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ModernPlantMetabolomics:AdvancedNaturalProductGeneDiscoveries,ImprovedTechnologies,andFutureProspectsJournal:NaturalProductReportsManuscriptID:NP-REV-05-2014-000072.
R1ArticleType:ReviewArticleDateSubmittedbytheAuthor:24-Sep-2014CompleteListofAuthors:Sumner,LloydW.
;TheSamuelRobertsnoblefoundation,PlantBiologyDivisionLei,Zhentian;TheSamuelRobertsNobleFoundation,PlantBiologyNikolau,Basil;IowaStateUniversity,Biochemistry,BiophysicsandMolecularBiology;U.
S.
DepartmentofEnergy,AmesLaboratorySaito,Kazuki;RIKENCenterforSustainableResourceScience,;ChibaUniversity,GraduateSchoolofPharmaceuticalSciencesNaturalProductReportsJournalNameRSCPublishingARTICLEThisjournalisTheRoyalSocietyofChemistry2013J.
Name.
,2013,00,1-3|1Citethis:DOI:10.
1039/x0xx00000xReceived00thJanuary2012,Accepted00thJanuary2012DOI:10.
1039/x0xx00000xwww.
rsc.
org/ModernPlantMetabolomics:AdvancedNaturalProductGeneDiscoveries,ImprovedTechnologies,andFutureProspectsLloydW.
Sumnera,ZhentianLeia,BasilJ.
Nikolaub,candKazukiSaitod,ePlantmetabolomicshasmaturedandmodernplantmetabolomicshasacceleratedgenediscoveriesandtheelucidationofavarietyofplantnaturalproductbiosyntheticpathways.
Thisreviewhighlightsspecificexamplesofthediscoveryandcharacterizationofnovelgenesandenzymesassociatedwiththebiosynthesisofnaturalproductssuchasflavonoids,glucosinolates,terpenoids,andalkaloids.
Additionalexamplesoftheintegrationofmetabolomicswithgenome-basedfunctionalcharacterizationsofplantnaturalproductsthatareimportanttomodernpharmaceuticaltechnologyarealsoreviewed.
Thisarticlealsoprovidesasubstantialreviewofrecenttechnicaladvancesinmassspectrometryimaging,nuclearmagneticresonanceimaging,integratedLC-MS-SPE-NMRformetaboliteidentifications,andx-raycrystallographyofmicrogramquantitiesforstructuraldeterminations.
Thereviewcloseswithadiscussiononthefutureprospectsofmetabolomicsrelatedtocropspeciesandherbalmedicine.
1.
IntroductionMetaboliteprofilingofbacterial,mammalian,andplantmetaboliteshasbeenanintegralcomponentofbiologicalstudiessincethe1960s.
Thecontinualrefinement,increasingscopeandlarger-scaleofplantmetaboliteprofilinghaveledtotheevolutionofmodernplantmetabolomics,whichhasmaturedasavaluabletoolforadvancingourunderstandingofplantbiologyandphysiology.
Thisreviewisnotintendedtobeacomprehensivereviewofplantmetabolomics,butinsteadwillfocusonsomeofthemorerecentadvancesthatcontinuetopropeltheutilityofplantmetabolomicsforthediscoveryandunderstandingofplantnaturalproductsorspecializedmetabolites.
Thisincludesexamplesofintegratedmetabolomicsandgenome-basedfunctionalcharacterizationsofplantnaturalproductsthatareimportanttomodernpharmaceuticaltechnology.
Thereviewfurtherhighlightsalargenumberofspecificexamplesillustratingtheutilityofmetabolomicsforthediscoveryandcharacterizationofnovelgenesandenzymesresponsibleforthebiosynthesisofnaturalproductssuchasflavonoids,glucosinolates,terpenoids,andalkaloids.
Thereviewthensummarizesrecenttechnicaladvancesinmassspectrometryimaging,nuclearmagneticresonanceimaging,integratedLC-MS-SPE-NMRformetaboliteidentifications,andx-raycrystallographyofmicrogramquantitiesforstructuraldeterminations.
Thereviewcloseswithasummaryonpotentialopportunitiesformetabolomicsrelatedtocropsandherbalmedicines.
2.
GenomicsandmetabolomicsminingofmedicinalplantsMedicinalplantshavealongassociationwithhumanhistory,beingpartofourfolkloreandthebasisofnaturalmedicinesthathavebeendiscoursedundersuchtopicsasherbalism,phytotherapy,ethnobotanyandethnopharmacy.
Historyisrepletewithexamplesofplantsbeingusedtoaffectthehumancondition;maybeafamousexamplebeingthatofhemlock,thefatalpoisonextractthatthecondemnedSocrateswasforcedtodrink.
1However,archaeologicalstudiesappeartoprovideevidenceofmedicinalplantusesome50,000yearsagointhePaleolithicage,andwrittenevidencecanbetracedtotheSumerians,about5,000yearsago.
2Atthestartofthe19thcentury,evidencebasedmedicalpracticesbegantoreplacesuchtraditionaloralternativemedicines,buttheuseofmedicinalplantsisstillalmostuniversalamongnon-industrializedsocieties,3andplantsstillserveasthesourceofmanynutraceuticalsandpharmaceuticals.
Plant-sourcedbiochemicalsareanimportantpartofourmodernpharmaceuticaltechnology.
Examplesofsuchcurrentmedicineswithalonghistoryareaspirin,theacetyl-esterofsalicylicacidthatwasoriginallyisolatedfromthebarkofthewillowtree;4andtheantimalarialalkaloid,quinineisolatedinthe19thcenturyfromthebarkoftreesofthecinchonagenus.
5Morerecentnaturalproductbasedpharmaceuticalsincludepaclitaxel(morecommonlyknownastaxolTM),isolatedfromthebarkofthePacificyewtree,andusedincancerchemotherapy,6,7andartemisinin,isolatedfromsweetwormwood(Artemisiaannua),andusedasananti-malarialdrug.
8Suchbioactivecompoundshavebeentraditionallyidentifiedandcharacterizedfollowingthefractionationandpurificationofextracts,guidedbybioactivityassays,astrategythathasbeenatthecoreofthefieldofnaturalproductchemistry.
9,10Manyofthesebioactivecompoundsareproductsofsecondaryorspecializedmetabolism,andassuchtheirtaxonomicdistributionisinrelativelynarrowphylogeneticcladeswithinPlantae.
Becauseofthenarrowphylogeneticdistributionofthesephytochemicals,thebiochemicalPage1of15NaturalProductReportsARTICLEJournalName2|J.
Name.
,2012,00,1-3ThisjournalisTheRoyalSocietyofChemistry2012andgeneticcomponentsthatsupporttheirbiosynthesiscannotbeidentifiedandcharacterizedbyhomologytoamodelsystem,buthavetobeinvestigateddirectlybystudyingtheorganismthatproducesthatphytochemical.
Theroleofmetabolomicsinsuchstudiesistwo-fold:1)toidentifythespatialandtemporaldistributionofthetargetphytochemicalasinfluencedbyplantdevelopmentandenvironmentalcues;and2)identifyrelatedcompounds,whichmaybeconsideredaseitherintermediatesofbiosynthesisoralternativeproductsofpromiscuousenzymesthatsupportthebiosynthesisofthetargetphytochemical.
Suchdata,whenintegratedwiththegeneralizableprinciplesoforganicsyntheticchemistry,canprovideavenuestodiscoveringbiosyntheticpathways.
Integratingmetabolomicsthereforewithgenome-basedfunctionalcharacterizationsofgeneproductsisprovidinganacceleratedpathtodiscoveringnovelbiosyntheticpathwaystospecializedmetabolites.
Withtheadventofadvancedgenomicscapabilities,associatedwiththeabilitytocomprehensivelyandquicklydetermineandassemblethegenomesofplants,therehasbeenalogicalneedtounderstandhowthisgeneticinformationisexpressedinacomprehensivemanner.
Thishasledtotheemergenceofsuchfieldsastranscriptomics,proteomicsandmetabolomics.
