HARNESSING PLANT BIOMASS FOR BIOFUELS AND BIOMATERIALS - PDF document

areinvolvedinwoundhealing,pestresistanceandsignal-ing,orhaveanti-microbialandanti-fungalactivity.LOXenzymeshavebeenclassiÞedwithrespecttotheirpositionalspeciÞcitywithregardtofattyacidoxygenation.OxygenationatC9(9-LOX)oratC13(13-LOX)ofthehydrocarbonbackboneleadstothe(9)-and(13peroxyderivatives,respectively.PlantLOXcanalsobegroupedintotwogenesub-familiesaccordingtotheiroverallsequencesimilarity.Enzymescarryingnoplastidictransitpeptideshowahighsequencesimilarity�(75%)tooneanotherandaredesignatedtype1LOX.Type2LOXharboranN-terminalextensionandhaveonlyamoderateoverallsequencesimilarity(approximately35%).Thethree-dimensionalproteinstructuresofsoybeanLOX-1and-3havebeenelucidatedandessentialaminoacidsidentiÞed(LiavonchankaandFeussner,2006).Invegetativetissues,LOXprovideshydroperoxidesthatcanbemetabolizedtocompoundsthatarecrucialelementsofplantdefense.ItislessclearwhyseedsandtubershavelargeamountsofLOX.GeneticremovalofspeciÞcLOXisoformsappearsnottocompro-miseplanthealth(BaysalandDemirdoven,2007).Intomato,ÞveLOXgenes(TomLoxA,B,C,DandE)areexpressedduringripening.AntisensesuppressionofTomLoxAandBintomatofruitresultedinnosigniÞcantchangesinthefruitßavor,butco-suppressionofTomLoxCstronglyaffectedtheproductionoffattyacid-derivedvolatiles(Chenetal.,2004).HPLcleavestheLOXproducts,resultingintheformation-oxoacidsandvolatileCandCaldehydes.SimilartoLOX,HPLcanbeclassiÞedintotwogroupsaccordingtosubstratespeciÞcity(Noordermeeretal.,2001).HPLisamemberofthecytochromeP450familyCYP74B/C,andactsonahydroperoxyfunctionalityinalipidperoxidewithoutanyco-factor.Recently,ahemi-acetalhasbeenidentiÞedasprimaryproductofHPL(Matsui,2006).Down-regulationofHPLhasbeenperformedinpotatoplants(Salasetal.,2005).SuchsilencingofHPLinducedanincreaseinLOXactivitybutadecreaseofmostoftheC-unsaturatedcarbonylfunctionalityintheHPLproductsispronetoisomerization,eitherenzymaticallycatalyzedbya3-enalisomeraseornon-enzymatically.Although3-enalisomeraseactivityhasbeendescribedinsoybeans(GlycinemaxL.)andalfalfa(MedicagosativaL.),neitheraproteinnoracorrespondinggenehasbeenclonedyet(Noordermeeretal.,1999;TakamuraandGardner,1996).andCaldehydescanbefurthermetabolizedbyADHtoformthecorrespondingalcohols.genesthataresuspectedtoparticipateintheproductionofaromasareexpressedinadevelopmentallyregulatedmanner,particu-larlyduringfruitripening(Manriquezetal.,2006).Over-expressionofthetomatogenehasledtoimprovedßavorofthefruitbyincreasingthelevelsofalcohols,particularly3-hexenol(Speirsetal.,1998).Anacyltransfer-asecatalyzestheformationof3-hexenylacetatefrom3Z-hexenolandacetylCoA,and2-alkenalreductasecanreduce-hexenaltohexanal(DÕAuriaetal.,2003;Manoetal. Figure7.Linolenicacid-derivedßavormolecules.AAT,alcoholacylCoAtransferase;ADH,alcoholdehydrogenase;AER,alkenaloxidoreductase;AOC,alleneoxidecyclase;AOS,alleneoxidesynthase;HPL,hydroperoxidelyase;JMT,jasmonatemethyltransferase;LOX,lipoxygenase;OPR,12-oxo-phytodienoicacidreductase;3-EI,3-enalisomerase.WilfriedSchwabetal.2008TheAuthorsJournalcompilation2008BlackwellPublishingLtd,ThePlantJournal,(2008),,712Ð732 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strawberry,furaneolisfurthermetabolizedbyantransferase(FaOMT)tomethoxyfuraneol(Weinetal.,2002).-diphenolicstructurewasidentiÞedasacommonstructuralfeatureoftheacceptedsubstrates,andisalsopresentinthedienolictautomeroffuraneol.Genetictrans-formationofstrawberrywiththesequenceintheantisenseorientation,underthecontrolofaconstitutivepromoter,resultedinaneartotallossofmethoxyfuraneol,demonstratingtheinvivomethylationoffuraneolbyFaOMTetal.,2006).However,thereducedlevelofmethoxyfuraneolwasonlyperceivedbyonethirdofthevolunteerpanelists,consistentwithresultsobtainedbyaromaextractdilutionassays.Norfuraneolandhomofura-neolhavebeenidentiÞedintomatoandmelonfruits,respectively,buttheirbiogeneticpathwaysandthatofmaltolremainunknown(SchwabandRoscher,1997).However,studiesintomatoandyeasthaveidentiÞedphosphorylatedcarbohydratesaspotentialprecursorsofthefuranones(Haucketal.,2003;Sasakietal.,1991).ThefuranonesaremutagenictobacteriaandcauseDNAdamageinlaboratorytests.However,theyarealsoveryeffectiveanti-carcinogenicagentsinthedietsofanimals,andtheirantioxidantactivityiscomparabletothatofascorbicacid(Slaughter,1999).Norfuraneolhasbeeniden-tiÞedasamalepheromoneinthecockroach(Walker),andfuraneoldetersfungalgrowth.Furaneolisalsooneofthekeyßavorcompoundsintheattractivearomaoffruits(Farineetal.,1994).Ithasbeenproposedthattheevolvedbiologicalfunctionofthefuranonesistoactasinter-organismsignalmoleculesinvarioussystems.The4-hydroxy-3()-furanonesassociatedwithfruitaromasacttoattractanimalstothefruit,whichensuresseeddispersal.Inthecaseofhumans,thecoinci-dentalchemicalsynthesisofthesecompoundsinfoodsduringpreparationresultsinthesefoodsappearingpartic-ularlyattractivethroughtransferredoperationoftheoriginalsignalingmechanisms(Slaughter,1999).Terpenoidpathway.Terpenoidsareenzymaticallysynthe-denovofromacetylCoAandpyruvateprovidedbythecarbohydratepoolsinplastidsandthecytoplasm.AlthoughfattyacidoxidationisoneofthemajorpathwaysproducingacetylCoA,thisprocessprobablydoesnotcontributetotheformationofterpenoidsasittakesplaceinperoxisomes.Terpenoidsconstituteoneofthemostdiversefamiliesofnaturalproducts,withover40000differentstructuresofterpenoidsdiscoveredsofar.Manyoftheterpenoidspro-ducedarenon-volatileandareinvolvedinimportantplantprocessessuchasmembranestructure(sterols),photosyn-thesis(chlorophyllsidechains,carotenoids),redoxchemis-try(quinones)andgrowthregulation(gibberellins,abscisicacid,brassinosteroids)(Croteauetal.,2000).Thevolatile Figure3.Glandularandnon-glandulartric-homesinOriganumdayiPost(Lamiaceae)(aÐc)Pelargoniumgraveolens(Geraniaceae)(dÐf).Leaves(a,d)andlightmicroscopyoftheleafsurface(b,e)andisolatedglandulartrichomes(c,f)isolatedaccordingtotheprocedureÞrstdevelopedbyGershenzonetal. Figure4.Carbohydrate-derivedßavormolecules,including4-hydroxy-2,5-dimethyl-3(2)-furanone(furaneol),2,5-dimethyl-4-methoxy-3(2)-furanone(methoxy-furaneol),4-hydroxy-5-methyl-3(2)-furanone(norfuraneol),2-ethyl-4-hydroxy-5-methyl-3(2)-furanone(homofuraneol),4-hydroxy-2-methylene-5-methyl-3(2furanone(HMMF)and3-hydroxy-2-methyl-4-pyran-4-on(maltol).WilfriedSchwabetal.2008TheAuthorsJournalcompilation2008BlackwellPublishingLtd,ThePlantJournal,(2008),,712Ð732 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normalintermediatesofthe-oxidation,donotformlac-tones.However,theyareconvertedtomethyl-orethyl-3-hydroxyestersinplantsandcontributetothearomaoffruits.Short-lengthmethylketones(C)arehighlypotentßavormoleculesthathavebeenfoundinnumerousplants,whilemedium-lengthmethylketones(C)arehighlyeffectiveinprotectingplantsfromnumerouspests.Recently,theÞrstmethylketonesynthasewasisolatedfromtomato,whichcatalyzesthehydrolysisandsubsequentdecarboxylationofCandC-ketoacylACPstogiveandCmethylketones,respectively(Fridmanetal.2005).Incontrast,infungi,methylketonesarederived-oxidativedegradationoffattyacidsthrough-ketoacylCoAintermediates(SchwabandSchreier,2002).Methylketonesareassumedtobeprecursorsofaroma-activesecondaryalcoholssuchas2-pentanoland2-heptanol,whichareimportantßavormoleculesproducedbypassionfruits(Figure8b)(Strohalmetal.,2007).Aminoacid-derivedavorcompoundsAlthoughaldehydesandalcoholsderivedfromthedegra-dationofbranched-chainandaromaticaminoacidsormethionineconstituteaclassofhighlyabundantplantvol-atiles,theirpathwayshavebeenbarelyanalyzedinplants.Especiallyimportantarebranched-chainvolatilesderivedfrombranched-chainaminoacids.Isoamylacetate,anesterwithastrongfruityodordescribedassimilartobananaorpear,isoneofthekeyconstituentsofbananaßavor(SurburgandPanten,2005).2-Methyl-butylacetatehasastrongapplescentandisassociatedwithapplevarietiesthatarerichinaromasuchasÔFujiÕ,ÔGalaÕandÔGoldenDeliciousÕ(DixonandHewett,2000;Hollandetal.,2005).Methyl2-methylbutanoatedeterminesthecharacteristicaromaofpricklypear(Weckerleetal.,2001),whileacombinationofseveralvolatileestersimpartstheuniquearomaofmelons,withisoamylacetateand2-methyl-butylacetatebeingprominentinmanyvarieties(BeaulieuandGrimm,2001;Jordanetal.2001;Shalitetal.,2001).Acids,alcohols,aldehydes,esters,lactonesandN-andS-containingavormolecules.Inmicro-organisms,thecatabolismofaminoacidshasbeenanalyzedindetailandisinitiatedbyaminotransferasesforming2-ketoacidsthatserveassubstratesforthreebiochemicalreactions:(i)oxidativedecarboxylationtocarboxylicacids,(ii)decarboxylationtoaldehydes,and(iii)reductionto2-hydroxyacids(Figure9a)(MarilleyandCasey,2004).Compoundsderivedfromleucinesuchas3-methylbutanal,3-methylbutanoland3-methylbu-tanoicacid,aswellasphenylacetaldehydeand2-phenyleth-anolformedfromphenylalanine,areabundantinvariousfruitssuchasstrawberry,tomatoandgrapevarieties(Aubertetal.,2005).Inaddition,alcoholsandacidsderivedfromaminoacidscanbeesteriÞedtocompoundswithalargeimpactonfruitodor,suchas3-methylbutylacetateand3-methylbutylbutanoateinbanana(Nogueiraetal.,2003).Genesencodingenzymesresponsibleforthedirectdecarboxylationofphenylalaninehavebeenisolatedfromtomato,petuniaandrose,showingthatalternativecatabolicpathwaysexistinplants(Figure9a)(Kaminagaetal.,2006;etal.,2006).Althoughtheenzymesdisplaysubtledifferencesinsequencesandenzymaticproperties,theirdown-regulationledtoreducedemissionof2-phenylacetal-dehydeand2-phenylethanol.Over-expressionoftheamino Figure9.Biosynthesisofaminoacid-derivedßavorcompounds.