbvwithatimeresolutionof1mininacontinuousgasstream.However,PAspectros-copyisstillaverysophisticatedon-linetechnology,whichneedssomefurtherdevelopmentstoÞnditswayintotherepertoireoftechniquescommonlyappliedinanalysisofbiogenicvolatiles.Inparticular,futureeffortsarenecessarytoreducethesizeoftheinstrumentsused,forgreatermobility,andtodemonstrateabroaderapplicationofthisspectroscopictechniquetotheanalysisofothervolatileetal.(1995)andLindingeretal.(1998a,b)reportedonthedevelopmentofprotontransferreactionÐmassspectrometry(PTR-MS),acombinationofaprotontransferreactiondrifttubeandaquadrupolemassspectrometer.Theinstrument,whichiscommerciallyavailablefromIoniconAnalytikGmbH,Austria,principallyallowsfastdetectionofmostVOCsincombinationwithlowdetectionlimits(10Ð100pptv).ProtontransferreactionÐmassspectrometrycanreplaceGC-MSinmanyapplicationsofVOCanalysisandachievearesponsetimemeasuredinsecondspercom-pound.ThefundamentaldifferencefromconventionalMSisthatPTR-MSusesÔsoftÕchemicalionizationofVOCmole-culesbyreactionwithhydroxoniumions(H)producedbyanexternalionsource.TheprincipleofoperationofthePTR-MSinstrumentisshownschematicallyinFigure6.Ahollowcathodedis-chargeactsasanionsource,whichproducesHfrompurewatervapor.PrimaryHionsenterthedrifttubethatisßushedcontinuouslywithambientairandundergonon-reactivecollisionswithanyofthecommoncomponentsinair(N,Ar,CO).Asmallfraction(typically1%)oftheprimaryHionstransfersitsprotonstoVOCswhicharepresentastracegasesinairandwhichhaveprotonafÞnitieshigherthanthatofwater.VolatileorganiccompoundsbecomeprotonatedinthefollowingDorotheaTholletal.2006TheAuthorsJournalcompilation2006BlackwellPublishingLtd,ThePlantJournal,(2006),,540Ð560 (withthereactionconstant).Unlikeconventionalelectronimpactionization,protonationdoesnotgenerallycauseafragmentationoftheionizedmolecule.Theionsaretrans-portedthroughthedrifttubebyahomogeneouselectricÞeld.Thisprovidesaconstantreactiontimeandenergy,sothattheprotontransferreactionsarewelldeÞned.Thefactordeterminingthetimeresponseisthetimetheairspendsinthedrifttube(sec).Primaryandproductionsenterasmallintermediatechamberwhereairispumpedoutandtheionsareextractedintothedetectionchamberwheretheirmassesareanalyzedusingaquadrupolemassspectro-meter.Thedetectionlimitatasignaltonoiselevelof2istypicallyintheorderofafew10pptvfora1secintegrationWhilePTR-MShasbeenusedprimarilyinÞeldcampaignsfortraceatmosphericmeasurementsofavarietyofcom-poundsincludingaromatics,isoprene,ketones,leafwoundaldehydesandalcohols(Falletal.,1999;HanselandWist-haler,2000;Sprungetal.,2001;Wisthaleretal.,2002),thistechnologyhasalsobecomeanalternativeandusefuladditiontoGCanalysisofplantenclosuremeasurements.ThefollowingVOCs,whicharerelevanttoplantphysiology,havebeenmeasuredbyPTR-MS:isoprenoids(isoprene,monoterpenes,sesquiterpenes),aldehydes,alcohols,ke-tonesandothers(e.g.Beauchampetal.,2005;Falletal.1999;Grausetal.,2004;Holzingeretal.,2000;Steeghsetal.2004).WhilePTR-MSdetectsmostvolatilesastheirmolecu-larmassplusone(e.g.methanolm33,acetaldehydem45,acetonem59,isoprenem69),someVOCsincludingCwoundalcoholsormonoterpenesundergosomedegreeoffragmentationwithintheinstrument.AlthoughthePTR-MSmethodhasfoundnumerousapplicationsandhasgreatlyexpandedthecapabilityforrelativelyfastmeasurementsofVOCsthereremainsigni-ÞcantweaknessessuchasdifÞcultiesinthedetectionofveryfast-reactingVOCspecies(e.g.monoterpenesandsesquiterpenes).Furthermore,interferenceinthedetectionofVOCsispossibleduetothelimitedselectivityobtainedfrommonitoringonlytheprotonatedparentmass.Forexample,PTR-MSmeasuresonlythetotalconcentrationofmonoterpeneorsesquiterpeneisomersandgivesnoinformationontheterpenecompositionofanairsample(Tanietal.,2003).Toenabledistinctionbetweenisobariccompoundslike,forexample,alcoholsandorganicacids,acombinationofaprotontransferreactiondrifttubewithaTOF-MSisrequiredwhichwilldramaticallyimprovethedutycycle(allmassescanbemonitoredsimultaneously)andaddahighmass-resolvingpower.Todistinguishevenbetweenisomers,MS-MScapabilityneedstobeadded,whichseemspossiblebyusingiontraportriplequadrupoleMStechnology.Atpresent,combinedexperimentsusingslowGCtechnology(providinghighselectivity)togetherwiththefastPTR-MSmethod(havingpoorselectivity)aremostsuitabletomonitorandidentifyfast-changingconcentrationsofVOCsattracelevels.VOCanalysistechniquesforstudyingthebiosynthesisofplantvolatilesandtheirroleinstressphysiologyTheuseofgasexchangecuvettesystemsCuvettesystemshavebecomeincreasinglypopularintheÞeldofon-lineanalysisofplantvolatiles,andespeciallyforVOCsemittedfromtrees.Inassociationwithsuitabledetectionsystems,cuvettemeasurementsallowpreciseandrapidmonitoringoftheaircompositionandthereleaseoruptakebyplantsofgaseouscompoundssuchasCOandVOCsenablingaccurateinvivostudiesoftherelation-shipsbetweenprimarymetabolism(photosynthesis)andsecondarymetabolism(VOCsynthesis).CuvettesystemsalsoallowtheinvestigationofphysicalconstraintstoVOCemissionssuchasthosecausedbystomatalopeningetal.,2004). Figure6.ProtontransferreactionÐmassspectrometrysystemforreal-timeanalysisofplantvolatiles.(a)SchematicrepresentationofthePTR-MSsystem.(b)InstrumentinuseforVOCcuvettemeasurementsfrompendunculateoak(inset).(PicturesbyJ.Holopainen,UniversityofKuopio,FinlandandJ.KrUniversityofFreiburg,Germany.)Plantvolatileanalysis2006TheAuthorsJournalcompilation2006BlackwellPublishingLtd,ThePlantJournal,(2006),,540Ð560 Twotypesofcuvettesareingeneraluse,leafandbranchcuvettes.Leafcuvettesaremadeofaframeinwhichaleaf,oraportionofit,issandwiched.TheygenerallyhaveaverysmallandÞxedvolume,rarelyexceeding0.5l.CuvettesusedformeasuringVOCemissionsfromtreebranches,havealargervolume�(2l)andareofteninßatable(withoutsolidframes)andofvariablevolume.AcollageofdifferentcuvettesisshowninFigure7.Cuvetteshavetransparentwindowsofglassorplastictoallowilluminationoftheenclosedplantmaterialontheadaxialand,occasionally,alsoontheabaxialsideoftheleaf.Lightisprovidedbyexternal,generallyartiÞcialsourcessuchaslight-emittingdiodes(LEDs)withwavelengthsneededforphysiologicallightreactionstooccur(Tennessenetal.,1994).Asacoldandeasymodularlightsource,LEDscangenerateveryhighanduniformlightintensitieswithoutoverheatingtheleafcuvette.However,LEDsarecurrentlydifÞculttouseinbranchcuvettesduetoinsufÞcientandinhomogeneous Pollutants? (off-line VOC measurements VOCsout ToGC-MSTo IRGAs(COTo GC or PTR-MS Figure7.ExamplesofcuvettesystemsformeasurementsofplantVOCsandphysiologicalparameters.