Althoughtechnicaldifficultieshavebeenassociatedwithimplementingtheseglobalgenomeexpression-profilingtechnologies,advancesinnucleicacidsequencinghasenabledtheacquisitionofnearcomprehensivetranscriptomicdatasets,foreventhemostcomplexgeneticorganisms.
Moreover,asaconsequenceoftheCentralDogma,andthenearlinearrelationshipbetweenthegenome,transcriptomeandproteome,oneisabletopredicatetheprimarystructuresofthesethreelevelsofgeneticexpressionfromanyoneother.
However,itisnotpossibletopredictthechemicalnatureofthemetabolomefromgenetic-baseddatasetsbecausetherelationshipbetweentheproteomeandmetabolomeisnotlinearbutredundant.
Despitethislimitation,thepastdecadehasseenconsiderableeffortsandsuccessesintheintegrationofgenomicsandmetabolomicsdatasetstouncovernewknowledgeconcerningthebiosynthesisofnaturalproductsthathavebioactivity,andthuspotentialapplicationsastemplatesfornewpharmaceuticalproducts.
Theseadvancesspanfromtheselectionandbreedingofelitegermplasmforenhancingtheproductionofthetargetphytochemical,totherecombinantproductionofthetargetphytochemicalinanon-plantbioengineeredhostsystem.
Theseareexemplifiedbythedevelopmentoftheartemisininanti-malarialdrug.
Itsproductivityisbeingenhancedviatraditionalbreedingapproacheswithnewhigh-yieldinghybridstoconvertA.
annuaintoarobustcroppingsystem,11,12andbythereconstitutionoftheartemisininbiosyntheticpathwayinare-engineeredmicrobialhost.
13Theselong-termeffortsthathavebeensupportedbytheBillandMelindaGatesFoundation(http://www.
gatesfoundation.
org)andareprovidingopportunitiesforentrepreneurialdevelopmentofasolutiontoaworld-healthissuebaseduponnaturalproductchemistry.
Theliteratureisabundantwithsuccessesindecipheringnewmetaboliccapabilitieswhenthistypeofintegratedmetabolomics-transcriptomicsstrategyhasbeenusedinthepast.
14-31Forexample,therelativeabundanceoftranscriptsencodingthealkaloidbiosyntheticenzymescorrelateswiththeinductionofbenzylisoquinolineaccumulationinPapaversomniferum.
14,27Comparisonoftranscriptomesandmetabolomes(particularlyfattyacidsandlipids)ofdevelopingseedsthataccumulate"unusual"fattyacidshasledtothediscoveryofaseriesofFAD2-relatedenzymes16-21thatareresponsibleforthegenerationofhydroxyfattyacids,16epoxy-fattyacids,17,18conjugatedfattyacids,19,20andacetylenicfattyacids.
17,21Hence,comparingtranscriptomicsandmetabolomicsdataisenablingtheaccurateannotationofawidevarietyofgenesinspecializedmetabolism.
Duetocontinuedtechnologyadvances,thisstrategyismuchmorereadilyapplicable,eveninthemostrecalcitrantsystems.
Databasestructuresthatenablethequeryingofbothdatasetsandprovideameansofgeneratingarobusttestablehypothesisconcerninggenefunctionsisimportanttotheintegrationofmetabolomicsandtranscriptomicsdatasets.
Atpresent,mostmetabolomicsdatabasescontainmetabolomics–onlydatasetsfromcarefullydefinedsampleswithacommonbiologicaltheme.
These"themed"metabolomicsdatabasescontainintriguingandusefuldata,32-40andtheymakeavailablethegeneralframeworkforsuchdatabases,includingtheimportanceofdataconsistencyanddepositionoffullmetadata.
41However,databasesthatintegratemetabolomicswithotherglobal–omicsdataarerequiredtoprovideaccuratepredictionsofgenefunctionsinmetabolicnetworks.
Aconsortiumofplantbiochemists,supportedbytheNationalInstitutesofHealthhasrecentlycompletedthetranscriptomeandmetabolomeevaluationofnearly20medicinalplantspecies.
Theresultingdatasetsareaccessibleinpublicallyavailabledatabases(http://medicinalplantgenomics.
msu.
edu/andhttp://metnetdb.
org/PMR/),andeachoffersdifferentfunctionalitiesforintegratedqueryingofthedatainordertocreatetestablehypothesesconcerninggene-functions.
42,43ThereisanincreasingliteraturebasethatexemplifiesthesuccessfuluseofthePlant&MicrobialMetabolomicsResource(PMR)structureanddatatoanalyzecombinedtranscriptomics-metabolomicsdatasetstoidentifygeneticelementsthatsupportthebiosynthesisofnovelmetabolitesinmedicinalplants.
Forexample,PMR-enabledanalysesfacilitatedtheidentificationofgenesassociatedwith:a)theCatharanthusroseuscytochromeP450thatcatalyzesareactioninthebiosynthesisofthealkaloid19-O-acetylhorhammericine;44b)threeDigitalispurpureagenescodingenzymesofcardenolidebiosynthesis:C4sterolmethyloxidase,progesterone5b-reductaseandcardenolidesynthase;45andc)identificationofValerianaofficinalisgenesencodingvalerena-1,10-dienesynthase38anditsroleinthesynthesisofnovelsesquiterpenes.
46Additionalanalogousdatafromothermedicinalspecies,suchasEchinaceapurpureaandHypericumperforatumarealsobeingusedtoidentifygenesthatencodefunctionalenzymesinthebiosynthesisofuniquepolyketidesthatoccurinthesespecies(e.
g.
,alkamides,acylphloroglucinolsandhypericin).
47,483.
MetabolomicsbaseddiscoveryandcharacterizationofnovelgenesandenzymesinvolvedinthebiosynthesisofnaturalproductsThroughthecorrelationofgeneexpressionandmetaboliteaccumulation,oneoftenobtainsgoodhintsforspecificgenesthatareinvolvedinthebiosynthesisofrelatedmetabolites.
49Thisisbasedonthepresuppositionofthemodeofco-responseofthebiosyntheticenzymetranscriptsandmetabolitesformedbytheseenzymes.
37,50Infact,thisisapowerfulstrategytoidentifynovelgenescommittedinaspecificbiosyntheticpathway.
Thefunctionsofnumerousgeneshavebeenidentifiedandcharacterizedbymetabolomicsandoftentogetherwithtranscriptomics.
51,523.
a.
FlavonoidsandrelatedcompoundsPage2of15NaturalProductReportsJournalNameARTICLEThisjournalisTheRoyalSocietyofChemistry2012J.
Name.
,2012,00,1-3|3Thegenesinvolvedinthebiosynthesisandaccumulationofflavonoids,someofthebestknownplantnaturalproducts,havebeendiscoveredwiththeaidofmetabolomics.
Naturally,thebiosynthesisofspecializedmetabolitesinArabidopsisthalianahasbeenextensivelyinvestigatedbymeansofintegratedmetabolomicsandtranscriptomics,becauseoftherichfunctionalgenomicsresourcesavailableforthismodelplant.
InA.
thaliana,atleast,54flavonoidmolecules(35flavonols,11anthocyaninsand8proanthocyanidins)havebeenobservedthroughextensiveLC-MSmetabolicprofilingofavarietyoftissues.
53Thegenesresponsiblefortheformationofscaffoldstructures(kaempferol,quercetinandisorhamnetinforflavonols;cyanidinforanthocyanins;andepicatechinforproanthocyanidins)havebeenisolatedmostlythroughtheanalysesofmutantswhichlackpigmentation.
54,55However,thegenesresponsibleforthetailoringreactions,whichmodifythesescaffoldsbyglycosylation,methylationandacylationandthussubsequentlyresponsiblefortheenormouschemicaldiversityofflavonoids,havebeenisolatedbyreversegeneticapproacheswheremetabolomicswasincorporated.
Metabolicprofilingofflavonoidsinaseriesofgene-insertionmutants56,57andtransgenicplantsoverexpressingatranscriptionfactor58havebeenusedtodecipherthefunctionofgenes.