(a)Catabolismofbranched-chainaminoacidsleadingtomethylbranchedßavorcompounds,and(b)postulatedbiosynthesisofsotolon.For-mationofaldehyde(a)fromaminoacidsrequirestheremovalofbothcarboxylandaminogroups.Thesequenceoftheseremovalsisnotfullyknownandcouldbetheoppositetothatshownoraldehydecouldbeformedinonestepbyaldehydesynthase(Kaminagaetal.,2006;etal.,2006).WilfriedSchwabetal.2008TheAuthorsJournalcompilation2008BlackwellPublishingLtd,ThePlantJournal,(2008),,712Ð732 oilsasarichsourceofßavormoleculesareusedwidelytopreventandtreathumandiseases(Edris,2007).Theses-quiterpeneartemisinin,derivedfromArtemisiaannua,hasattractedspecialattentionduetoitsanti-malarialactivityetal.,2007).Alternativesourcesforthesupplyoftheanti-malarialcompoundhavebeenproposedduetoshortageoftherawmaterial(Changetal.,2007).Inaddition,volatilesmayalsobeusedtoeffectivelymanipulaterumenmicrobialfermentationtoreducetheformationofammoniaandmethane(Calsamigliaetal.,2007).Thus,ßavorcom-poundshavenumerousfunctionalpropertiesandexerttheiractioninmammalsaswellasinotherorganisms.Theywillcontinuetobeindispensablenaturalingredients.AcknowledgementsWethankAlonZaxforhelpinthepreparationofFigure3.W.S.thanksDegussaAGforfunding.Abanda-Nkpwatt,D.,Krimm,U.,Coiner,H.A.,Schreiber,L.andSchwab,W.(2006a)PlantvolatilescanminimizethegrowthsuppressionofepiphyticbacteriabythephytopathogenicfungusBotrytiscinereainco-cultureexperiments.Environ.Exp.Bot.Abanda-Nkpwatt,D.,Musch,M.,Tschiersch,J.,Boettner,M.andSchwab,W.(2006b)Molecularinteractionbetweenteriumextorquensandseedlings:growthpromotion,methanolconsumption,andlocalizationofthemethanolemissionsite.J.Exp.Bot.,4025Ð4032.Aharoni,A.,Keizer,L.C.P.,Bouwmeester,H.J.etal.(2000)IdentiÞ-cationofthegeneinvolvedinstrawberryßavorbiogenesisbyuseofDNAmicroarrays.PlantCell,647Ð662.Aharoni,A.,Giri,A.P.,Deuerlein,S.,Griepink,F.,deKogel,W.J.,Verstappen,F.W.A.,Verhoeven,H.A.,Jongsma,M.A.,Schwab,W.andBouwmeester,H.J.(2003)Terpenoidmetabolisminwild-typeandtransgenicArabidopsisplants.PlantCell,2866Ð2884.Aharoni,A.,Giri,A.P.,Verstappen,F.W.A.,Bertea,C.M.,Sevenier,R.,Sun,Z.,Jongsma,M.A.,Schwab,W.andBouwmeester,H.J.(2004)Gainandlossoffruitßavorcompoundsproducedbywildandcultivatedstrawberryspecies.PlantCell,3110Ð3131.Ahmed,M.A.,ElÐMawla,A.andBeerhues,L.(2002)BenzoicacidbiosynthesisincellculturesofHypericumandrosaemum,727Ð733.Aubert,C.,Baumann,S.andArguel,H.(2005)OptimizationoftheanalysisofßavorvolatilecompoundsbyliquidÐliquidmicro-extraction(LLME).Applicationtothearomaanalysisofmelons,peaches,grapes,strawberries,andtomatoes.J.Agric.Food,8881Ð8895.Bagchi,G.D.(2000)Essentialoilproducingglandsinsomeimpor-tantaromaticplants:structureandprocessofsecretion.J.Med.AromaticPlantSci.,605Ð615.Bak,S.,Paquette,S.M.,Morant,M.etal.(2006)Cyanogenicglycosides:acasestudyforevolutionandapplicationofcytochromesP450.Phytochem.Rev.,309Ð329.Baker,A.,Graham,I.A.,Holdsworth,M.,Smith,S.M.andTheodoulou,F.L.(2006)Chewingthefat:-oxidationinsignallinganddevelopment.TrendsPlantSci.,124Ð132.Baldwin,E.A.,Scott,J.W.,Shewmaker,C.K.andSchuch,W.Flavortriviaandtomatoaroma:biochemistryandpossiblemechanismsforcontrolofimportantaromacomponents.HortScience,1013Ð1022.Barghini,P.,DiGioia,D.,Fava,F.andRuzzi,M.(2007)VanillinproductionusingmetabolicallyengineeredEscherichiacoliundernon-growingconditions.Microb.CellFact.,13Ð24.Basear,K.H.C.andDemirci,F.(2007)Chemistryofessentialoil.InFlavorsandFragrances(Berger,R.G.,ed.).Heidelberg:Springer,pp.43Ð86.Bayrak,A.(1994)VolatileoilcompositionofTurkishrose(damascenaJ.Agric.Food.Chem.,441Ð448.Baysal,T.andDemirdo¬ven,A.(2007)Lipoxygenaseinfruitsandvegetables:areview.EnzymeMicrob.Technol.,491Ð496.Beaulieu,J.C.andGrimm,C.C.(2001)IdentiÞcationofvolatilecompoundsincantaloupeatvariousdevelopmentalstagesusingsolidphasemicroextraction.J.Agric.FoodChem.Beekwilder,J.,Alvarez-Huerta,M.,Neef,E.,Verstappen,F.W.A.,Bouwmeester,H.J.andAharoni,A.(2004)Functionalcharacter-izationofenzymesformingvolatileestersfromstrawberryandbanana.PlantPhysiol.,1865Ð1878.Bernreuther,A.andSchreier,P.(1991)Multidimensionalgaschro-matography/massspectrometry:apowerfultoolforthedirectchiralevaluationofaromacompoundsinplanttissues.2.Linaloolinessentialoilsandfruits.Phytochem.Anal.,167Ð170.Beuerle,T.andPichersky,E.(2002)PuriÞcationandcharacterizationofbenzoate:coenzymeAligasefromClarkiabreweriBiochem.Biophys.,258Ð264.Boatright,J.,Negre,F.,Chen,X.,Kish,C.M.,Wood,B.,Peel,G.,Orlova,I.,Gang,D.,Rhodes,D.andDudareva,N.(2004)Under-standinginvivobenzenoidmetabolisminpetuniapetaltissue.PlantPhysiol.,1993Ð2011.Bohlmann,J.andKeeling,C.(2008)Terpenoidbiomaterials.PlantJ.,656Ð669.Bohlmann,J.,Meyer-Gauen,G.andCroteau,R.(1998)Plantterpe-noidsynthases:molecularbiologyandphylogeneticanalysis.Proc.NatlAcad.Sci.USA,4126Ð4133.Bones,A.M.andRossiter,J.T.(2006)Theenzymicandchemicallyinduceddecompositionofglucosinolates.Bood,K.G.andZabetakis,I.(2002)Thebiosynthesisofstrawberryßavor(II):biosyntheticandmolecularbiologystudies.J.FoodSci.,2Ð8.Buck,L.andAxel,R.(1991)Anovelmultigenefamilymayencodeodorantreceptors:amolecularbasisforodorrecognition.,175Ð187.Buseman,C.M.,Tamura,P.,Sparks,A.A.etal.(2006)Woundingstimulatestheaccumulationofglycerolipidscontainingoxophy-todienoicacidanddinor-oxophytodienoicacidinArabidopsisleaves.PlantPhysiol.,28Ð39.Calsamiglia,S.,Busquet,M.,Cardozo,P.W.,Castillejos,L.andFerret,A.(2007)EssentialoilsasmodiÞersofrumenmicrobialfermentation.J.DairySci.,2580Ð2595.Chang,M.C.Y.,Eachus,R.A.,Trieu,W.,Ro,D.-K.andKeasling,(2007)EngineeringEscherichiacoliforproductionoffunctionalizedterpenoidsusingplantP450s.Nat.Chem.Biol.Chase,T.,Jr(2000)Alcoholdehydrogenases:identiÞcationandnamesforgenefamilies.PlantMol.Biol.Rep.,333Ð350.Chen,S.andAndreasson,E.(2001)Updateonglucosinolatemetabolismandtransport.PlantPhysiol.Biochem.,743Ð758.Chen,F.,DÕAuria,J.C.,Tholl,D.,Ross,J.R.,Gershenzon,J.,Noel,J.P.andPichersky,E.(2003)AnArabidopsisthalianageneformethylsalicylatebiosynthesis,identiÞedbyabiochemicalgenomicsapproach,hasaroleindefense.PlantJ.,577Ð588.Biosynthesisofplant-derivedavorcompounds2008TheAuthorsJournalcompilation2008BlackwellPublishingLtd,ThePlantJournal,(2008),,712Ð732 microbialfermentation,biotransformationusingwholecells,biocatalysisusingisolatedenzymes,planttissuecultureandtransgenicplants.Byvolume,short-chainacidsandalcoholsarethemostprominentaroma-activecompoundsproducedbybiotech-nology.Thestrongoxidativecapabilitiesofstrainsareharnessedfortheproductionofaceticacid,propionicacid,butanoicacid,2-methylpropa-noicacid,2-methylbutanoicacidand3-methylbutanoicacid,andthederivedshort-chainalcoholsconstitutecheapby-productsduringthedistillationofÔbioethanolÕ(Schrader,2007).Thesenaturalacidsandalcoholsareofgreatimpor-tancetotheßavorindustry(US$100perkg),eitherbecauseoftheirintensesmellandsourtaste,inthecaseoftheacids,orassubstratesforenzymaticsynthesisofßavorestersetal.,2004).ThederivedaldehydesareefÞcientlyproducedbyoxidationofthecorrespondingalcoholsusingalcoholoxidase-oralcoholdehydrogenase-expressingmicro-organisms,e.g.PichiapastorisMicrobialbiotransformationandbiosynthesisofßavorandfragrancechemicalsoffertheadditionalpotentialben-eÞtsofproducingopticallyactiveisomersthatoftenhavemarkeddifferencesinßavorandfragrancequalityandsensoryintensity.4decanolide(-decalactone),whichimpartsafruity,peach-likearoma,hasanannualmarketvolumeofseveralhundredtonnes.Natural4wasanextremelyexpensive,naturalaromachemical(US$10000perkg)intheearly1980s.Today,itisprimarilyproducedbyYarrowialipolytica,resultinginadropofthepricetoUS$300perkg.Theyeastdegradesricinoleicacidoctadecenoicacid),thedominantfattyacidofcastoroil,totheenantiomericallypurelactone(Schrader,2007;Schraderetal.,2004).Alternatively,opticallypurearomachemicals,e.g.