(a)AsimpliÞedsketchofatypicalcuvetteallowingmeasurementsoftheconcentrationofCOandHO(byinfraredanalysis)andVOCemissionsbyon-lineanalysisviaPTR-MSorGC,oroff-lineanalysisviacartridgetrappingandsubsequentanalysisbyGC-MS.(b)Specialcuvetteforsimultaneousmeasurementsofgasexchangeandßuorescencepropertiesoftheleaf.(c)Cuvetteforanalyzingozoneexchangebyleaveswithoutinterferenceofcuvettematerial(thiscuvetteisentirelymadeoutofTeßon).(d)CuvettesystemusedtomonitorgasexchangesfromentirebranchesunderÞeldconditions(courtesyofR.Baraldi,CNR-IBIMET,Bologna,Italy).(e)CuvettesystemforgasexchangeanalysisfromwholeplantsofArabidopsisthalianaAllcuvettes[except(d)]allowfullcontroloftheprincipalenvironmentalparameters(temperature,lightintensityandquality,windspeedandrelativehumidity)andcanthereforebeusedtoparameterizethedependenceofVOCemissionsfromkeyenvironmentalandphysiologicalfactors.IRGA,infraredgasanalyzerDorotheaTholletal.2006TheAuthorsJournalcompilation2006BlackwellPublishingLtd,ThePlantJournal,(2006),,540Ð560 illuminationoverlargeandunevensurfaces.LeafcuvettesaregenerallythermoregulatedbyPeltierresistancesor,moresimplybutlesseffectively,bywatercirculatingintheirbodywallsatthedesiredtemperature.Inbothcases,theactualleaftemperatureisconstantlymeasuredbythermo-couplesappressedtothesideoftheleafwhichisnotdirectlyexposedtoincidentlight.Large-volumebranchcuvettesareoftennotthermostatedandtemperatureissimplymonit-oredbyoneormorethermocouplesappressedtoleavesinstrategicpositions.Inallcuvettesystems,aircirculatesinsidethecuvetteenteringfromoneormoreinletholesandgenerallyexitingthroughonlyoneoutlet.Thegeometryofthecuvettemustfavorrapidandturbulentairmotiontoavoidairstagnation,whichmayinturncausecondensationofwateroncoldspotsofthecuvetteandtheformationofaboundarylayerresistancerestrainingtheexchangeofgasesbetweentheleafandtheatmosphere.Therefore,built-infansareoftenusedtostirtheairinsidethecuvettes.Ingeneral,thevolumeofairmustturn-overatleasttwiceperminutetoavoidtheabove-mentionedproblems.Thehumidityoftheairinthecuvettecanbeeasilycontrolledwhenworkingwithlabor-atoryequipmentbybubblingairintowaterandsettingthedesiredrelativehumiditybyabatingtheexcessintoacondenser(asimplecontainer)maintainedatalowertemperature.ThissystemisdifÞculttouseinÞeldexperi-ments,butexcesshumiditycanbecontrolledwiththeuseofdesiccantssuchasmagnesiumperchloriteandsilicagel.Laboratoryequipmentalsoallowsexcellentcontrolofthecompositionoftheairbymixingaircomponentsfromgascylinders.Althoughmeasurementsundercontaminant-freeconditionsmaynotreßectÔreallifeÕconditions,theyaredesiredtodeterminespecies-speciÞcemissionratesofVOCs,andfortheirparameterizationonthebasisofsingleenvironmentalorphysiologicalfactors.ToeliminaterapidreactionsofVOCswithozonepresentintheinßowingair,ozone-freesyntheticairfromcylinderscanbeusedorozonemaybescavengedfromambientairbyappropriatechemicaltrapsorphysicaldevicessuchasasimplepieceofmetal.Inaddition,specialcareshouldbetakeninthechoiceofozone-freefans.Tosimulatethepresenceofcontaminantsorpollutantssuchasozoneoranthropogenichydrocarbons,thesegasescanbesimplyaddedtotheairßow.Thisalsoallowsthefumigationwithenrichedisotopes(typically),tostudytheirincorporationinmetabolites(seeLikeotherdevicesusedinthecollectionofVOCs,cuvettesshouldbeentirelyconstructedwithmaterialswhichareinerttoVOCs,includingglassorspecialplasticandtransparentmaterialssuchasTedlarorTeßonÞlm(Figure7b).Ifmetalistobeused,themetalparts(generallythecuvetteframe)canbewrappedinTeßonÞlmorsprayedwithaliquidlayerofTeßon.Inaddition,theuseofsoftTeßonO-ringsisrecom-mendedtoavoidwoundingthesandwichedleafandeliminatewound-inducedVOCemissions.Incaseoffumi-gatingleaveswithveryreactivecompoundssuchasozone,Þttingsandpipelinesexternaltothecuvetteshouldconsistofinertmaterials(Figure7c).CuvettemeasurementsareafavoriteinvivomethodforstudyingVOCbiosynthesis.TheyhavebeenemployedforthesimultaneousdetectionofisoprenoidemissionsandCOuptakebyphotosynthesisinresponsetochangesinlightandCOandtheapplicationofphotosynthesisinhibitors.ThesestudiesledtotheearlypredictionthatisoprenoidVOCscanbeformedfromphotosynthesisintermediates(LoretoandSharkey,1990).ConcurrentmeasurementsofphotosynthesisandVOCemissionalsoallowedcalculationsofthefractionofphotosyntheticallyÞxedcarbonthatisdirectlymetabolizedinVOCbiosynthesis(Kreuzwieseretal.2002;SharkeyandLoreto,1993).Simultaneousmeasure-mentsarebestperformedwhenairleavingthecuvetteissplittoreachaninfraredgasanalyzerforCOdetectionandafastsensorforVOCemissionssuchasPTR-MS,allowing,inaddition,theexaminationoflabeledcompounds.Specialleafcuvettesallowintegratedmeasurementsofgasexchangeandphotochemicalcharacteristicsoftheleafbychlorophyllßuorescencedetection(Figure7b).Therationaleforthesemeasurementsisthat(i)partoftheelectrontransportratedrivingphotosynthesisandphoto-respirationmayalsodriveVOCbiosynthesis,(ii)theemis-sionofsomeVOCsmayberelatedtothedissipationofexcesslightand(iii)VOC-inducedphotoprotectionmaybemonitoredwitharapid,non-invasiveandremotetechnique.Simultaneousmeasurementsofgasexchangeandchloro-phyllßuorescencesuggestedthatthephysiologicalroleofVOCs(mainlyisoprene)isnotrelatedtoenergydissipationmechanisms(LoretoandSharkey,1990),assupportedbytheearlyworkofJonesandRasmussen(1975).Measure-mentsofgasexchangeandchlorophyllßuorescence,ontheotherhand,enabledcalculationofthefractionofphotosyn-theticelectrontransportneededforbiosynthesisofterpenevolatilesandallowedmodelingofmonoterpeneandiso-prenesynthesisonthebasisofthisphysiologicalparameterratherthanbytheindirectrelationshipwithenvironmental(empirical)variables(Niinemetsetal.,2002).FluorescencewassuccessfullyusedtomonitorVOC-inducedprotectionofphotosynthesisinresponsetostress,whengasexchangemeasurementsarepreventedbyfumigationwithtoxiccompounds(e.g.ozone,LoretoandVelikova,2001;Loretoetal.,2001b)orwhenremotescreeningofphotochemicalefÞciencywassufÞcienttodetectprotectionfromheatdamagebyisoprenes(SharkeyandSingsaas,1995).Finally,specialcuvettesallowrapidfreeze-clampingoftheenclosedleavesforbiochemicalanalysisofenzymeactivitiesandmetaboliteswithveryrapidturnover.InasystemdevelopedbySharkey(LoretoandSharkey,1993)theleafsandwichedinthecuvetteisfrozenextremelyquickly(sec)bysmashingitbetweentheplasticPlantvolatileanalysis2006TheAuthorsJournalcompilation2006BlackwellPublishingLtd,ThePlantJournal,(2006),,540Ð560 