Generally,thepredictedcatalyticactivitieswereconfirmedusingrecombinantproteinsandinvitrobiochemicalassays.
This'reversegenetics'strategyforgeneidentificationhasbeenmostpowerfulwhenbioinformaticpredictionofcandidategenesobtainedthroughaco-expressionnetworksprecededtheexperimentalanalysisofthegeneknock-outmutants.
PublictranscriptomedatasetsforA.
thaliana,e.
g.
AtGenExpress,40areparticularlyusefulforco-expressionanalyses,andusuallyinclude'bait'genesknowntobeinvolvedinaparticularpathwayforthediscoveryofothernovelgenesinthesameco-expressionframework.
Severaltailoringenzymes,glycosyltransferases,59-62acyltransferases,63,64andmethyltransferase57,65havebeencharacterizedbythisorasimilarapproach.
TheAtMetExpressdatabasewasdevelopedbyextendingtheintegratedanalysesofgeneexpressionandmetabolicprofilingdatafromspecificArabidopsistissues.
AtMetExpresscontainsLC-MS-baseduntargetedmetabolomicsdatacollectedfromavarietyofA.
thalianatissues66andecotypes.
67Datawereacquiredfromsamplesforwhichtranscriptomeandsinglenucleotidepolymorphismdataareavailable.
Fromtheintegrateddatasets,testablehypothesescouldbegenerated.
Forexample,thenovelfunctionofadirigentproteinwassuggestedtoberesponsiblefortheformationofaneolignanmetabolite,anddetailedexperimentssubsequentlyconfirmedthisfunctionforthisprotein(Yonekura-Sakakibaraetal.
,inpreparation).
66Anotherdatabase,MetaboliteProfilingDatabaseforKnock-OutMutantsinArabidopsis(MeKO),providesinformationfor50plantgrowthmutants,imagesofmutants,metaboliteaccumulationandinteractiveanalysistools.
68Non-processeddata,includingchromatograms,massspectra,andexperimentalmetadatathatfollowtheguidelinessetbyMetabolomicsStandardsInitiative(MSI)arefreelydownloadablefromhttp://prime.
psc.
riken.
jp/meko/.
Proof-of-conceptanalysessuggestthattheMeKOdatabaseishighlyusefulforgenefunctionhypothesisgenerationandforimprovinggeneannotation.
Rice(Oryzasativa)isamajorcropcriticaltosustainingthehumanpopulation,andasaresult,richexperimentalresourceshavebeengeneratedbypastbreedingprograms.
Metabolomicsanalyseshavealsobeenperformedonaseriesofgeneticallydefinedriceinter-crossedlinestoidentifymetabolicquantitativetraitloci(QTL).
QTLanalysesof87back-crossed,inbredlinesofanindicaandjaponicariceledtotheidentificationandcharacterizationofthelocusonchromosome6,whichisresponsibleforthebiosynthesisoftheflavoneglycoside,apigenin-6,8-di-C-α-L-arabinoside.
69Usingadensergeneticmapincluding210recombinantinbredlinesfromtwoeliteindicavarieties,severalgenecandidatesinvolvedinthebiosynthesisofflavoneO-andC-glycosideswerealsocharacterized.
70Usingthisapproach,candidategenesinthenarrowedQTLregioncouldbefunctionallyverifiedthroughadditionalinvitrobiochemicalassays.
Inparticular,thegenesrelatedtothebiosynthesisofflavonoidC-glycosides,whicharecharacteristicmetabolitesinrice,wereofspecialinterest.
71Proanthocyanidins,alsoknownascondensedtannins,areflavanolpolymersandsynthesizedfromthemonomericbuildingunits,flavan-3-ols(i.
e.
cathechinorepicathechin).
72ThemolecularanalysesandtargetedmetabolicprofilingofArabidopsismutantsexhibitingapaleyellowseedcoatduetothelackofproanthocyanidinsrevealedthemechanismandgenesinvolvedinthisbiosyntheticpathway.
Akeyenzyme,anthocyanidinreductase,catalyzesthereductionofcyanidintoepicatechinintheArabidopsiscytosol.
73Theepicatechinisthenmostlikelyglycosylated74andtransportedintovacuole,75wherepolymerizationbyalaccase-likeprotein(s)takesplacetoforminsolubleproanthocyanidins.
76However,manyquestionsregardingthisprocessstillremaintobesolved.
773.
b.
GlucosinolatesOneofthefirstexampleswheremetabolomicsplayedakeyroleintheidentificationofbiosynthesticgenesrelatedtoplantnaturalproductswasforglucosinolatebiosynthesisinArabidopsis.
IntegrationofA.
thalianatranscriptomeandmetabolomeanalysesforplantssubjectedtosulfate-deficientstressandbioinformaticmultivariatedata-mining78enabledthepin-pointingofgenesencodingasulfotransferase79andaMYBtranscriptionfactor.
80Batch-learning,self-organizingmapping(BL-SOM)analysesofconcatenatedmatricescontainingtranscriptomeandmetabolomedatawasefficientlyusedforselectingcandidategenesinvolvedinthepathway.
Co-expressionnetworkanalyseswithpublicDNA-chipmicroarraydatasetshavealsobeenutilizedasakeytechnologyforselectionofcandidategenes.
81GeneticanalysisofArabidopsiscombinedwithtargetedanalysisofglucosinolatesenabledtheidentificationofgenescommittedintheirbiosynthesis.
82MetabolicQTL83andco-expressionnetworkanalysesledtotheidentificationofanovelflavin-monooxygenaseresponsibleforS-oxygenationinthealiphaticglucosinolatebiosynthesis.
84Metabolicprofilingofglucosinolates,camalexinandtheirglutathione(GSH)-conjugatesintheArabidopsisγ-glutamylpeptidasemutantsresultedintheidentificationoftwocytosolicplantγ-glutamylpeptidasesinvolvedintheprocessingofGSHconjugatesintheglucosinolateandcamalexinpathways.
853.
c.
TerpenoidsTerpenoidsarerecognizedasoneofthemoststructurally-diverseclassesofnaturalproducts,presumablybecauseofthehugevariationinthepolymerizationofisopreneunits,referredas'scaffoldformation',andsubsequentmodificationbyoxygenationandglycosylation,referredtoas'tailoringreactions'.
Herewediscussandprovideexamplesofhowmetabolomics-basedgenediscoveryhasbeenusedtobetterunderstandthebiosynthesisoftriterpenesaponins.
Formorecomprehensivediscussions,readerscanrefertoseveralexcellentreviewarticles.
86-88Page3of15NaturalProductReportsARTICLEJournalName4|J.
Name.
,2012,00,1-3ThisjournalisTheRoyalSocietyofChemistry2012β-amyrinisproducedbycyclizationof2,3-oxidosqualenebyspecificoxidosqualenecyclases,andservesastheentrypointintotriterpenesaponinsbiosynthesis.
β-amyrinisthenoxygenatedatseveralcarbonatomsbycytochromeP450sandthesegenerallyserveasthefirst'tailoringreactions'.
Anelegantinvestigationcombiningexpressedsequencetaganalyses,geneexpressionanalysesandmetabolicprofilingledtotheidentificationtwoP450s,CYP88D689andCYP72A15490,whichcatalyzeoxidationofβ-amyrintoglycyrrhetinicacidinGlycyrrhizauralensis(licorice).
CYP88D6catalyzestwosequentialoxidationsattheC-11methylenetoproduceaketo-group,andCYP72A154performsathreestepoxidationatC-30methyltoyieldacarboxylicacid.
(SeeFigure1).
AsequencehomologofCYP72A154,CYP72A63fromMedicagotruncatula,wasalsoabletocatalyseC-30oxidationofβ-amyrinevenmoreefficientlythanCYP72A154,suggestingafunctionfortheCYP72Asubfamilyproteinsastriterpene-oxidizingenzymes.