()-mentholmaybecommerciallysynthesizedbyenantio-selectivehydrolysisofracemicsub-strates,e.g.menthylbenzoate,byrecombinantlipase(MenzelandSchreier,2007).Natural3hexenol(US$3000perkg)isalsoproducedcompetivelycomparedtoitsisolationfrompeppermintoilbyaseriesofbiocatalyticsteps.Theindustrialprocessmimicstheplantbiochemicalpathwaystartingfromlino-lenicacidandexploitingcrudeplantenzymepreparationsoflipoxygenase(soyßour)andhydroperoxidelyase(guavafruithomogenate)toproducethedesiredaldehyde.Addi-tionally,bakerÕsyeastisusedasreducingcatalysttoconvertthealdehydetothecorrespondingalcohol(Figure7)(Schrader,2007).Biosyntheticvanillinhasbeenmarketedsince2000,andispreparedbytheactionofmicro-organismsonferulicacidextractedfromricebran(Barghinietal.,2007).AtUS$1000perkg,thisproductisnotcost-competitivecomparedwithpetrochemicalvanillin,whichsellsforaroundUS$11perkg,butitcanbelabeledasnaturalßavoring.Naturalvanillinderivedfromisonlyavailableinverylowamountsandisthereforelimitedtoafewselectedfoodapplications.V.planifoliacellcultureshavebeenstudiedextensively,noeconomicalfeasiblevanillinproductionhasresultedfromthis(KorthouandVerpoorte,2007).Theexamplesshowthatbiotechnologicalproductionofßavorcompoundsisamaturedisciplineinthechemicalindustry.About100aromachemicalsinthemarketarealreadyproducedbyenzymaticormicrobialprocesses,includinggeneticallyengineeredmicro-organisms(Schrad-er,2007).Theseproductionlinesrelyonplantsasthesourceoftheßavorprecursorsandmostoftheinvolvedgenesandenzymesoriginatefrommicrobialsources,exceptforbiotechnologicalproductionoftheCcompounds.Success-fulmetabolicengineeringofthepathwaysofanumberofplantßavormoleculeshasalreadybeenachievedinplants,butthesetransgencisarenotyeteconomicallycompetitiveetal.,2007;Lavyetal.,2002;Lewin-etal.,2001;MahmoudandCroteau,2001;Munetal.,2006).Insomecases,geneticmanipulationledtochangedphenotypes,indicatingthedepletionofessentialintermediatesthroughredirectionofthecarbonßux(Aharonietal.,2003).Thus,moreresearchisnecessarytoelucidatetheregulationofßavorformationinPlantvolatileoilsaresynthesized,storedandreleasedbyavarietyofspecializedepidermalormesophyllstructures,probablybecausemostoftheßavormoleculesarephyto-toxic.Theregulationandformationofthesestructuresisnotunderstood,buttheirmanipulation,whetherachievedthroughconventionalmeansormodernmolecularapproaches,maybeanadditionalpotentialapproachforchangingoilcompositionandquality.PossibleusesofavormoleculesAroma-activevolatilesareimportantsecondaryplantmetabolitesthataretraditionallyusedfortheirßavorandfragranceinthefood,pharmaceuticalandperfumeryindustriestoimprovetheattractivenessoftheproducts(Guentert,2007).However,theyalsoserveasprecursorsourcesfortheproductionofaromachemicals,drugsandplantprotectionagents,particularlyenantiomers,andareusefulchiralbuildingblocksinchemicalandbiotechnolog-icalsyntheses(DeCarvalhoanddaFonseca,2006).IdentiÞ-cationofthebiologicalandpharmacologicalactivitiesofßavorcompoundshasbeengatheringmomentuminrecentyears.Thecommercializationofplantvolatilesisalsofocusedontheirbioactivities,includinganti-microbial,anti-viral,anti-oxidative,analgesic,digestive,anti-carcinogenic,semiochemicalandotheractivities(Korochetal.,2007).Aromatherapyisaformofcomplementaryoralternativemedicinethatusesessentialoilsandotherscentedplantcompoundsfortherapeuticpurposes,affectingapersonÕsmoodorhealth(Kiecolt-Glaseretal.,2008).Thus,essentialWilfriedSchwabetal.2008TheAuthorsJournalcompilation2008BlackwellPublishingLtd,ThePlantJournal,(2008),,712Ð732 sedativeandhasbeenusedasacosmeticadditive(Shojietal.,2000).Thebiosynthesispathwayisthoughttobeginwithphloroglucinolandincludesthreemethylationsteps.Manymodernrosevarietiessynthesizearelatedcompound,3,5-dimethoxytoluene(Flamentetal.,1993),fromorcinol(3,5-dihydroxytoluene)bytwosuccessivemethylations.The-methyltransferasesresponsibleforthesereactionshavebeencharacterized(Lavidetal.,2002;Scallietetal.2002).Orcinol-methyltransferasescanalsocarryoutthelasttwomethylationstepsof1,3,5-trimethoxybenzenebio-synthesisbutareincapableofefÞcientlymethylatingphloroglucinol,explainingtheabsenceof1,3,5-trimethoxy-benzenefromtheßoralscentofmodernrosevarieties.Anovelphloroglucinol-methyltransferasethatmethylatestheÞrststeptoproducetheintermediate3,5-dihydroxyani-solehasbeenisolatedfromrosepetals,andthetwopreviouslydescribedorcinol-methyltransferasescatalyzethesubsequentsteps(Wuetal.,2004).Methylsalicylateandmethylbenzoatearecommoncomponentsofßoralscentandarebelievedtobeimportantattractantsofinsectpollinators(Dobson,1994;DudarevaandPichersky,2000;Dudarevaetal.,1998,2000).Enzymesthatcatalyzetheformationofmethylsalicylateandmethylbenzoatefromsalicylicacid(SA)andbenzoicacid(BA),respectively,havebeencharacterizedfromßowersof,snapdragon(Antirrhinummajus),petunia(ArabidopsisthalianaStephanotisoribundaetal.,2003;MurÞttetal.,2000;Negreetal.,2002;Pottetal.,2002;Rossetal.,1999).Whiletheseenzymesuse-methionineasthemethyldonorasdomanypreviouslycharacterizedmethyltransferasesthatactonavarietyofsubstrates(e.g.DNA,protein,phenylpropanoids),theseSAandBAcarboxylmethyltransferaseshaveprimaryaminoacidsequencesthatshownosigniÞcantsequenceidentitytoothermethyltransferases.Interestingly,agroup-methyltransferasesinvolvedinbiosynthesisofthealkaloidcaffeine,includingtheobrominesynthase,sharesequencesimilaritywiththeSAandBAcarboxylmeth-yltransferases(DÕAuriaetal.,2003).TheseenzymeswerethereforegroupedintoanewclassofmethyltransferasesdesignatedtheSABATHmethyltransferases,andthisfamilynowalsoincludesjasmonicacidmethyltransferaseetal.,2001),indole-aceticacidmethyltransferaseetal.,2003)andcinnamic/-coumaricacidmethyl-transferase(Kapteynetal.,2007).Therecentlyobtainedthree-dimensionalstructureofC.breweriSAcarboxylmethyltransferase(Zubietaetal.,2003),combinedwithmodelingoftheactivesitepocketintheS.oribundaenzymes(Pottetal.,2004),alsoindicatesthattheseenzymeshaveauniquestructurethatisdistinctfromthoseofunrelatedmethyltransferasesfoundinplants(Noeletal.,2003).Vanillin(4-hydroxy-3-methoxybenzaldehyde)isthemostwidelyusedßavorcompoundintheworld.Itistheprincipalßavorcomponentofthevanillaextractobtainedfromcuredpods(beans)oftheorchidVanillaplanifoliaVanillinaccumulatesinthesecretionaroundtheseedsinthematurefruits.Auniquesecretorytissuecomposedofcloselypackedunicellularhairsislocatedinthreegapsbetweentheplacentasalongthecentralfruitcavity.Thesecellsseemtoberesponsibleforvanillinsecretion(Joeletal.2003).Vanillaextractisvaluedasanaturalßavor,but,becauseofitscostandlimitedavailability,lessthan1%oftheannualworlddemandforvanillinisisolatedfromitsnaturalsource(Waltonetal.,2003).Mostofthevanillinusedbytheßavorindustryoriginatesfromchemicalmethodsthatuseguaiacol,eugenolorligninasstartingmaterials(RaoandRavishankar,2000).Vanillinisbelievedtobesynthe-sizedfromphenylpropanoidprecursors,andvariousbio-syntheticpathwayshavebeenproposed.Athree-steppathwayforvanillinbiosynthesisfrom4-coumaricacidhasbeenproposedbasedonprecursoraccumulationandonfeedingcellculturesofV.planifoliawiththeproposedprecursors(Havkin-Frenkeletal.,1999).Inthispathway,4-coumaricacidisÞrstconvertedto4-hydroxybenzaldehydeby4-hydroxybenzaldehydesynthasethroughachain-short-eningstep(Podstolskietal.,2002).Then,hydroxylationatposition3ontheringisperformedby4-hydroxybenzalde-hydesynthase,converting-hydroxybenzylalcoholto3,4-dihydroxybenzylalcoholoraldehyde.TheÞnalenzymaticstepwasshowntobecatalyzedbyamultifunctional-methyltransferasefromV.planifoliathathasabroadsubstraterange,including3,4-dihydroxybenzaldehyde(Paketal.,2004).BiotechnologicalprocessesNaturalaromachemicalshavebeenusedformillenniaandarestillusedtoday.However,overthepast50years,dueinparttotheexpenseofnaturalingredients,theßavorandfragranceindustryhasdevelopedandusessyntheticprod-uctscomprisingnature-identicalodorantsandartiÞcalcompoundsthatarenotfoundinnature.Today,90%ofthenewlyintroducedfragranceingredientsaresyntheticaromachemicals(DePreville,2006).However,thedemandfornaturalaromachemicalsisgrowingfast,inresponsetobothconsumers,whoareaskingforareturntonature,aswellasperfumersandßavorists,whoarelookingfornovelcreativeingredients.Therefore,thepercentageofnaturalßavorswithrespecttoalladdedßavorshasincreasedto90%(EU)and80%(USA)inbeverages,to80%(EUandUSA)insavoryfoods,andto50%(EU)and75%(USA)indairyfoods(Schrader,2007).Increasingoilpriceshaveputadditionalpressureonthistrend.However,thequalityandsupplyoftraditionalnaturalßavorandfragrancechemicalsarelim-ited.Inadditiontoextractionfromnaturalsources,viablealternativeandinnovativewaystoprovideßavorandfragrancechemicalsincludebiotechnologicalroutes,i.e.Biosynthesisofplant-derivedavorcompounds2008TheAuthorsJournalcompilation2008BlackwellPublishingLtd,ThePlantJournal,(2008),,712Ð732 