windowsofthecuvetteunderhighpressure�(100bar)withmetallicdrumspreviouslyfrozeninliquidnitrogen.Thissystemwaseffectivelyused,withslightmodiÞcation,todeterminepoolsofdimethylallyldiphosphate(DMADP),thelastintermediateinisoprenebiosynthesis(Loretoetal.,2004;Wolfertzetal.,2003),andtomeasuretheactivitiesofisopreneandmonoterpenesynthases(Loretoetal.,2001a).TheuseofPTR-MSinanalyzingtheoriginofcarbonandmetabolicintermediatesinVOCbiosynthesisOneofthemostpowerfulandvaluableabilitiesofPTR-MSaswellasPAspectroscopyÐthefullpotentialofwhichhasnotyetbeentappedÐisthedetectionofstableisotopesallowingstudyofthemetabolicoriginandßuxofinter-mediatesinthebiosynthesisofplantvolatiles.On-lineanalysisusingeitherstableC-(e.g.Karletal.,2002;etal.,2004a)ordeuterium-labeledprecursors(e.g.Wolfertzetal.,2003,2004)havedemonstratedtheversatilityofPTR-MSandPAspectroscopyforelucidatingtheoriginofcarbonandmetabolicintermediatesinplas-tidicisoprenebiosynthesisandprovidingnewinsightsintothedynamicsofmetabolicturnover.Figure8showsanexampleofC-labelingofisopreneinthepresenceoffollowedbyalossoftheC-labeledisotopebysubsequentexposuretoinpoplar()leavesmeasuredbyPTR-MS.Dependingonthesci-entiÞcquestionandtheavailabilityofstableisotopeprecursors,thisapproachcanbeappliedtostudythebiosynthesisofmanyotherplantvolatilessuchasmono-terpenes,alcoholsandaldehydes.DeterminingthebiosyntheticoriginofVOCsbyisotoperatioInsomeVOCanalyses,CisotoperatioshavebeendeterminedbycouplingGCwithisotope-ratiomassspec-trometryafterconversionoftheelutingcompoundsintoCOandHO(Gleixneretal.,1998).TheisotopiccarbonratiosofplantVOCsdependonthedifferentialincorporationofCisotopesinthecourseofdifferentenzymaticreac-tions.Therefore,determinationoftheisotoperatiosofplantvolatilescangivevaluablehintsabouttheirbiosyntheticandsubcellularorigin.Earlyuseofthismethodindicatedthatvolatileisopreneandcarotenoidssharethesamebiosyn-theticpathwayandthatenvironmentalstressescouldincreasethesupplyofcarbontoisoprenefrompathwaysotherthanphotosyntheticcarbonÞxation(Sharkeyetal.1991).RecentCisotoperatiostudiesofvolatileterpenesconÞrmedthedifferentbiosyntheticoriginofmonoterpenesandsesquiterpenesfromtheplastidicmethylerythritolphosphate(MEP)pathwayandthecytosolicmevalonatepathwayrespectively,butalsoindicatedthemetaboliccrosstalkbetweenbothpathways(Juxetal.,2001).AnalysisofvolatileplantenzymeproductsTechniquesusedforanalyzingVOCscanalsobeappliedtodeterminetheactivityofenzymescatalyzingtheformationofplantVOCs.SincethesolubilityofmanyVOCssuchasterpenehydrocarbonsisextremelylowinthehydrophilicphase,thesecompoundsrapidlyevaporatefromanaque-ousenzymesolutionandcanbeanalyzedbyheadspacecollectiontechniques.ThesensitivemethodofSPME(see min50 mi min –5min50 mi min m74m73m71m70m69Emission rate (nmol sSum of isoprene isotopes VMR (ppm(a)(b) 0246 m74m73m72m71m70m69Emission rate (nmol sSum of isoprene isotopes 0200300 VMR (ppm