M.
truncatulaCYP716A12hasbeensuggestedtobeamultifunctionalP450catalyzingtheoxidationatC-28positionsofβ-amyrin,α-amyrinandlupeol.
91,92Twohomologsfromgrape(CYP716A15andCYP716A17)alsocatalyzetheoxidationatC-28oftriterpenestoproduceoleanolicacid,ursolicacidandbetulinicacid.
92Glycosyltransferasescatalyzingthenext'tailoringreaction'oftriterpenesaponinshavebeenidentifiedwiththeaidofmetabolomics.
Fromasetofco-expressedgenesinM.
truncatula,UGT73F3wasidentifiedasanenzymethatglucosylateshederageninattheC-28position.
93UGT73C10andUGT73C11fromBarbareavulgariscatalyze3-O-glucosylationofthesapogenins,oleanolicacidandhederagenin.
94BiochemicalanalysisofsoybeanmutantsledtotheidentificationoftwoglycosyltransferasesforC-22positionofthesoyasapogenolAaglycone.
UGT73F2wasidentifiedasaglucosyltransferaseandUGT73F4asaxylosyltransferase,exhibitingaclosesimilarityasallelicgenes.
95Thereareincreasinglinesofevidencethatgenesinvolvedinsomeplantsecondarymetabolicpathwaysareclusteredinthegenome.
96,97Thetriterpenoidpathwayisthebestcharacterized.
Thesetsofgenesresponsibleforavenacinbiosynthesisinoat(Avenastrigosa)98,forthesynthesisofthalianol99-andmarneral100-derivedtriterpenesinA.
thaliana,andbiosynthesisofsteroidalglyco-alkaloidsintomatoandpotato101havebeenidentifiedasbeingclusteredintheirrespectivegenomes.
However,therecentlyidentifiedsterolsidechainreductase2genesfromtomatoandpotatoinvolvedinsteroidalglycoalkaloidbiosynthesisarelocatedapartfromtheseclusters.
1023.
d.
AlkaloidsThebiosynthesisofmorphineinopiumpoppy(Papaversomniferum)isoneofthebestcharacterizedplantsecondarymetabolicpathways.
Mostgenesinvolvedinthepathwayhavenowbeenidentified103,104bytakingadvantageofomicstechnologiesincludingtargeted-metaboliteprofiling.
Therehasalsobeenasuggestionforthepresenceofageneclusterinthegenomeofopiumpoppythatmayencodethesemetabolicfunctions.
105Monoterpenoidindolealkaloids,representedbyanti-neoplasticdimericalkaloidsinCatharanthusroseus,areformedfromtryptamineandsecologaninthroughthecombinationoftheaminoacid,tryptophan,andthemonoterpenoid,iridoid,pathways.
ANAD(P)H-dependentoxido-reductase-likeenzymecatalyzestheformationoftheiridoidscaffold.
106A7-deoxyleganeticacidglucosyltransferasecontributestothefurtherstepsofsecolaganinbiosynthesisinC.
roseus.
107Veryrecently,identificationofallgenesfortheseco-iridoidpathwayinC.
roseushasbeencompletedbythecombinedeffortsoftranscriptomeandmetabolomefollowedbyverificationbyheterologousexpressionofcandidategenes.
108,109Figure1–ProposedPathwayforBiosynthesisofGlycyrrhizin.
Thestructuresofpossiblebiosyntheticintermediatesbetweenβ-amyrin(1)andglycyrrhizin(8)areshown:(2),11-oxo-β-amyrin;(3),30-hydroxy-β-amyrin;(4a),30-hydroxy-11-oxo-β-amyrin;(5),11-deoxoglycyrrhetinicacid;(6),glycyrrhetaldehyde;and(7a),glycyrrhetinicacid.
Solidblackarrowsindicateadimerizationreactionoftwofarnesyldiphosphate(FPP)moleculescatalyzedbysqualenesynthase(SQS)originatingsqualene,oxidationbysqualeneepoxidase(SQE)to2,3-oxidosqualene,orcyclizationcatalyzedbybeta-amyrinsynthase(bAS).
Adashedarrowbetweenmevalonicacidandfarnesyldiphosphateindicatesmultipleenzymereactions.
ThebluearrowindicatesasingleoxidationreactioncatalyzedbytheCYP88D6enzyme(Sekietal.
,2008);theredarrowindicatesasingleoxidationreactioncatalyzedbytheCYP72A154enzyme,asdescribedherein;thedottedarrowssignifyundefinedoxidationandglycosylationsteps.
UGATs,UDP-glucuronosyltransferases.
FigurefromSekiHetal.
PlantCell2011;23:4112-4123;www.
plantcell.
org;CopyrightAmericanSocietyofPlantBiologists.
AnexcellentexampleofintegratedanalysesoftranscriptomeandmetabolomedataforpathwayelucidationofalkaloidbiosynthesisisexhibitedinthestudyofcamptothecinsynthesisinOphiorrhizapumilacellcultures.
DifferentialanalysesPage4of15NaturalProductReportsJournalNameARTICLEThisjournalisTheRoyalSocietyofChemistry2012J.
Name.
,2012,00,1-3|5ofgeneexpressionandmetaboliteaccumulationbetweencamptothecin-producinghairyrootsandnon-producingcellsuspensionculturesledtotheidentificationofdifferentiallyaccumulatedmetabolitesandgenespotentiallyinvolvedintheirbiosynthesis.
110MetabolomicsinvestigationofRNAinterferencelines,inwhichthegenesencodingtheenzymesthatcatalysethefirstcommittedreactionsinthebiosyntheticpathways(tryptophandecarboxylaseandsecologaninsynthase)aresuppressed,delineatedtheintermediarymetabolitecandidatesinthebiosyntheticpathway.
111Bytakingtheadvantageofultra-highresolutioncapacityofFT-ICR-MS,thechemicalcompositionofthemetabolomicpeaksthatwererevealedintheLC-MSanalysesweredetermined,andenabledtheinferenceofthemetaboliteannotationsfollowingdatabasequeries.
112Lysinedecarboxylase,whichcatalyzestheformationofcadaverinefromlysine,isthefirstcommittedenzymeinthebiosynthesisofthemajorityoflysine-derivedalkaloids.
113LysinedecarboxylasecDNAwasidentifiedthroughthecomprehensivedifferentialgeneexpressionanalysesofanalkaloid-accumulating'bitter'varietyofLupinusangustifoliusandanalkaloid-less'sweet'variety.
114TheinvivofunctionoftheisolatedgenewasconfirmedbymetabolomicprofilingoftransgenicA.
thalianaandtobaccocellsoverexpressinglysinedecarboxylasecDNA.
Cadaverineandcadaverine-derivedtobaccoalkaloidswerenewlydetectedintransgenicArabidopsisandtobacco,respectively.
4.
Recenttechnicaladvancesinplantmetabolomics4.
a.
SpatiallyResolvedMetabolomicsPlantsasmulticellularorganismsintegratenumerousbiochemicalprocessesthataredistributedamongdifferentcell-typesandamongdifferentsubcellularcompartmentsthatcomposeacumulativeanddynamicmetabolicnetwork.
Thestructureofthedistributedmetabolicnetworksisusuallyinferredbytheasymmetricdistributionofenzymes(proteins)ormRNAsassociatedwiththeindividualcomponentswithinthenetwork.
Technologiesarecurrentlyavailablethatprovidehighspatialandtemporalresolutionimagesofthedistributionofthesemacromoleculeswithinindividualcellsandevensubcellularcompartments.
Theseimagingtechnologiesoftenutilizespecificmacromolecularinteractionsbetweenthetargetedmoleculeandareporter-molecule(e.
g.
,antibodies,nucleicacidhybridizations).
Technologiesthatcanlocatethepositionofmetabolitesatsuchahighspatialresolutionlevelcoulddirectlydemonstratethenatureofthenetworkanditsregulation.
Theutilityofsuchdataisillustratedwithmetabolitesthatinteractwithelectromagneticwaves,andarethusvisibly"colored".