whicharesynthesizedfromcertainaminoacids,aresulfur-rich,nitrogen-containingthioglycosidesthat,uponhydroly-sisbyendogenousthioglucosidases,producevolatileproductssuchasisothiocyanates,thiocyanatesandnitriles(Figure10c)(BonesandRossiter,2006;ChenandAndreas-son,2001).Thesearetheactivesubstancesthatserveasdefensecompoundsorattractantsfortheplant.Forhumans,theyfunctionascancer-preventingagents,biopesticidesandßavorcompounds(HalkierandGershenzon,2006).Cyanogenicglycosides,whichare-glycosidesof-hydroxynitriles,areanothergroupofaminoacid-derivedßavorprecursors(Figure10d)(Baketal.,2006).Cyanogen-esisistheprocessbywhichhydrogencyanideandvolatileketonesoraldehydesarereleasedfromcyanogenicglyco-sidesandisdependentonglycosidaseactivities(Vetter,2000).Enzymatichydrolysisyieldsanunstablehydroxynitrileintermediatethatspontaneouslydecomposesundercertainconditionstohydrogencyanideandacarbonylcompound.Alternatively,theintermediatecanbebrokendownenzy-maticallyby-hydroxynitrilelyase.Ecologicalstudieshaveshownthatcyanogenicglycosidescanactaseitherfeedingdeterrentsorphagostimulants,dependingontheinsectspecies(Vetter,2000).Phenylpropenesandotheraromaticderivatives.andphenylpropanoidvolatilecompounds,primarilyderivedfromphenylalanine,contributetothearomasandscentsofmanyplantspeciesandplayimportantrolesinplantcom-municationwiththeenvironment(DudarevaandPichersky,2006;KnudsenandGershenzon,2006;Picherskyetal.2006).SeveralenzymesthatcatalyzetheÞnalstepsinthebiosynthesisofthesecompoundshavebeenisolatedandcharacterized.However,theearlystepsleadingtotheformationofthebenzenoidbackboneremainunclear(BeuerleandPichersky,2002;SchneppandDudareva,2006;Wildermuth,2006).Ingeneral,biosynthesisofbenzenoidsfromphenylalaninerequiresshorteningofthecarbonskeletonsidechainbyaCunit,whichcanpotentiallyoccurviaeitherthepathwayornon-oxidatively(Boatrightetal.,2004).Experi-mentswithstableisotope-labeledprecursorsintobaccoNicotianatabacum)leaves(Ribnickyetal.,1998)suggestedthatbenzoicacidisproducedfromphenylalanine-derivedcinnamicacidviathe-oxidativepathway,ÞrstyieldingbenzoylCoA,whichcanthenbehydrolyzedbyathioesterasetofreebenzoicacid.Incontrast,labelingexperiments,togetherwithinitialenzymecharacterization,incellcultures(Ahmedetal.,2002)supportedtheexistenceofapathwayfornon-oxidativeconversionofcinnamicacidtobenzaldehydewithsubsequentformationofbenzoicacid,whichcanbefurtherconvertedtobenzoylCoA(BeuerleandPichersky,2002).InvivoisotopelabelingandmetabolicßuxanalysisofthebenzenoidnetworkinpetuniaPetuniahybrida)ßowersrevealedthatbothpathwaysyieldbenzenoidcompounds,andthatbenzylbenzoateisanintermediatebetween-phenylalanineandbenzoicacidetal.,2004).Transgenicpetuniaplantsweregeneratedinwhichexpressionofbenzoyl-CoA:phenyletha-nol/benzylalcoholbenzoyltransferase(BPBT),thegeneencodingtheenzymethatusesbenzoylCoAandbenzylalcoholtomakebenzylbenzoate,wasreducedoreliminated.Eliminationofbenzylbenzoateformationdecreasedtheendogenouspoolofbenzylacidandmethylbenzoateemissionbutincreasedemissionofbenzylalcoholandbenzylaldehyde,conÞrmingthecontributionofbenzylben-zoatetobenzoicacidformation(Orlovaetal.,2006).Labelingexperimentswith-phenylalaninerevealedadilutionofisotopicabundanceinmostmeasuredcompoundsinthedark,suggestinganalternativepathwayfromaprecursorotherthanphenylalanine,possiblyphenylpyruvate.Phenylpropenessuchasanethole,eugenolandisoeu-genolareproducedbyplantsasdefensecompoundsagainstanimalsandmicro-organismsandasßoralattrac-tantsofpollinators.Moreover,humanshaveusedphenyl-propenessinceantiquityforfoodpreservationandßavoringandasmedicinalagents(Grossetal.,2002).Glandulartrichomesofsweetbasil(Ocimumbasilicum),whichsyn-thesizeandaccumulatephenylpropenes,containanenzymethatcanuseconiferylacetateandNADPHtoformeugenoletal.,2006).Petunia(Petuniahybridacv.Mitchell)ßowers,whichemitlargeamountsofisoeugenol,possessanenzymehomologoustothebasileugenol-formingenzymethatalsousesconiferylacetateandNADPHassubstratesbutcatalyzestheformationofisoeugenol.Thebasilandpetuniaphenylpropene-formingenzymesbelongtoastructuralfamilyofNADPH-dependentreductasesthatalsoincludespinoresinol/lariciresinolreductase,isoßavonereductaseandphenylcoumaranbenzylicetherreductaseetal.,2006).Phenylpropenesandbenzoidsarefurthersubjectedtomethylationcatalyzedbyplant-methyltransferases(OMTs)(Ibrahimetal.,1998).VariousOMTsinvolvedinthebiosyntheticpathwaysofßoralscentcomponentshavebeenidentiÞedandcharacterized.Forexample,-methionine(iso)eugenolOMT,whichcatalyzesthemeth-ylationofeugenolandisoeugenoltoformthevolatilesmethyleugenolandisomethyleugenol,hasbeenisolatedClarkiabrewerietal.,1997).EugenolOMTandchavicolOMT,whichconverteugenolandchavicoltomethyleugenolandmethylchavicol,respectively,havebeenidentiÞedinOcimunbasilicumetal.,2002;Lewin-etal.,2000).Similarly,enzymaticactivitiesabletoanoleto-anetholeandchavicoltoestagolehavebeendemonstratedinFoeniculumvulgaretissues(Grossetal.,2002,2006).Anotherbenzylalcoholderivative,1,3,5-trimethoxyben-zene,hasbeenidentiÞedasakeycomponentoftheodorofChineserose(Yomogida,1992).ThisvolatileisaneffectiveWilfriedSchwabetal.2008TheAuthorsJournalcompilation2008BlackwellPublishingLtd,ThePlantJournal,(2008),,712Ð732 potential(WinterhalterandRouseff,2002).Thesynthesisof-ionone,geranylacetoneand6-methyl-5-hepten-2-oneintomatofruitsincreases10Ð20-foldduringfruitripening,andthesecompoundswereproducedbytheactivityofthegenesandLethatwereisolatedfromtomatofruits(Simkinetal.,2004).Intomatofruit,-iononeispres-entatverylowconcentrations(4nll),butduetoitslowodorthreshold(0.007nll)isthesecondmostimportantvolatilecontributingtofruitßavor(Baldwinetal.,2000).SilencingofLeandLeresultedinasigniÞcantdecreaseinthe-iononecontentofripefruits,implyingaroleforthesegenesinC13norisoprenoidsynthesisinvivoetal.,2004).ReductionofPetuniahybridaCCD1transcriptlevelsintransgenicplantsledtoa58Ð76%decreasein-iononesynthesisinthecorollasofselectedpetunialines,indicatingasigniÞcantroleforthisenzymeinvolatilesynthesis(Simkinetal.,2004).Also,apotentialCCDgenewasidentiÞedamongaVitisviniferaL.ESTcollection,andrecombinantexpressionofVvconÞrmedthatthegeneencodesafunctionalCCDthatcleaveszeaxanthinsymmetricallyyielding3-hydroxy--iononeandaCdehyde(Mathieuetal.,2005).CCDswerealsofoundtobeinvolvedintheformationofimportantaromacompoundsinmelon(Cucumismelo)(Figure6).TheproductoftheCCD1gene,whoseexpressionisup-regulateduponfruitdevelopment,wasshowntocleavecarotenoids,generatinggeranylacetonefromphytoene,pseudoiononefrom-iononefrom-carotene,and-iononeandpseudoiononefrom-carotene(Ibdahetal.,2006).Fattyacid-derivedandotherlipophylicavorcompoundsThemajorityofplantvolatilesonaquantitativeandquali-tativebasisoriginatefromsaturatedandunsaturatedfattyacids.Fattyacid-derivedstraight-chainalcohols,aldehydes,ketones,acids,estersandlactonesarefoundubiquitouslyintheplantkingdomathighconcentrations,andarebasicallyformedbythreeprocesses,-oxidationandthelipoxygenasepathway(SchwabandSchreier,2002).Inplants,fattyacidsarestoredastriacylglyceridesandthere-foreenzymaticoxidativedegradationoflipidsisprecededbytheactionofacylhydrolase,liberatingthefreefattyacidsfromacylglycerols.However,identiÞcationofanumberofoxylipin-containingphosphatidylglycerols,monogalacto-syldiacylglycerolsanddigalactosyldiacylglycerolsdemon-stratedthatdirectoxidationofthefattyacidsidechaininacylglyceridesispossible(Busemanetal.,2006).Lipoxygenasepathway(in-chainoxidation).SaturatedandunsaturatedvolatileCandCaldehydesandalcoholsareimportantcontributorstothecharacteristicßavorsoffruits,vegetablesandgreenleaves.TheyarewidelyusedasfoodadditivesbecauseoftheirÔfreshgreenÕodor.Theshort-chainaldehydesandalcoholsareproducedbyplantsinresponsetowoundingandplayanimportantroleintheplantsdefensestrategiesandpestresistance(Matsui,2006;StumpeandFeussner,2006).Atleastfourenzymesarein-volvedinthebiosyntheticpathwayleadingtotheirforma-tion:lipoxygenase(LOX),hydroperoxidelyase(HPL),3enalisomeraseandalcoholdehydrogenase(ADH)(Figure7).LOXisanon-heme,iron-containingdioxygenasethatcatalyzestheregio-andenantio-selectivedioxygenationofunsaturatedfattyacids(e.g.linoleicand-linolenicacid)containingoneormore1-pentadienoicmoieties(Lia-vonchankaandFeussner,2006).NumerousplantLOXhavebeencharacterizedbecausetheyareessentialcomponentsoftheoxylipinpathway,convertingfattyacidsintohydro-peroxidesandÞnallyßavorssuchas3-hexenol,2and2-nonadienal.ProductsoftheLOXpathway