Figure8.ProtontransferreactionÐmassspectrometrymeasurementsofC-labeledisopreneisotopes.KineticsofClabelingofisoprenefollowingexposureto(a)andwash-outoflabelingafterreturntonormal(b)inpoplarleaves.Toppanel:EnclosureconcentrationsofasresponsetoswitchingfromoneCOsourcetotheotherat0.Bottompanel:Leafemissionratesofisopreneisotopespecies(m69,unlabelled;m70,singlelabeled;m71,doublelabeled;m72,triplelabeled;m73,fourfoldlabeled;m74,fullylabeled)andthesumofallisopreneisotopes.DataareredrawnfromGraus(2005).VMR,volumemixingratio.DorotheaTholletal.2006TheAuthorsJournalcompilation2006BlackwellPublishingLtd,ThePlantJournal,(2006),,540Ð560 above)wassuccessfullyappliedtoqualitativeanalysisofterpenevolatilesproducedinvitrobyrecombinantplantterpenesynthases(Chenetal.,2003,2004;Koetal.2004;Tholletal.,2005).ArelevantprotocolisprovidedasProtocolS1.AutomatedSPMEextractionmaybeappliedfortherapidanalysisofalargenumberofassays,forexampletoevaluatedifferentproductproÞlesofrecombinantenzymesobtainedinasite-directedmutagenesisapproach.QuantitativeanalysiswithSPMEmaybepossiblebutrequiresextremelycarefulcalibration.Directheadspaceanalysishasbeenusedasanalternat-ivemethodformeasuringtheactivityofterpenesynthases.Thistechniquewassuccessfullyusedfortheanalysisoftheactivityofisoprenesynthase(Schnitzleretal.,1996;SilverandFall,1991)andhasbecomethestandardapproachfordeterminingtheactivityofthisenzymefrommanyplantspecies.Theprincipleoftheenzymeassayisrathersimple:Proteinextractscontainingappropriatecon-centrationsofsubstrate(DMADP)andco-factorsareincu-batedingas-tightvialsunderoptimizedconditions.Toterminatetheenzymereaction,theliquidphasecontainingtheenzyme,substrateandco-factorsisremovedbyagas-tightsyringewhiletheevaporatedenzymeproductre-mainstrappedintheheadspaceofthegas-tightvial(Figure9).Analiquotoftheheadspaceistheninjectedbyagas-tightsyringeforGCanalysis.Analyticalpre-requi-sitesfortheapplicationofautomatedheadspaceanalysisareapre-column,ÞlledwithabsorbentmaterialsuchasTenaxTAfortrappingthevolatilecompoundsupstreamoftheGCcolumn,andatemperature-programmedinjectionsystemforfocusedcompoundinjection.Inasimilarapproach,theactivityofmonoterpenesynthaseshasbeenqualitativelyandquantitativelydeterminedincell-freeproteinextractsofotherplantspecies(Piceaabiescusilex;Fischbachetal.,2000;Loretoetal.,2001a)withahighlylineardetectionrangeofmonoterpenevolatilesintheassayheadspaceandadetectionlimitof1.5ppmvetal.,2000).Themethodhasalsobeenexploi-tedfortheanalysisofcellularDMADPlevelsviaquantita-tiveconversionofDMADPtoisopreneunderacidicandhigh-temperatureconditions(BruggemannandSchnitzler,2002b;Fisheretal.,2001;Loretoetal.,2004;Rosenstieletal.,2002,2003).Thesametechniquecanbeusedtomeasuretheactivityofisopentenyldiphosphate:dimethylallyldiphosphate(IDP)isomerases(Figure9,ggemannandSchnitzler,2002a).EvaluationofthemethodsusedforVOCanalysisAlthoughthelargearrayofavailabletechniquesforvolatileanalysisallowsselectionofthemethodmostsuitableforstudyingspeciÞcresearchproblems,italsoleadstothedifÞcultyofcomparingresultsbiasedbyeachindividualmethod.AßoralorleafvolatileproÞlemayvarydependingonthetechniqueused.Unfortunately,almostnocompar-ativestudiesofdifferentmethodshavebeenperformed.Someofthemostcriticalfactorsinßuencingvolatiledataacquisitionaretheenvironmentalconditionsinthecollec-tionchamberorcuvette,includinglightintensity,tem-peratureandrelativehumidity,sincetheydirectlyinßuencevolatilemetabolismandthephysiologyoftheplantinclu-dingphotosynthesisandtranspiration.Theeffectsoflightintensityandtemperaturehavebeenintensivelyinvesti-gatedforisopreneaswellasothervolatilesemittedfromleavesandßowers.Controloftheairßowiscriticalforregulatingtemperatureandrelativehumidityandproviding 05101520 IsoprenesynthaseMonoterpene synthases IDP isomerase Isopreneormonoterpenes Isoprene 05101520 IsoprenesynthaseMonoterpene synthases IsoprenesynthaseMonoterpene synthases IDP isomerase IDP isomerase+ DMADPorGDPAcidichydrolysisof Isopreneormonoterpenes Isoprene Isoprene30°C or 40°C 30°C70°C Figure9.Schemeforheadspaceanalysisofterpenesynthaseandisopentenyldiphosphate(IDP)isomeraseactivities.Enzymesareincubatedwiththeirsubstratesandco-factorsinsealedGCvials.Afterterminationofthereaction,thewaterphaseiswithdrawnformthevialandVOCproductsareanalyzedviaGCbyautomatedheadspaceinjection.Plantvolatileanalysis2006TheAuthorsJournalcompilation2006BlackwellPublishingLtd,ThePlantJournal,(2006),,540Ð560 optimalgasexchangebetweentheplanttissueandthesurroundingair.Anothercauseofqualitativeandquanti-tativevariationofvolatileresultsobtainedbydifferentanalysisprotocolsistheuseofdifferentvolatileadsorbentsanddesorptionmethods.RagusoandPellmyr(1998)ob-servedsigniÞcantvariationinvolatileblendsfromßowersC.breweriduetotheuseofdifferentsorbentsandor-ganicsolventsforcompoundelution.Inaddition,theyreportedontheinßuenceofhigherßowratesintrappingcompoundswithlowervolatility,butsimultaneouslycausingincreasedcontaminantbackgroundlevelsinGCAnothervariableinvolatileanalysisisthetimeatwhichvolatilesarecollectedinheadspacesamplingtechniques.Thetimeforvolatilecollectionshouldalwaysbeoptimized,sinceemissionsareaffectedbydiurnalandcircadianrhythms.ChangesinvolatileproÞlesinresponsetoherb-ivoryormimickedherbivory(RoseandTumlinson,2005;etal.,1990)canoftentakeseveralhourstoseveraldays,dependingontheplantspeciesandthepartoftheplantchosenforvolatilemeasurements(RoseandTumlin-son,2004).Initialcollectionofvolatilesmaybeperformedoverseveraldayswith24-hcollectionintervals,andsubse-quentlycomparedwithresultsfromseveralshortersamp-lingintervals.Forlow-emittingplantsinparticular,longercollectiontimesareappliedtotrapenoughvolatilesforanalysis.Tofurtherenhancethedetectabilityofvolatilecompounds,onecanincreasetheamountofplantmaterial,decreasethevolumeofsurroundingairspaceandincreasetheairßow,althoughlimitationssuchasincreasedtranspir-ation(inthecaseoftoomuchplanttissue)oraircontam-ination(inthecaseofhigherßowrates)havetobeconsidered.Ariskwillremainthatsomevolatilesmaybeemittedinquantitiesbelowtechnicaldetectability,whichmaybefunctionallyrelevant,forexample,forinsectattrac-ResultsobtainedfromvolatilecollectionsunderÞeldconditionswillalwaysvaryfromthoseacquiredinthelaboratoryduetoßuctuatingenvironmentalparametersandthedifferentialreactivityofplantVOCswithreactivemole-culesintheatmosphere,suchasozone,whenambientairisIntersamplevariabilityofaparticularmethodoccursduetotheplantmaterialbeinginvestigated.Evenifclonallinesareused,caremustbetakentostandardizeplantage,development,light,nutritionalandsoil-moistureconditionsaswellasthetimeofdayforvolatilesampling.Furthermore,ithastobeconsideredthattheemissionofVOCsisstronglyaffectedbyinsectandmicrobialdamageorevenpesticidetreatment,andplantshavetobeselectedormonitoredaccordinglypriortovolatileanalysis.Overall,researchersareadvisedtoperformtheirvolatileanalyseswithmorethanÞvereplicatesandtotestseveraldifferentparametersusing,ifpossible,morethanonemethodofanalysis.ExperimentalconditionsshouldbereportedascarefullyaspossibletofacilitatecomparisonofWithinthepastdecadewehaveseenenormousprogressintheanalysisofplantvolatilesthathasbeenstimulatedbyverydifferentareasofresearchsuchasplantbiochemistry,ecologyandatmosphericchemistry.Particularachieve-mentshavebeenmadeinreal-timeVOCanalysisinmonit-oringrapidandsubtlechangesofvolatileemissionsinresponsetoabioticorbioticfactors.TheseadvanceshavecreatedopportunitiesfordetailedviewsonthetimecoursesofVOCemissions,whethertheyareinvolvedintheattrac-tionofpollinatorsorthedeterrenceofherbivores.Further-more,thesenewanalyticalcapabilitiescanmatchorexceedthetimescaleandsensitivityofcontemporarygenomicandproteomictechniquestoprovidemoreprecisecorrelationswiththebiochemistryunderlyingthebiosynthesisandregulationofVOCs.Therecentdevelopmentsingaschro-matographyandmassspectrometrydescribedherehavereduceddetectionlimitsandimprovedseparationefÞcien-ciesandtimeresponsefactors,andthesewillfacilitatehigh-throughputscreeningsofplantstodetecttransgenics,mutantsornaturalvariantswithalteredVOCemissionpro-Þles.DespitethisprogressinVOCanalysis,traditionalVOCcollectiontechniquesandoff-lineGCanalyseswillremainessentialformanylaboratoriesandespeciallyforremoteÞeldstudies.Therefore,thisreviewhasdescribedsomeimportantguidelinesandparametersregardingVOCcol-lectiondevicesandGCseparationtechniques.Insummary,themethodspresentedhereshouldgivetheVOCresearchertheßexibilitytoinvestigatemultipleaspectsandstillunex-ploredareasofthebiosynthesis,emissionandfunctionofplantVOCs.Forexample,littleisknownsofaraboutthebiosynthesisofVOCsinrootsandtheirroleintherhizo-sphereandsoilecology.Furthermore,integrativestudiesofprimarymetabolismandVOCbiosynthesishavemostlybeenconductedwithisopreneandshouldbeamodelforcomparativeinvestigationsofthebiosynthesisofßoralandwound-inducedvolatiles.AcknowledgementsWearegratefultoJonathanGershenzonandEranPicherskyforsupportandinspiration.WethankJimTokuhisaforhelpfulcommentsandcriticalreviewofthemanuscript.Theexperi-mentalworkpresentedbyD.T.,W.B.andU.RwassupportedbyfundsoftheMaxPlanckSociety(toJonathanGershenzonandW.B.)andNationalScienceFoundationgrantIBN-0211697(toEranPichersky,UniversityofMichigan).WethankMartinGrausandRobertFischbachforscientiÞcsupportinpresentingPTR-MSresultsandheadspaceanalysisprotocolsforterpenesynthaseactivityassays.WearethankfultoNataliaDudarevaandAngelaDorotheaTholletal.2006TheAuthorsJournalcompilation2006BlackwellPublishingLtd,ThePlantJournal,(2006),,540Ð560 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