Forexample,ithasbeenknownformanyyearsthatmanyredandbluecoloredanthocyaninsarelocatedinepidermalcellsusingcytochemicalormicrospectrophotometricmethods,115,116andthishasenabledthedecipheringofgenenetworksthatprogramandintegratecellulardifferentiation,environmentalcuesandmetabolism.
117-119Moregeneralizedtechniqueshavebeendevelopedthatcombinemicro-dissectionwithcouplingreactionsthatamplifythedevelopmentofmetabolite-dependentcoloredproducts.
120Othermethodsthatutilizetheisolationofprotoplasts121,122ornon-aqueousfractionation123ofsubcellularcompartmentshavealsobeenusedtodeterminelevelsofmetabolitesindifferentcompartments.
However,theseprotocolsaresomewhatdifficulttorecapitulateandit'snotalwaysclearifthesemethodshave"trapped",in-placetheintermediatesofmetabolism,andthusthelocationofmetaboliteshasbeendifficulttocorroborate.
Morerecentlylaser-basedmicrodissectiontechniqueshavebeenappliedtoplantsystemstoharvestpopulationsofcellsthatmorphologicallyappearanidenticaldevelopmentalstate,butmostofthesetechniqueshavebeenappliedtoevaluatethetranscriptomeandproteomeoftheisolatedcellpopulations.
124Thus,fasterandmoreefficientmethodsareneededtobetteranalyzeandunderstanddynamicandspatiallyresolvedmetabolism.
4.
b.
MassSpectrometryImagingChemicalimagingisanimportantapproachtovisualizinglocalizedmetabolism.
Recenttechnicaladvanceshaveenabledmetaboliteimagingusingmassspectrometry(MS)125-128andnuclearmagneticresonance(NMR).
129-132Massspectrometryimagingtoolshavealsobeenreferredtoasmassmicroscopesandsimilarly,NMRimagingasNMRMicroscopes.
129Massspectrometryimaging(MSI)isperformedthroughthelocalizedionizationofmetabolites,peptides,and/orproteinsfromspecifictwo-dimensional133andmorerecentlythree-dimensional134,135coordinatesofabiologicaltissue.
Thus,enablingthevisualizationofthespatialdistributionofproteinsandmetabolites.
Agreaterproportionofthecurrentliteraturerelatestoimagingmassspectrometryofproteins,butimagingofplantsmallmoleculesandmetabolitesisgainingpopularity136-138.
Variousionizationtechniqueshavebeenusedinmassspectrometryimaging.
Theoldestissecondaryionmassspectrometry(SIMS)139whichwaspopularizedforelementalandsurfaceanalyses.
140Secondaryionizationistypicallyachievedusingahighenergymonoatomic(e.
g.
Ga+,Cs+)orpolyatomic(e.
g.
C60+)primaryionbeamswhicharefocuseduponasurface,anduponimpact,desorbandformsecondaryionsfromthatsurfaceformassanalysis.
TheprimaryionsusedinSIMSaretypicallyhighenergythatresultsinsubstantialfragmentationofbiologicalmolecules.
Thus,SIMSimaginghasfoundthemostutilityinimagingofelemental/atomicspecies.
SIMSimaginghasbeenrecentlyusedtoevaluatealuminiumdistributioninsoybeanroots,141elementalnutrientdistributioninPhaseolus,142andArandSiinriceroots.
143Overtime,SIMSimagingofmetabolitessuchasflavonoidsinPisumsativumandA.
thalianahasbeenachievedusingpolyatomicprimaryionsources144thatdistributetheenergyoverabroadersurfaceandresultinlessdamage.
AprimaryadvantageofSIMSistheabilitytofocustheprimaryionbeameitherelectrostaticallyormagneticallytoachieveveryhighspatialresolutions.
CurrentlynanoSIMShasbeenreportedwithaspatialresolutionof50nanometersinplants.
145However,SIMSisplaguedbyverylowionizationefficiencies.
Thus,ithasrelativelylowsensitivityforbiologicalmoleculesandothermethodshavebecomemorepopular.
Laserdesorptionionization(LDI)MSItechniquesarecurrentlythemostwidelyusedtechniques.
Laserdesorptionionizationcanbeperformeddirectlyorassistedwithamatrix.
DirectLDI-MSIrequiresmetaboliteswithhighlyabsorbingchromaphoreswhichabsorbsufficientenergyfromthelasersourcetoenabledirectdesorptionandionization.
ExamplesofPage5of15NaturalProductReportsARTICLEJournalName6|J.
Name.
,2012,00,1-3ThisjournalisTheRoyalSocietyofChemistry2012suchmoleculesincludephenolics,flavonoids,phloroglucinols,quinonesandnapthoanthrones.
Hlscherandcolleaguesrecentlypresentedareportontheuseofultraviolet-LDItostudythedistributionofsecondarymetabolitesinArabidopsisandHypericumspecies.
146LDIusingcolloidalgoldorsilvertoassistintheionizationprocesshasalsobeenrecentlyreportedtoimageepicuticularwaxesfromArabidopsisleaves147andothertissues.
136TheVertesgrouphasfurtherpioneeredlaserablationelectrosprayionization(LAESI),148-154includingthedevelopmentofanetchedtipopticalfiberthatcanprobesinglecells.
153,154Matrixassistedlaserdesorptionionization(MALDI)iscurrentlythemostpopularMSItechniqueduetoitsgenerallybettersensitivity.
MALDI-MSIwasfirstintroducedforproteins,128buthasbeenrapidlyadoptedtosmallmolecules155suchasdrugs125andplantmetabolites.
138,156MALDI-MSIhasbeenusedinavastnumberofplantapplications.
123,157-159Ithasbeenusedtoimageagrochemicalsinsoy,160carbohydratesinwheat,161lipids,162flavonolsinapple,163glycoalkaloidsinpotato,164glucosinolatesinArabidopsis,165anthocyaninsinblueberry166andrice,167andalargearrayoforganicacids,aminoacids,sugars,lipids,flavonoidsandtheirconjugatesinM.
truncatularootnodules.
168Althoughinitialapplicationstoplantsystemswerefocusedonmetabolitesthatoccurontheexteriorsurfacesofplants(i.
e.
,epicuticularlipids)169-171,histologicalpreparativemethodsarealsobeingevaluatedtoimage"interior"metabolitesofplanttissues.
136,172Desorptionelectrosprayionization(DESI)isanotherionizationtechniquethatisbeingdeveloped173-176andhasfounddiverseapplicationsinmetaboliteimaging.
157,177-184DESIimagingisachievedbydirectinganelectrosprayaerosolontoanambientsurface.
Secondaryionsoriginatingfromthesurfacearethengeneratedandusedformassspectralimaging.
Alternatively,plantmetabolitescanbetransferredorimprintedontoaseparateporouspolytetrafluoroethylene(PTFE)surfaceandsimilarlyimagedfromthePTFEsurfacetoenhancethesignalintensity.
157DESI-MSIhasbeenusedtoimagediterpeneglycosidesinStevialeaves185andalkaloidsinvarioustissuesofpoisonoushemlock(Coniummaculatum),jimsonweed(Daturastramonium)andnightshade(Atropabelladonna).
186LaskinandcolleaguesdramaticallyimprovedthespatialresolutionofDESIimaging,usingananoDESItechniqueandachievedspatialresolutionofabout12m.
187ThespatialresolutionofearlyMALDI-MSIwasapproximately150x150m,whichismulti-cellularformostorganisms.
ThespatialresolutionofMALDI-MSIispredominantlydictatedbythelaseropticsandspotsize.
However,improvementsinlaseropticshaveresultedinimprovedspatialresolutions,andmanycommercialsystemsarenowavailablewitharesolutionofapproximately10x10m.
Thus,therehasbeenanalmost10-foldincreaseinthespatialresolutiontopositionimagedmetabolitesrelativetoknowncellularstructures,fromapproximately100-mresolutionin2005,171toapproximately10-mresolutionin2010.