Figure6.Carotenoidsandtheirdegradationproducts.Carotenoidsubstrates(left)areoxidativelycleavedtoyieldtheapocarotenoidderivatives(right).Biosynthesisofplant-derivedavorcompounds2008TheAuthorsJournalcompilation2008BlackwellPublishingLtd,ThePlantJournal,(2008),,712Ð732 HARNESSINGPLANTBIOMASSFORBIOFUELSANDBIOMATERIALS Biosynthesisofplant-derivedßavorcompounds WilfriedSchwab 1,* ,RachelDavidovich-Rikanati 2 andEfraimLewinsohn 2 1 BiomolecularFoodTechnology,TechnicalUniversityMunich,85354Freising,Lise-Meitner-Straße34,Germany,and 2 DepartmentofVegetableCrops,NeweYa’arResearchCenter,AgriculturalResearchOrganization,POBox1021, RamatYishay,Israel Received30November2007;revised1February2008;accepted4February2008. * Forcorrespondence(fax+498161712950;e-mailschwab@wzw.tum.de). Summary Plantshavethecapacitytosynthesize,accumulateandemitvolatilesthatmayactasaromaandßavor moleculesduetointeractionswithhumanreceptors.Theselow-molecular-weightsubstancesderivedfrom thefattyacid,aminoacidandcarbohydratepoolsconstituteaheterogenousgroupofmoleculeswith saturatedandunsaturated,straight-chain,branched-chainandcyclicstructuresbearingvariousfunctional groups(e.g.alcohols,aldehydes,ketones,estersandethers)andalsonitrogenandsulfur.Theyare commerciallyimportantforthefood,pharmaceutical,agriculturalandchemicalindustriesasßavorants,drugs, pesticidesandindustrialfeedstocks.Duetothelowabundanceofthevolatilesintheirplantsources,manyof thenaturalproductshadbeenreplacedbytheirsyntheticanaloguesbytheendofthelastcentury.However, theforeseeableshortageofthecrudeoilthatisthesourceformanyoftheartiÞcalßavorsandfragranceshas promptedrecentinterestinunderstandingtheformationofthesecompoundsandengineeringtheir biosynthesis.AlthoughmanyofthevolatileconstituentsofßavorsandaromashavebeenidentiÞed,manyof theenzymesandgenesinvolvedintheirbiosynthesisarestillnotknown.However,modiÞcationofßavorby geneticengineeringisdependentontheknowledgeandavailabilityofgenesthatencodeenzymesofkey reactionsthatinßuenceordivertthebiosyntheticpathwaysofplant-derivedvolatiles.Majorprogresshas resultedfromtheuseofmolecularandbiochemicaltechniques,andalargenumberofgenesencoding enzymesofvolatilebiosynthesishaverecentlybeenreported. Keywords:aroma,ßavor,terpenes,phenylpropanoids,fattyacidderivatives,furanones. Introduction FlavorisdeÞnedasthecombinationoftasteandodor.Itis, however,inßuencedbyothersensationssuchaspain,heat, coldandtactilesensations,oftenreferredtoastheÔtextureÕ offoods(Thomson,1987).Duetothemultitudeofmolecules thatconveyßavor,thisreviewwillonlyfocusonßavor compoundsthatprimarilyimpartsmell,althoughseveralof themmightalsointeractwithtastereceptors.Odorantsare volatilechemicalcompoundsthatarecarriedbyinhaledair totheolfactoryepitheliumlocatedinthenasalcavitiesofthe humannose(BuckandAxel,1991).Theodorantmustpos- sesscertainmolecularpropertiesinordertoproducea sensoryimpression.Itmusthaveacertaindegreeoflipo- philicityandsufÞcientlyhighvaporpressuresoitcanbe transportedtotheolfactorysystem,somewatersolubilityto permeatethethinlayerofmucus,andmustoccurata sufÞcientlyhighconcentrationtobeabletointeractwithone ormoreoftheolfactoryreceptors. Theknowledgeanduseofplantsasßavoringandseason- ingtoenhancethequalityoffoods,beveragesanddrugsisas oldasthehistoryofmankind.Plantsusedasspicesand condimentsareusuallyaromaticandpungentowingtothe presenceofvaryingtypesofessentialoils.Inaddition,people havealsousedperfumeoilsandunguentsmadefromplants ontheirbodiesforthousandsofyearsinlesserorgreater amountsdependentonfashionwhims.TheÞrstperfumes wereallnatural.Inthe19thcentury,thecommercializationof ßavorsandfragrancesonanindustrialscalestartedwiththe isolationofsinglechemicalsresponsibleforthecharacteris- ticaromaofnaturalproducts(e.g.cinnamaldehydeisolated fromcinnamonoilandbenzaldehydefrombitteralmondoil) 712 ª 2008TheAuthors Journalcompilation ª 2008BlackwellPublishingLtd ThePlantJournal (2008) 54 ,712Ð732doi:10.1111/j.1365-313X.2008.03446.x sumerpreference.Thefunctionoffruitvolatilesasasignalofripenessandasanattractantforseed-dispersingorgan-ismsissupportedbythefactthatsomesubstancesarespeciÞcallyformedbyripefruitsbutareabsentinvegetativetissuesandnon-ripefruit.Unlikeripefruitsandßowers,vegetativetissuesoftenproduceandreleasemanyofthevolatilessensedasßavorsonlyaftertheircellsaredisrupted.Thesevolatileßavorcompoundsmayexhibitanti-microbialactivityandhaveanti-canceractivitiesbutcanbetoxicathighdoses(GoffandKlee,2006).Fromthechemicalperspective,ßavormoleculesconstituteaheterogeneousgroupofcompounds,withstraight-chain,branched-chain,aromaticandheteroaromat-icbackbonesbearingdiversechemicalgroupssuchashydroxyl,carbonyl,carboxyl,ester,lactone,amine,andthiolfunctions.Morethan700ßavorchemicalshavebeenidentiÞedandcatalogued(SurburgandPanten,2005;http://www.ßavornet.org/index.html).Mostarefromvariousplantsourcesofdiverseplantfamiliesandaremajorconstituentsofessentialoils.Thebiosyntheticpathwaysofimportantplantvolatileshavebeentracedbackuptointermediatesofprimarymetabolism(CroteauandKarp,1991).Ithasbeenshownthatcarbohydrates,fattyacidsandaminoacidsrepresentthenaturalcarbonpoolsforßavorcompounds,whichcanalsobeliberatedfromtheirpolymers(Figure2).BiosyntheticpathwaysAsmanyplantßavorcompoundsareaccumulatedandbio-synthesizedinspecializedanatomicalstructures(Figure3)(Bagchi,2000;Croteauetal.,2000),thedevelopmentoftechniquesforisolationofthesecretorycellsinsuchstruc-tures(Gershenzonetal.,1992)hasproventobeofcrucialimportanceinourunderstandingofthekeybiosyntheticpathwaysandtheirregulation.Moreover,asthesetissuescontainmanyoftheenzymesandsigniÞcantlyexpressmanyofthegenesinvolvedintheproductionofsuchmetabolites,theisolationofsecretorycellshasgreatlycontributedtocharacterizationofmanyoftheenzymesandgenesinvolvedintheformationofmanyplantnaturalproductsinpepper-mint,sweetandlemonbasil,aswellastomatoandothercrops(Fridmanetal.,2005;Gangetal.,2002;Iijimaetal.2004a,b;McConkeyetal.,2000).Thefollowingsectionsde-scribethebiosynthesisofplant-derivedßavormoleculesgroupedbytheirbiogeneticorigin.Carbohydrate-derivedavorcompoundsFuranonesandpyrones.Furanonesandpyrones(Figure4)areimportantfruitconstituentsorhavebeenisolatedfromthebarkandleavesofseveraltreespecies(SchwabandRoscher,1997).AlthoughhexosesandpentosesaretheprimaryphotosyntheticproductsandserveasexcellentßavorprecursorsintheMaillardreaction,onlyalimitednumberofnaturalvolatilesoriginatedirectlyfromcarbohydrateswithoutpriordegradationofthecarbonskeleton.Suchcompoundsincludethefuranonesandpyrones(BoodandZabetakis,2002).Substituted4-hydroxy-3(2)-furanonesandthepyronemaltolconstituteanuncommongroupofßavormoleculeswithexceptionallowodorthresholds.Furanoneshavebeendetectedinafewplantspeciesinwhichtheyareemittedonlybythefruits.MaltolhasbeenisolatedfromthebarkandleavesofLarixdeciduasEvodiopanaxinnovansphyllumjaponicumandfourkindsofPinaceaeplants(TiefelandBerger,1993).Incorporationexperimentsusinglabeledprecursorsrevealedthatfructose-1,6-diphosphateisanefÞcientbiogeneticprecursoroffuraneol.Instrawberry)andtomato(Solanumlycopersicumthehexosediphosphateisconvertedbyanasyetunknownenzymeto4-hydroxy-5-methyl-2-methylene-3(2none,whichservesasthesubstrateforanenoneoxidore-ductaserecentlyisolatedfromripefruit(Kleinetal.,2007;etal.,2006).AhighlysimilarsequencewasidentiÞedinanESTcollectionforpineapple(Ananascomosusanotherspecieswhichproducesfuraneolinitsfruits.In Figure2.Naturalcarbonpoolsfortheproduc-tionofßavorcompounds,andthepathways.Biosynthesisofplant-derivedavorcompounds2008TheAuthorsJournalcompilation2008BlackwellPublishingLtd,ThePlantJournal,(2008),,712Ð732 threeprenyltransferases.Terpenesynthasesarethepri-maryenzymesresponsibleforcatalyzingtheformationofhemiterpenes(C),monoterpenes(C),sesquiterpenes)orditerpenes(C)fromthesubstratesDMADP,GDP,FDPorGGDP,respectively.PrenyltransferasescatalyzetheadditionofIDPunitstoprenyldiphosphateswithallylicdoublebondstothediphos-phatemoiety.MostoftheprenyltransferasesacceptDMADPastheinitialsubstrate,buttheyalsobindGDPorFDPdependingontheparticularprenyltransferase(GreenhagenandChappell,2001;Tarshisetal.,1994,1996;WithersandKeasling,2007).TheavailabilityofGDPandFDPareoftenthekeyfactorintheproductionofmonoterpenesandsesquiterp-enesinplants.Thisproblemwaselegantlyovercomeinmetabolicengineeringexperimentsbytheco-expressionofGDPandFDPsynthaseswithappropriatemonoterpeneandsesquiterpenesynthasesover-expressedintobacco(Wuetal.2006).Thisstrategy,togetherwithtargetingoftheover-expressiontotheplastidcompartment,resultedinincreasedsynthesisofthesesquiterpenesamorpha-4,11-dieneandpatchoulolandthemonoterpenelimonene(Wuetal.,2006).ThethirdphaseofterpenevolatilebiosynthesisinvolvesconversionofthevariousprenyldiphosphatesDMADP,GDP,FDPandGGDPtohemiterpenes,monoterpenes,sesquiterpenesandditerpenes,respectively,bythelargefamilyoftheterpenesynthases.Triterpenes(andsterols)andtetraterpenes(suchascarotenoids)arederivedfromthecondensationoftwomoleculesofFDPorGGDP,respectively.Planthemiterpene,monoterpene,sesquiter-peneandditerpenesynthasesareevolutionarilyrelatedtoeachotherandarestructurallydistinctfromtriterpeneortetraterpenesynthases.Muchoftheprogressachievedinrecentyearsinterpenoidmetabolismisdescribedelsewhereinthisissue(BohlmannandKeeling,2008).Manyterpenesynthaseshavebeenisolatedandcharacterizedfromvariousplantspecies(Bohlmannetal.,1998;Tholl,2006).Whilemanyterpenevolatilesaredirectproductsofterpenesynthases,manyothersareformedthroughtrans-formationoftheinitialproductsbyoxidation,dehydro-genation,acylationandotherreactions(CroteauandKarp,1991;Croteauetal.,2000;Dudarevaetal.,2004;Picherskyetal.,2006).Forexample,()-menthol,theprin-cipalmonoterpeneofcommercialpeppermintessentialoilandthecomponentresponsibleforthefamiliarcoolingsensationofpeppermintanditsproducts,isformedbyeightenzymaticstepsinvolvingmonoterpenesynthases,isome-rasesandreductases(Ringeretal.,2005;TurnerandCroteau,2004).Thebiosynthesisstartswiththeformationof4limonenefromGPPandendswiththereductionof(menthoneto(Menthaarvensisistheprimaryspeciesofmintusedtomakenaturalmentholcrystalsandnaturalmentholßakes.Aswithmanywidelyusedaromachemicals,theannualdemandformentholof6300tonnesgreatlyexceedsthesupplyfromnaturalsources.Metabolicengineeringoftheterpenoidpathwayisaconstantlyimprovingtool,usedforthefundamentalstudyofterpenoidbiosynthesis(Luetal.,2001,2004;Oharaetal.,2003).Inaddition,thistoolisbeingusedmoreandmorefortheunderstandingofchemicaldiversityincropsetal.,2004;Portnoyetal.,2008),aswellasimprovementoftraitsincropssuchasdiseaseandpestresistance(Kappersetal.,2005;Schneeetal.,2006;Wuetal.,2006),enhancedandalteredaromaformation(Lavyetal.,2002;Lewinsohnetal.,2001;MahmoudandCroteau,2001)andproductionofmedicinalcompounds(Wuetal.2006).MostoftherecentprogressinthisÞeldhasbeensummarizedbyLuetal.(2007).Arecentexampleinwhichßavorengineeringwasdetectedbynon-trainedtestpanelistsinvolvedectopicexpressionofthelemonbasilgeraniolsynthasegeneunderthecontrolofthefruitripening-speciÞctomatopolygalacturonasepromoteretal.,2007).Thiscauseddiversionoftheplastidialterpenoidpathwayforproductionoflycopenetotheaccumulationofhighlevelsofgeraniolandabouttennovelgeraniolderivatives,andhadaprofoundimpactontomatoßavorandaroma,asevaluatedorganoleptically.Apocarotenoidformation.Carotenoidsaretetraterpenoidpigmentsthataccumulateintheplastidsofleaves,ßowersandfruits,wheretheycontributetothered,orangeandyellowcoloration.Inadditiontotheirrolesinplantsasphotosyntheticaccessorypigmentsandcolorants,carote-noidsarealsoprecursorsofapocarotenoids(alsocallednorisoprenes)suchasthephytohormoneabscisicacid,thevisualandsignalingmoleculesretinalandretinoicacid,andaromaticvolatilessuchas-ionone.Evidence,basedoncomparativegenetics,hasindicatedthatcarotenoidpig-mentationpatternshaveprofoundeffectsontheapocarot-enoidandmonoterpenearomavolatilecompositionsoftomatoandwatermelonfruits(Lewinsohnetal.,2005a,b).Thisworkindicatedthatthevariousßavorsandaromasofotherwisesimilarfruitofdifferentcolorshavearealchem-icalbasisandarenotsolelyduetopsychologicalprecon-ception.Indeed,enzymescapableofcleavingcarotenoidsatspeciÞcsiteswerefoundtobeinvolvedinthesynthesisofanumberofapocarotenoids.Carotenoidcleavagedioxygen-ases(CCDs)catalyzetheoxidativecleavageofcarotenoids,resultinginproductionofapocarotenoids(Schmidtetal.2006).CCDsoftenexhibitsubstratepromiscuity,whichprobablycontributestothediversityofapocarotenoidsfoundinnature.Apocarotenoidsarecommonlyfoundintheßowers,fruits,andleavesofmanyplants(WinterhalterandRouseff,2002),andpossessßavoraromapropertiesto-getherwithlowaromathresholds.Theyarefoundamongthepotentßavorcompoundsinwinesandcontributetoßoralandfruityattributes(WinterhalterandSchreier,1994).Therefore,theyhavebeensubjecttoextensiveresearchinrecentyearswithregardtotheirstructureandßavorWilfriedSchwabetal.2008TheAuthorsJournalcompilation2008BlackwellPublishingLtd,ThePlantJournal,(2008),,712Ð732 Selectedvolatilesubstancesevenreachannualconsump-tionratesofmorethan5000tonnes(Table2).About40%ofthefragrancechemicalsarealsousedinmakingßavors,but80%oftheglobalconsumptionofvanillin,menthol,euge-nol,limonene,andestersofloweralcoholsandlowerfattyacidsisusedinmakingßavors.Today,duetothehighcostorlackofavailabilityofnaturalßavorextracts,mostcommercialßavorantsareÔnature-identicalÕ,whichmeansthattheyarethechemicalequiva-lentofnaturalßavorsbutarechemicallysynthesized,mostlyfrompetroleum-derivedprecursors,ratherthanbeingextractedfromthesourcematerials.Becausechemicalsynthesisoftenusesenvironmentallyunfriendlyproductionprocessessuchasheavymetalcatalysts,andcrudeoilrepresentsalimitedsource,itisdesirabletoswitchtobioproduction,includingtheextractionfromnaturaldenovomicrobialprocesses(fermentation),andbioconversionofnaturalprecursorsusingmicro-organismsorisolatedenzymes(Guentert,2007).BiologicalfunctionsofplantvolatilesEssentiallyallplantpartssuchasleaves,ßowers,fruitsandrootsemitvolatiles,whichhavemultiplefunctionsthatarenotalwayssolelyrelatedtotheirvolatility(PicherskyandGershenzon,2002).Becauseplantvolatilesareinvolvedinspecies-speciÞcecologicalinteractionsandareoftenrestrictedtospeciÞclineages,theyhavebeenconsideredtobeassociatedwithdefensiveandattractiveroles(Picherskyetal.,2006).Itisbelievedthattheyarenotessentialforplantsurvivalbutprovideadaptivecharacteristicsunderstrongenvironmentalselection.Compoundsemittedbyßowersmostprobablyservetoattractandguidepollinators,butonlyafewstudieshavedemonstratedtheabilityofindividualsubstancestoattractspeciÞcpollinators(Dudarevaetal.,2004).However,vola-tilesmightalsoprotectthecarbohydrate-richnectarbyinhibitingmicrobialgrowth.Similartohumans,itisprob-ablythequalitativeandquantitativecompositionoftheßavormoleculesthatimpartsthespeciÞcsensoryimpres-sionforthepollinatorsratherthanthepresenceofacertainindividualcompound.Volatilesmaybeabettersignalatnightthanßoralcolororshapetodrawinsectpollinators.Becausevolatilesshowanti-microbialandanti-herbivoreactivity,itisbelievedthattheyservetoprotectvaluablereproductivepartsofplantsfromenemies.Forexample,onemonoterpenol(linalool)anditsderivativessigniÞcantlyrepelledanagriculturalpestÐtheaphidMyzuspersicaeÐindual-choiceassays(Aharonietal.,2003).Isoprene,aubi-quitousvolatilehydrocarbon,actstoincreasethetoleranceofphotosynthesistohightemperaturebystabilizingthethylakoidmembranesorquenchingreactiveoxygenspeciesetal.,2004).Ageneralpropertyofvegetativeplanttissueisthereleaseofvolatilesfollowingherbivoredamage(DeBruxellesandRoberts,2001;PicherskyandGershenzon,2002).Someofthesesubstanceshavebeendemonstratedtoserveasindirectplantdefensesthroughmulti-trophicinteractionsbecausetheyattractarthropodsthatpreyuponorparasitizetheherbivores,thusminimizingfurtherdamagetoplanttissue.However,volatilesalsoactasdirectrepellentsortoxicantsforherbivoresandpathogens,