169,188Consideringthatplantcellsareapproximately30-50minsize,thislevelofspatialresolutionisenablingthelocalizationofmetabolitestothelevelofindividualcellularstructuresandthespatialresolutioncontinuestoincrease.
Anultra-highspatialresolutionversionofmatrix-assistedlaserdesorptionionization(MALDI)method,calledscanningmicroprobeMALDI(SMALDI)hasbeendeveloped,anditcandeterminethepositionofanalytestoaresolutionoffewmicrons189,190.
OthercustomMSIinstrumentswithresolutionsof1marealsobeingbuiltinresearchlabs,butcommercialsystemswithcomparablehighspatialresolutionsarenotyetreadilyavailable.
Asthespatialresolutionincreasesduetodecreasedlaserspotsize,thenumberof'pixels'neededtoimagethesameareaincreasesandsodoestheimagingtime.
Theincreasedimagingtimeshavebeenmediatedbyadditionalimprovements/increasesinlaserfrequencieswith1kHzlasersnowbeingcommon.
However,continualimprovemetnsinincreasedspatialresolutionandimagingspeedsarestilldesiredtoenablegreaterresolutionofsmallercellsandsub-cellularmetabolitesinshortertimes.
Unfortunately,wemaybenearingthepracticalspatiallimitsofcurrentMSItechnologyduetothereducedquantitiesofmetabolitesinsmallervolumes(i.
e.
,sensitivity),andtheRaylieghlimitsoftheopticalUVlasers,whichareontheorderofseveralhundrednanometers.
CurrentMSIalsofacesthechallengesofcompetitiveionizationandionsuppressionmakingitmoredifficult.
4.
c.
NMRImagingNuclearmagneticresonanceimaging,oroftenreferredtoassimplymagneticresonanceimaging(MRI),isanotherchemicallyspecific,spatialimagingtechnologywhichmeasurestheresonanceintensityofparamagneticnucleifoundinmanybiologicalmetabolitestogenerateimages.
191SeeIshidaetal.
129andKockenbergeretal.
130forearlierreviewsofMRI.
OneofthemajorbenefitsofMRIisthatitisnon-destructiveandcanimageintact,livingplanttissues.
MRIisachievedbymeasuringtheresonanceintensitywhilevaryingamagneticgradientalongthexandyaxesatspatiallyfixedvalues.
129-131Duringthisprocess,theresonancesignalisrecordedforonlyasmallspatiallydistinctpartofthesampleandsensitivitycanbeasignificantchallenge.
Asthedesiredspatialresolutionincreases,thesignaltonoiseratioisfurtherdecreased.
Thus,manyMRIexperimentsutilizethe1Hresonancesignalfromwaterduetoitsrelativelyhighabundanceresultinginhighsensitivity.
Asaresult,MRIhasbeenextensivelyusedtostudywatercontentandmovement/transportthroughoutplants.
192-196Theabundant1Hresonancesignalfromwaterisalsopreferredandnecessarytoobtainthehighestresolutionimages.
Theoreticalresolutionsofupto1mhavebeenpredictedforMRIwithrealisticthreedimensionalresolutionsof3.
7x3.
3x3.
3m3achieved.
197Itislikewisepossibletoimage1Hsignalsoriginatingfrommetabolitessuchassugarsandaminoacids,198,199aswellastoimageotherparamagneticnucleisuch13C,15N,17O,19F,23Na,31P,and39K.
200,201SomeofthemostimpressiverecentMRIofplantshasbeenfocusedonimaginglipiddistributioninseeds.
202,203MRIisawell-developedtechnologythatcontinuestoimprovewiththeimprovedsensitivitiesenabledthroughhigher-fieldmagnetsandcryogenicallycooledprobes.
However,itscurrentresolutionisontheorderof1-10minthex,yandzplanesandvariesdependentupontherelativesensitivityoftheselectednuclearresonancesignal.
Similartomassspectrometryimaging,theprimarylimitationtoMRIresolutionisthelowconcentrationofmetaboliteslocalizedwithinsmallerspatialregions.
BothMSIandMRIcanachieveapproximatecellularresolutionformanyplants,buttheirspatialresolutionsultimatelyfallshortofmostopticalimagingtechnologies.
However,bothMSIandMRIprovidechemicallyPage6of15NaturalProductReportsJournalNameARTICLEThisjournalisTheRoyalSocietyofChemistry2012J.
Name.
,2012,00,1-3|7specificspatialdetailsthatsimplycannotbeachievedwithopticaltechniques.
Thus,bothMSIandMRIarecontributingtoourunderstandingofthedifferentialanddynamicmetabolismthatisoccurringthroughoutplantsatanevergrowingrate,andaredestinedtobekeymetabolomicstechnologiesinthefuture.
MSIhasthecurrentadvantagethatitcandifferentiallyprofileandimagealargernumberofm/zvaluesorpotentiallydifferentmetabolitessimultaneouslyrelativetoMRI,butMRIhasthepredominantadvantagethatitisnon-destructiveandcanimageintact,livingorganisms.
4.
d.
LC-MS-SPE-NMRThelarge-scaleprofilingofplantmetabolites(i.
e.
metabolomics)isadvancingourfundamentalunderstandingofplantbiochemistry,yieldingdiscoveryofnovelmetabolitesandgenefunctions,andprovidinganadvancedmechanisticunderstandingofplantresponsestobiotic,abiotic,andenvironmentalstimuli.
SeeSection3fordetails.
However,thecurrentdepth-of-coverageisstilllimitingandchemicalannotationisthenumberonechallengefortheMetabolomicsCommunity.
204-206Thus,thereisacriticalneedtoidentifymoremetabolitesbothsystematically(i.
e.
logicalprogressiontowardsidentificationofallmetabolites)andinabiologicallydirectedmanner(i.
e.
thoseobservedtobedifferentiallyaccumulatedduringcomparativeexperiments)soastoincreaseourdepth-of-coverageandtoenhancethebiologicalcontextandcontentofmetabolomicsexperiments.
TheMetabolomicsCommunityhascometoageneralconsensusthataminimumoftwoorthogonalanalyticaldataarenecessaryforconfidentidentificationrelativetoanauthenticstandard.
207Intheabsenceofauthenticstandards,itisnecessarytoreturntothetraditionalstandardsoftheorganicchemistryjournalsthatincludeNMRandaccuratemassmeasurementsforelementalanalyses.
Historically,massspectrometryandNMRworkflowshavebeensegregated,andtheidentificationofmetabolitesbyNMRhasbeenalengthyprocess.
However,MSandNMRmethodsarenowbeingbroughttogetherinamoreintegratedmanner.
OneapproachistocoupleHPLCdirectlytoNMRbytransferringtheHPLCeluenttoaNMRflow-probethatcanbeoperatedincontinuous-floworstopped-flowmanner.
208However,thepracticalimplementationandutilityoftheseapproachesischallengingduetolowsensitivities,reducedtimeforNMRspectralacquisition,andhighcostsofdeuteratedHPLCsolvents.
TheselimitationscanbecircumventedthroughtheautomatedpurificationandconcentrationofoneormanyHPLCseparatedmetabolitesviapost-column,solid-phaseextraction(SPE).
HPLCisoftencoupledtoparallelorin-linedetectorssuchasUVandMSresultinginacomplexinstrumentalensemble,i.
e.
HPLC-UV-MS-SPE(Figure2).
PurifiedsamplescollectedfromrepetitiveHPLC-UV-MS-SPEseparationscanthenbeelutedwithdeuteratedsolventsintotraditionalNMRtubesorintoaflow-probeforNMRanalyses.
209-211TheauthorsofthisarticleareconvincedthatsuchintegratedMSandNMRtechniquesarenecessaryfor'higher'through-putmetaboliteidentificationsandlarge-scaleplantmetabolomics.
Thus,theauthorsareadvocatingthisconceptbyintegratingultra-highpressureliquidchromatography(UHPLC,18,000psi)withhighresolutionquadrupoletime-of-flightmassspectrometry(QTofMs,resolution~40,000)andSPEfortheautomatedandhigherthrough-putpurificationandconcentrationofchemicallyundefinedplantmetabolites.