andsomehavethepotentialtoeliminatereactiveoxygenspecies.Thisalsoincludesroot-emittedvolatiles,whichmayfunctionasanti-microbialoranti-herbivoresubstancesorexhibitallelopath-icactivitiesthatincreasetheecologicalcompetitivenessoftheplant(Steeghsetal.,2004).Accordingly,plantvolatilescanminimizethegrowthsuppressionofepiphyticbacteriabythephytopathogenicfungusBotrytiscinereaandthusaffectpopulationdynamicsonleafsurfaces(Abanda-Nkp-etal.,2006a),whilesimplealcoholsemittedbyleavesmayprovideacarbonandenergysourceforepiphyticmethylotrophs(Abanda-Nkpwattetal.,2006b).Volatilesalsoattractfemaleinsectstolayeggsonßowerbudsandberries(Tasinetal.,2007).Infruits,volatileemissionandaccumulationhaveprob-ablyevolvedtofacilitateseeddispersalbyanimalsandinsects.Forhumans,volatilesinfruitshaveaconsiderableeconomicimpact,asparametersoffoodqualityandcon- Table2EstimatedworldconsumptionofselectedaromachemicalsinßavorandfragrancecompositionsVolume(tonnes)2-Phenylethylalcoholandesters7000140001tonnenatural;costofnatural:US$1000perkgLinaloolandesters60008000Estersoflowerfattyacids5800Vanillin5500100001800tonnesnatural;costofnatural:US$1000perkg;costofsynthetic:US$11perkgBenzylacetate400012000Menthol40006300Geraniolandesters350012000Ionones350012000Anethole3200Cinnamaldehyde20003000Aliphaticaldehydes1450Benzylsalicylate9508000Benzylbenzoate7504000Jasminederivatives7008000compoundsandesters40010tonnesnatural;costofnatural:US$3000perkgMaltol300Somogyi(1996);http://salesmanagement-info.blogspot.com/2007/07/top-30-aroma-chemicals-used-in-making.html,exceptSchraderetal.(2004)andhttp://de.wikipedia.org/wiki/Menthol.WilfriedSchwabetal.2008TheAuthorsJournalcompilation2008BlackwellPublishingLtd,ThePlantJournal,(2008),,712Ð732 Thereactionsequenceleadingfrom-linolenicacidtothesignalingmoleculejasmonicacidinvolvestheenzymes13-LOX,alleneoxidesynthase,alleneoxidecyclaseand12-oxo-phytodienoicacidreductase,followedbythreesuc--oxidationsteps(Figure7)(HoweandSchilmiller,2002;Lietal.,2005).Jasmonicacidanditsvolatilemethylesteractasphytohormonesandareinvolvedinplantresponsestostressanddevelopmentalprocesses.Inaddi-tion,methyljasmonateisthemaincomponentofthescentofjasmineßowersandcontributestothepreciousßavorsofandlemonoil.mone,whichactsaseitheranattractantorarepellentforvariousinsects,isadecarboxylatedderivativeofjasmonicacidgeneratedbyoxidativedegradationofjasmonateetal.,2005).-andAlthoughthedegradationofstraight-chainfattyacidsby-and-oxidationisamajorprocessfortheformationofßavormoleculesinallorganisms,thespeciÞcpathwaysinplantsarenotwellunderstood.Thefattyacid-oxidationmechanisminplantsinvolvesfreefattyacids(C)thatareenzymaticallydegradedviaoneortwointermediatestoC()long-chainfattyaldehydesand.Adual-function-dioxygenase/peroxidaseandNADoxidoreductasecatalyzethe-oxidationoffattyacidsinplants(Saffertetal.,2000).-OxidationresultsinsuccessiveremovalofCunits(acetylCoA)fromtheparentfattyacid.Thedetailedmechanismsofconventional-oxidationarewellestablished(GoepfertandPoirier,2007).Forwardandreversegeneticscreenshaverevealedtheimportanceof-oxidationduringplantdevelopmentandinresponsetostress(Bakeretal.,2006).Combinationsofmutationsshowmuchstrongerphenotypes,butitisunclearwhetherthenecessityfor-oxidationistoprovideanenergysourceoralipid-derivedsignalmolecule.Short-andmedium-chainlinearcarboxylicacidsthatareformedbyrepeated-oxidativecyclesfollowedbytheactionofanacylCoAhydrolasehavebeenfoundinmanyessentialoilsisolatedfromdifferentplantsources(Figure8a).Asasecondpathway,denovosynthesisandhydrolysisofacylacylcarrierprotein(acylACP)canalsoprovidevolatileacids.AliphaticacidsuptoCplayasigniÞcantroleinßavorsduetotheirsharp,butteryandcheese-likeodors,notonlyontheirown,butparticularlyassubstratesintheformoftheiracylCoAsforbiosynthesisofotherßavors.Aliphaticshort-andmedium-chainaldehydesandalcoholsareemittedbyvariousplantpartsandareprobablyformedbyenzymaticreductionoftheparentacylCoAs(Flaminietal.,2007).Alternatively,alcoholscanalsobeformedbyADH-mediatedhydrogenationofaldehydes,andmedium-chainaldehydesareintermediatesofthe-oxidationcyclestartingwithcommonfattyacids(Hambergetal.,1999).However,alco-holsarelessimportantasßavormoleculesduetotheirhighodorthresholdsincomparisonwiththeiraldehydehomo-logues.MostplantADHsareZn-dependentmedium-chaindehydrogenasesthatarethoughttobeinvolvedintheresponsetoawiderangeofstresses,includinganaerobiosisandelicitors(Chase,2000).AnADHwithspeciÞcsubstratepreferencehasbeenisolatedfrommelons(Manriquezetal.2006).SpeciÞcally,ßavoresterproductionreliesuponthesupplyofacylCoAsformedduring-oxidationandalcohols.Alcoholacyltransferases(AAT)arecapableofcombiningvariousalcoholsandacylCoAs,resultinginthesynthesisofawiderangeofesters,thusaccountingforthediversityofesters.Numerousgeneshavebeenisolatedandcharacterizedinfruitandvegetables(Aharonietal.,2000;etal.,2004;El-Sharkawyetal.,2005).Aliphaticesterscontributetothearomaofnearlyallfruitsandareemittedbyvegetativetissues.Someareresponsibleforaparticularfruitaromaorforthesmellofaßower.However,manyoftheseesterspossessanon-speciÞcfruityodor.Anothermajorgroupoffattyacid-derivedßavormoleculesarealkanolides,whichhave-(4-)or-(5-)-lactonestructures(Figure8a).Sensoryimportantlactonesusuallypossess8Ð12carbonatomsandsomeareverypotentßavorcompo-nentsforavarietyoffruits(BasearandDemirci,2007).ThefactthatboththeopticalpurityandtheabsoluteconÞgura-tionvaryforidenticallactonesisolatedfromdifferentsourcessupportstheideaofdifferentbiosyntheticpathways.How-ever,alllactonesoriginatefromtheircorresponding4-or5-hydroxycarboxylicacids,whichareformedbyeither(i)reductionofoxoacidsbyNAD-linkedreductase,(ii)hydrationofunsaturatedfattyacids,(iii)epoxidationandhydrolysisofunsaturatedfattyacids,or(iv)reductionofhydroperoxidesttlerandBoland,1996).EnzymesandgenesspeciÞcallyinvolvedintheformationhavenotyetbeenreported.Incontrastto4-and5-hydroxyfattyacids,3-hydroxyacids,the Figure8.Biosynthesisof(a)short-chainacids,aldehydes,alcohols,estersandlactones,and(b)methylketones.AAT,alcoholacylCoAtransferase;MKS,methylketonesynthase;ACP,acylcarrierprotein.Biosynthesisofplant-derivedavorcompounds2008TheAuthorsJournalcompilation2008BlackwellPublishingLtd,ThePlantJournal,(2008),,712Ð732 aciddecarboxylaseintomatoresultedinfruitswithupto10-foldincreasedlevelsof2-phenylacetaldehyde,2-pheny-lethanoland1-nitro-2-phenylethane(Tiemanetal.,2006).Themodulationoftheemissionof2-phenylethanoland2-phenylacetaldehydeisimportantbecausethesesubstancesexertadualeffect.Atlowconcentrations,bothcompoundsareassociatedwithpleasantsweetßowerynotes,whileathighlevels,thepungentaromaof2-phenylacetaldehydeisnauseatingandunpleasant(Tadmoretal.,2002).4,5-Dimethyl-3-hydroxy-2(5)-furanone(sotolon)isthemajorßavor-impactcompoundofdriedfenugreekseedsTrigonellafoenum-graecumL.,andisprobablyformedfrom-isoleucine(Slaughter,1999).Thesupposedbiogeneticoriginissupportedbystereochemicalconsider-ations(Figure9b).Sulfur-containingßavorcompoundsoriginatingfrommethionineandcysteineareresponsiblefortheodorofgarlic(methanethiol,dimethyldisulÞde,-methylthioace-tate),onions(propanthial-oxide),boiledpotato(methion-al)andcookedcabbage(methanethiol)(Jonesetal.,2004).InArabidopsis,thecleavageofmethionineiscatalyzedby-lyase,resultingintheproductionofmetha-nethiol,2-ketobutyrateandammonia(Reetal.,2006).Inonion(Alliumcepa)andgarlic(A.sativum),aseriesofvolatilesulfurcompoundsisgeneratedbycleavageof-alk(en)ylcysteinesulfoxideßavorprecursorscatalyzedbytheenzymesallinaseandlachrymatory-factorsynthase(Jonesetal.,2004;Lanzotti,2006).Thebiosynthet-icpathwayinvolvesalk(en)ylationofthecysteineingluta-thione,followedbycleavageandoxidationtoformthesulfoxidesor(thio)alk(en)ylationofcysteineorserine.Oncetheplanttissueisdamaged,theßavorprecur-sorsareenzymaticallycleavedbyallinasetogiveaseriesofvolatilesulfurcompoundsthatundergofurthervapor-phasechemicaltransformations(Figure10a).Becausethelevelsoftheßavorprecursorsamountto1Ð5%dryweightincertainspecies,itissupposedthattheyplayamajorrolefortheplant.Tworolesthathavebeenascribedaredefenseagainstpestsandpredation,particularlyintheover-winter-ingbulb,andcarbon,nitrogenandsulfurstorageandtransport(Jonesetal.,2004).Volatilebiogenticaminesareanothergroupofßavormoleculesthataresynthesizedfromaminoacidsortheirprecursors.Thevolatileindolisformedinmaizebythecleavageofindole-3-glycerolphosphate,anintermediateintryptophanbiosynthesis(Figure10b)(Freyetal.,2000).Cruciferousvegetablessuchasmustard,broccoli,caulißower,kale,turnips,collards,Brusselssprouts,cab-bage,radishandwatercresscontainglucosinolates,whicharenaturalprecursorsofßavormolecules.Glucosinolates, Figure10.Biosyntheticpathwaysfor(a)thiosulf-inatesandtheirdegradationproducts,(b)for-mationofindoleinmaize,(c)volatilesproducedfromglucosinolates,and(d)cyanogenicgluco-Biosynthesisofplant-derivedavorcompounds2008TheAuthorsJournalcompilation2008BlackwellPublishingLtd,ThePlantJournal,(2008),,712Ð732 terpenoidsÐhemiterpenoids(C),monoterpenoids(Csesquiterpenoids(C)andsomediterpenoids(C)Ðareinvolvedininteractionsbetweenplantsandinsectherbi-voresorpollinatorsandarealsoimplicatedingeneraldefenseorstressresponses(Dudarevaetal.,2004;Picher-skyandGershenzon,2002;Picherskyetal.,2006).Terpe-noids,mainlytheCandCmembersofthisfamily,werefoundtoaffecttheßavorproÞlesofmostfruitsandthescentofßowersatvaryinglevels(Figure5).Citrusfruitaromaconsistsmostlyofmono-andsesquiterpenes,whichaccu-mulateinspecializedoilglandsintheßavedo(externalpartofthepeel)andoilbodiesinthejuicesacs.Themonoter-limonenenormallyaccountsforover90%oftheessentialoilsofthecitrusfruit(Weiss,1997).Thesesquit-erpenesvalenceneand-and-sinensal,althoughpresentinminorquantitiesinoranges,playanimportantroleintheoverallßavorandaromaoforangefruit(Maccaroneetal.1998;Voraetal.,1983;Weiss,1997).Nootkatone,aputativederivativeofvalencene,isasmallfractionoftheessentialoils,buthasadominantroleintheßavorandaromaofgrapefruit(MacLeodandBuigues,1964;ShawandWilson,1981),whilethemonoterpenelinaloolwasfoundtobeanimportantgeneralstrawberryaromacompound(Aharonietal.,2004;LarsenandPoll,1992)andisfoundinmanyotherfruitsincludingpeaches,guavas,nectarines,papayas,mangoes,passionfruits,tomatoes,litchi,oranges,pricklypearsandkoubos(Baldwinetal.,2000;BernreutherandSchreier,1991;FlathandTakahashi,1978;Idsteinetal.1985;Ninioetal.,2003;OngandAcree,1998;VisaiandVanoli,1997).Thecombinationofthemonoterpenesgera-niol,citronellolandroseoxideisakeycomponentofthecharacteristicaromaofaromaticmuscatgrapesaswellasthespecialscentofroses(Bayrak,1994;DunphyandAllcock,1972;Luanetal.,2005).Terpenoidsarealsotheprimaryconstituentsoftheessentialoilsofmanytypesofherbs.Thepeltateglandulartrichomesofpeppermintproducecopiousamountsofacommerciallyvaluable,menthol-richessentialoil,composedprimarilyofmenthanemonoterpenes(TurnerandCroteau,2004).Theglandulartrichomesofsweetbasil()arerichinphenylpropenesaswellasmonoterp-enesandsesquiterpenes(Iijimaetal.,2004a).Lemon-scentedherbsofvariousplantfamilies,suchaslemonbasil,Lamiaceae),lemongrass(,Poaceae)andlemonverbena(AloysiacitriodoraVerbenaceae),accumulatecitral,amixtureoftheisomericmonoterpenealdehydesneralandgeranialetal.,1998;Iijimaetal.,2004a,b;Giletal.2007).Therefore,manyterpenoidsarecommerciallyimpor-tantandarewidelyusedasßavoringagents,perfumes,insecticides,anti-microbialagentsandimportantrawmate-rialforthemanufactureofvitaminsandotherkeychemicals.Manyterpenoidshavemedicinalproperties;consequentlytheyareofinteresttothepharmaceuticalindustryasanti-retroviralagentsoranti-malarialcompounds(Modzelewskaetal.,2005).Asaresult,modulationofterpenoidbiosyn-thesisinmedicinalandaromaticplantshasreceivedmuchinterest(Goetal.,2007;MahmoudandCroteau,2001,2002;Mahmoudetal.,2004;Munetal.2006,2007;TadmorandLewinsohn,2007).Syntheticvaria-tionsandderivativesofnaturalterpenesandterpenoidsalsogreatlyexpandthevarietyofaromasusedinperfumeryandßavorsusedinfoodadditives.Despitetheirdiversity,allterpenoidsderivefromthecommonbuildingunitsisopentenyldiphosphate(IDP)anditsisomerdimethylallyldiphosphate(DMADP)(CroteauandKarp,1991;Croteauetal.,2000;McGarveyandCroteau,1995).Inplants,bothIDPandDMADParesynthesizedviatwoparallelpathways,themevalonate(MVA)pathway,whichisactiveinthecytosol,andthemethylerythritol4-phosphate(MEP)pathway,whichisactiveintheplastids(Lichtenthaler,1999;Rodriguez-ConcepcionandBoronat,2002;Rohdichetal.,2002;Rohmer,2003).Itisgenerallyrecognizedthatthecytosolicpathwayisresponsibleforthesynthesisofsesquiterpenes,phytosterolsandubiquinone,whereasmonoterpenes,gibberellins,abscisicacid,carote-noidsandtheprenylmoietyofchlorophylls,plastoquinoneandtocopherolareproducedinplastids(Lichtenthaler,1999;nandBoronat,2002;Rohdichetal.2002;Rohmer,2003),butindicationsofcross-talkbetweentheplastidicandcytosolicpathwayshavebeenfoundintobacco,Arabidopsisandsnapdragonpetals(Aharonietal.2004;Dudarevaetal.,2005;Oharaetal.,2003).Thedirectprecursorsofterpenoids,lineargeranyldiphosphate(GDP,),farnesyldiphosphate(FDP,C)andgeranylgeranyldiphosphate(GGDP,C),areproducedbytheactivitiesof Figure5.Importantplant-derivedvolatileterpenoids.Biosynthesisofplant-derivedavorcompounds2008TheAuthorsJournalcompilation2008BlackwellPublishingLtd,ThePlantJournal,(2008),,712Ð732 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atatimethatischaracterizedbysigniÞcanttechnologicalbreakthroughs,largelyinchemistry.TheÞrstßavorandfragrancecompanieswerefoundedbyentrepreneurialscientistsorbusinesspeople,andmanystillexist.Thesynthesisofaromachemicalsthatimpartthecharacteristicodorandtasteofnaturalproductsbecamecommonplaceandgaverisetoanewbranchofchemicalspecialities.Today,thetotalmarketforßavorsandfra-grancesisestimatedatUS$18billion,withmarketsharesbetweentheßavorandfragrancebusinessesbeingalmostequal(Guentert,2007).ThelargestmarketsareinEurope(36%)andNorthAmerica(32%),followedbytheAsianPaciÞcregion(26%).Eightmajorglobalcompaniesshare60%oftheworldmarket.Theßavorandfragranceindustryisacompositeoffourcloselyinter-relatedandoverlappingbusinesssectors(Figure1).EssentialoilsandothernaturalextractsareusuallydeÞnedasaromaticmaterialsobtainedfrombotan-icaloranimalsourcesbydistillation,coldpressing,solventextractionormaceration.Essentialoilsrepresentcomplexaromamixturesofpotentiallyhundredsofchemicalcon-stituents.AromachemicalsareorganiccompoundswithadeÞnedchemicalstructure.Theyareproducedbyorganicorbiocatalyticsynthesisorisolatedfrommicrobialfermenta-tions,plantsoranimalsources,andareusedtocompoundßavorsandfragrances.Flavorblendsandfragranceblendsarecomplexformulationsofaromaticmaterialssuchasessentialoilsandtheirnaturalderivativesaswellasaromachemicalscontainingupto100constituents.Formulatedßavorsareusedbythefoodandbeverage,tobaccoandpharmaceuticalindustries,whileformulatedfragrancesareusedtogivepleasantscentstoÞnefragrances,personalcareandhouseholdproducts.Limitedinformationisavailablepubliclyonthesizeofthemarketforessentialoilsandplantextracts.Becauseßavorandfragrancetradingisamulti-billiondollarbusiness,thecompanieskeepthecompositionsoftheirproductsconÞ-dential,thustheconsumptionvaluesforaromachemicalsareonlyroughestimatesandvaryconsiderablyfromsourcetosource.InformationonthelevelsoftheworldÕstop20essentialoilsinvolumetermswascompiledsomeyearsago(Table1)(Lawrence,1993).Orange(Citrussinensis)oilproductionwas26000tonnes,followedbyoilsfromcorn-mint(Menthaarvensis)andeucalyptus(Eucalyptusglobu-)(cineole-type).Recentvaluesforcornmintoilshowthatthemarkethasgrownsubstantiallysincethen(Table1).Expertsforecastgrowthinglobaldemandforßavorandfragrancesof5.4%peryear,andpredictthatgrowthindemandforessentialoilsandnaturalextractswilloutpacethatforsyntheticaromachemicalsoverthenextseveralyears(Short,2002).Worldwidetransactionsinthemarketsforessentialoilsshowanannualaverageincreaseof10%forexports(Figure1).Flavorandfragranceblendsthatarecomposedofessentialoilsandsyntheticaromachemicalsarestillthelargestproductsegments(intermsofsales)fortheßavorhouses(Figure1).Although2800individualaromachemicalsareusedinßavorandfragrancecompo-sitions,onlyafewhundredareofferedonthemerchantmarketandusedinquantitieslargerthan50tonnesperyear. Figure1.Overviewoftheßavorandfragranceindustry. Table1WorldÕstopessentialoilsinvolumetermsEssentialoilSpeciesVolumeCitrussinensis(L.)Osbeck2600058.5MenthaarvensisL.f.Malinv.exHolmes4300(20000)34.4Eucalyptuscineole-typeEucalyptusglobulusLabill.,E.polybracteaR.T.BakerandotherEucalyptusspecies372829.8CymbopogonwinterianusJowittandC.nardue(L.)Rendle283010.8L.236728.4Lawrence(1993),exceptSrivastavaetal.Biosynthesisofplant-derivedavorcompounds2008TheAuthorsJournalcompilation2008BlackwellPublishingLtd,ThePlantJournal,(2008),,712Ð732 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