SpecificmetabolitestargetedforannotationarerepetitivelycollectedfrommultipleUHPLC-UV-MSseparationsontothesameSPEcartridgewhichallowsfortherecoveryoflargerquantitiesnecessaryforNMRanalyses.
Thissystemenableshighresolutionseparationsofmetabolitesthatcanthenbefurtherisolatedusingacombinationofmass,retentiontime,andUVsignalstoinitiateSPEpurificationandconcentration.
PurifiedandconcentratedtargetmetabolitesisolatedonSPEcartridgesaredriedtoremoveprotonatedsolventsandelutedwithdeuteratedsolventsforfurtherNMRanalyses.
StructuralidentificationsarethenmadefromthecombinationofUHPLCretention,UV,MS,MS/MS,andNMRdata.
Targetedrecoveryamountsof1-5garetypicallysufficientfor1Dand2DNMRusingaBruker600MHzNMRwitha1.
7mmTCIcryoprobe.
Page7of15NaturalProductReportsJournalNameRSCPublishingARTICLEThisjournalisTheRoyalSocietyofChemistry2013J.
Name.
,2013,00,1-3|8Figure2–SchematicofanintegratedUHPLC-UV-MS-SPE-NMRsystem.
Comparativemetabolomicsisperformedusinglarge-scalemetaboliteprofilingbyUHPLC-UV-MS.
Differentiallyaccumulatedmetabolitesorotherwisetargetedmetabolitesarethenpurifiedandconcentratedbysolid-phaseextraction(SPE)frommultipleUHPLC-UV-MSseparations.
TheUHPLC-UV-MSpeaksofinterestcanbetargetedforSPEpurificationbaseduponretentiontime,m/z,andUVabsorbance.
Oneormanytargetedmetabolite/peakscanberepetitivelyisolatedondifferentSPEcolumns(upto2x96usingaBruker/SparkHollandProspekt2).
SPEpurifiedandconcentratedmetabolitesarethendriedtoremoveprotonatedsolventsandelutedusingdeuteratedsolventsintotraditionaltubesorintoaflow-probeforNMRanalyses.
Metabolitesarethenultimatelyidentifiedbaseduponretention,UVabsorbance,accuratemass,tandemmass,1Dand/or2D1H/13CNMRspectra.
4.
e.
TandemMassSpectralDatabaseandComputationalMSAsnotedabove,themajorchallengeinplantmetabolomicsismetaboliteannotation.
Typicallythousandsofmetabolites(ormassfeatures)canbedetectedinanuntargetedmetabolomicsexperiment.
Identificationofthosemetabolitesisverychallengingbecause(1)manynaturalproductshaveasubstantialnumberofisomerswhichcannotbedifferentiatedsolelybasedupontheirm/zvalues,and(2)foreachsinglemassfeature,multipleempiricalformulascanoftenbegeneratedwithinasmallm/zvariancewindow,i.
e.
,<5ppm.
Tandemmassspectrometryisthereforeoftenemployedtoprovideadditionalstructuralinformationthatcanbeusedtoannotatemetabolitesbymatchingtandemspectraofexperimentalsamplestothoseofauthenticcompoundswithinadatabase.
Overthepastyears,anumberofonlinepublictandemmassspectraldatabaseshavebeendeveloped.
TheseincludeMassBank,212HMDB,213Metlin,214,215GolmMetabolomeDatabase(GMD),216thePlatformforRIKENMetabolomicsPage8of15NaturalProductReportsJournalNameARTICLEThisjournalisTheRoyalSocietyofChemistry2012J.
Name.
,2012,00,1-3|9(PRIMe),217,218MeltDB,219andMadisonMetabolomicsConsortiumDatabase(MMCD).
220Manyofthesedatabasesfocusuponspecificorganisms.
Forexample,thePRIMedatabasefocusesprimarilyonplantmetabolomes,theHMDBisdedicatedmoretowardsthehumanmetabolome,andMetlinisdirectedmoretowardspharmaceuticalresearchandbiomarkerdiscovery.
Amongthesepublicdatabases,MassBankisuniqueinthatitalsoservesasapublicrepositoryforthemetabolomicsandthenaturalproductresearchcommunities.
Itcurrentlyhasmorethan40,000spectracontributedby28differentinstitutesworldwide.
Itisalsooneofthefirstpublicmassspectraldatarepositoriesthatallowuserstosharetheirspectraldata.
Thisisveryimportantasmany"unknowns"encounteredinuntargetedmetabolomicsareoften"knownunknowns".
Identificationofthese"knownunknowns"canbedifficultastheirstructuralinformationisoftenscatteredthroughouttheliteratureandvariousdatabases,someofwhichmaybedifficulttoassess.
Providingapublicplatformforresearcherstodepositandsharetheirmassspectraldatacansignificantlyimprovemetaboliteannotationwithintheplantmetabolomicscommunity.
Inadditiontopublicdatabases,commercialdatabasesarealsoavailablesuchastheAgilentFiehnGC-MSMetabolomicsLibrary,AgilentMetlinPersonalMetaboliteDatabaseandWileyRegistry/NISTMassSpectralLibrarythatnowcontainsbothelectronionization(EI)andtandemMS/MSspectra.
UnlikeEIspectra,electrosprayionizationMS/MSspectraarehistoricallylessreproducible.
Differentinstrumenttypes(oreventhesameinstrumenttypebutfromdifferentvendors),differentionopticsanddifferentcollisionenergiescanproducesubstantialvariationswithintheMS/MSspectra.
221Thisoftenleadstounsuccessfulidentificationsandevenmisidentifications.
Inaddition,theoverallmetabolomecoverageoftheavailableMS/MSdatabasesisstillverylimitedduetothelackofauthenticstandardsandthecomplexityofthemetabolomes.
Thus,theidentificationofmetabolitesnotincludedinthedatabasesischallengingandtypicallyrequiresadditionalcomputationalMS(orMSinformatics)orempiricalmethods,ieNMR.
ComputationalannotationtoolsbaseduponMSdatahavebecomeanimportantpartofthemetabolomicsworkflowowingtotheirrapiddevelopmentsoverthepastdecade.
222InsilicogeneratedMS/MSdatabasesareexpectedtosignificantlyimprovethesuccessofmetaboliteannotationwhenexperimentalMS/MStrainingdataofauthenticstandardsbecomeavailable.
Severalpublicorcommercialsoftwarepackagesandwebsitesarenowavailable,includingMetFragandMetfusion,223,224FragmentIdentificator(FiD),225lipidblast,226SIRIUS,227MSinterpreter(NIST),228MSfragmenter(ACDlab)andMassFrontier(HighChem).
Insilicofragmentationispredictedbaseduponfragmentationrulesgeneralizedfromliteraturedata229,230orcombinatorialapproachesthatusebondenergiestopredictfragmentationchemistries.
223-225Whilerule-basedapproachescanbehighlyspecific,especiallyforthesameclassofmetabolites,theytypicallycannotcorrectlypredictfragmentsformetabolitesforwhichfragmentationrulesareabsent.
Combinatorialmethodsrequireextensivecomputationwhenthecandidatemetabolitehasalargenumberofchemicalbondsandfunctionalgroups,andtheylackthespecificityoftherule-basedapproaches.
Somesoftware,suchasMassFrontier,containmanuallycuratedfragmentationpatternsfromseveralthousandpublications.
Insilicofragmentationpredictionhasbeensuccessfullyusedwithcompoundsconsistingofsimilarrepeatingunitssuchaspeptides,231lipids,226,232andglucans.
233,234However,itsuseonarbitrarysmallmoleculesisstillverychallengingduetothestructuraldiversityofsmallmoleculesandtheircomplexfragmentationpatterns.
Forexample,weobservedthathydroxylationpositionrendersfragmentationofstructurallysimilarflavonoidscompletelydifferent.
Thusanalgorithmthatcombinesrule-basedapproaches,combinatorialmethods,publishedfragmentationmechanismsandmachinelearningarelikelytobemoresuccessfulinpredictingfragmentationofsmallmolecules.
4.
f.
Large-scaletargetedmetabolomicsviaMRMLarge-scaletargetedmetabolomicsaimstodetectandquantifydozenstohundredsofknowncompoundsinacomplexsamplemixture.
Itcanbeusedtoassessmetabolicchangesresultingfromgeneticmanipulationand/orenvironmentalperturbationbyselectivelymonitoringasubsetofmetabolitesassociatedwithcertainspecificpathways.
UltrahighperformanceliquidchromatographycoupledtoatriplequadrupoleMS(UPLC-QqQ-MS)isidealfortargetedmetabolomicsduetoitsgoodsensitivity,reproducibility,robustquantificationandbroaddynamicrange.
ItistypicallyoperatedinMRM(MultipleReactionMonitoring)modeinwhichcollisionenergiesandotherparametersforeachindividualtargetcompoundshavebeenpre-optimizedwithauthenticstandardstoenhancesensitivityandselectivity.
235DuringaMRMexperiment,ametaboliteprecursorisfirstresolvedandisolatedbythefirstquadrupole(Q1)andfragmentedinthesecondquadrupole(Q2)whichfunctionsasacollisioncell.
Thethirdquadrupole(Q3)servesasafinalmassfiltertomonitorspecificproductions.
Theidentityofmetabolitescanbeensuredwhencombinedwithknownchromatographicretentiontimesbecauseindividualmetaboliteshavespecificprecursor/productions,whicharealsoknownasatransitionpair.
Quantificationisperformedusingthemorespecificandabundanttransitionpairforeachtargetedmetabolite.
Large-scaleMRMhasbecomeaworkhorsefortargetedmetabolomicsduetoitshighsensitivityandselectivity.
TheutilityofMRMintargetedplantmetabolomicshasbeenwelldemonstrated.
236-238Typically,twoMRMtransitions,i.
e.
,aquantitativetransitionandaqualitativetransition,aremonitoredtoincreasetheconfidenceincompoundannotation.
However,duetothecomplexityofthemetabolomes,someisomericmetabolitesmightnotbeadequatelyseparatedbyLCandtheymightproducethesameproductionsthatareusedformonitoringthetargetcompounds.
235Thiscanleadtofalsepositiveresults.
239,240Somesolutions,rangingfromtheuseofprobability-basedcomputationalapproachestoadifferentinstrumentsetup,havebeenproposedtominimizereportingoffalsepositiveresultsandconfirmationofidentities.
241,242TriggeredMRMorMRM-EPI(MRM-triggeredenhancedproductionscan)thatinitiatetheacquisitionofaproductionspectrumwhenthesignaloftheMRMtransitionexceedsapre-setthresholdcanbeveryusefulinconfirmingtheidentityofthemetabolites.
243However,ifaproductionspectrumisacquiredforeverytargetmetabolite,thetotalnumberoftransitionscanincreasesubstantially.
Thiscanleadtopoorquantificationaccuracyandlowsensitivityduetocompressedacquisition/dwelltimesfortheindividualMS/MStransitions.
AnotherchallengeinMRMtargetedmetabolomicsisthattheproductionspectracanbedominatedbyonemajorfragmentforsomemetabolites.
OtherfragmentshaveverylowsignalPage9of15NaturalProductReportsARTICLEJournalName10|J.
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,2012,00,1-3ThisjournalisTheRoyalSocietyofChemistry2012intensitiesandthusarelessselectiveandsensitiveifusedasMRMtransitions.
Inaddition,forsomemetabolites,neutrallossesofH2O,CO2,methylorglycosylgroupsarethemajorfragmentions.
Ionsresultingfromtheseneutrallossesarelessselectiveandcommonlygiverisetomatrixinterferences.
4.
g.
X-rayusingporouscomplexesTheuseofX-raycrystallographyinstructuraldeterminationshasalonghistory,andthefirstcrystallographicstructuralelucidationofasmallorganicmoleculewasperformedin1923.
244EarlyuseofX-raycrystallographyfocusedoninorganiccompoundsandminerals.
However,it'sutilityinthedeterminationoforganicandbiologicalrelevantmoleculeseventuallyevolved,andtheNoblePrizeinChemistrywasawardedtoDorothyC.
Hodgkinin1964forsolvingthestructuresofimportantbiologicalmoleculessuchascholesterol,penicillinandvitaminB12.
245However,overthepast20yearsorso,crystallographyhasfallenfromfavourasastructuraldeterminationtoolforsmallmolecules.
Thishasbeenprimarilyduetothelengthytimeneededtogeneraterelativelylargequantitiesoftheanalyterequiredfortheoftennumeroustrial-and-errorattemptstoproducequalitycrystalsrelativetoothertechnologiessuchasMSandNMR.
Inaddition,notallmoleculescanformthecrystallinestructuresnecessaryforX-rayanalysis.
Fortunately,transformativenewtechnologyisnowmakingX-raystructuraldeterminationofgtongquantitiesofbiomoleculesareality.
246,247Inokumaandcolleagueshavedevelopedamethodthatremovesthebottleneckneededtoproducerelativelylargesinglecrystalsforsingle-crystalX-raydiffraction(SCD)analysesandallowsforthestructuraldeterminationofnon-crystallinematerials.
Theirmethodinsteaddissolvestargetedanalytesinasolventthatisthenslowlyinfusedintoaporous,crystallinehostcomplex.
Astheanalyteinfusesintothehoststructureitabsorbsontotheporesurfacesvianoncovalentchemicalinteractions.
Thisproducesanorderedstructureoftheanalytewithintheorderedhostcrystalasthesolventisslowlyevaporated.
Thestructureoftheanalyteisthensolvedwithinthestructureofthecrystallinehostmatrixusingtraditionalinstrumentation.
Theauthorsreportthattheirstandardprotocolutilizesapproximately5gofanalyte,butfurtherdemonstratetheirapproachbysolvingthecrystalstructurefromaslittleas80ngofguaiazuleneusingstandardlaboratoryinstrumentation;i.
e.
,BrukerAPEX-IICCDwithaMoradiationsourceorRigakuVariMaxwithaCuradiationsource.
Theauthorsfurtherproposethatstructurescouldbedeterminedfromaslittleas10ngorlessusingasynchrotronradiationsource.
InokumaandcolleaguesadditionallydemonstratedthattheirmethodwascompatiblewithHPLCpurifiednaturalproductsfromorangepeel(Citrusunshiu).
246Inthisapproach,hostcrystal'sponges'wereaddeddirectlytoHPLCfractionscollectedinvialsfollowedbysubsequentX-raycrystallography.
UsingtheirHPLC-SCDapproach,theydeterminedthestructuresofthreepolymethoxyflavonesandconcludedthat'LC–SCDanalysiswillbeapowerfultoolfortherapidcharacterizationofmultiplecomponentswithmuchhigherstructuralreliabilitythanliquidchromatographymassspectrometryandLC–NMRtechniques'.
Overall,thesensitivityofthereportedHPLC-SCDtechniquerivalsthatofmassspectrometryandsurpassesthatofNMR.
Thus,itisveryconceivablethatHPLC-SCDcouldbeapowerfulandrealisticalternativeforhigherthrough-putmetaboliteidentifications.
IntegrationofSCDwithHPLC-MS-SPE-SCDisalsoeasilyvisualized.
5.
FutureprospectsforcropbreedingandherbalmedicineAlargeproportionofthesuccessofplantmetabolomicshasbeenobtainedusingmodelspecies.
However,therearegreatneeds,opportunitiesandchallengesassociatedwithexpandingtheutilityofmetabolomicsincropspecies.
Amajorchallengeisthecomplexgeneticsgenerallyassociatedwithcrops.
However,metabolomicshassuccessfullyusedforenhancedbreedingofimportantcrops.
248Asabasisforfuturebreedingprograms,theidentificationofgenomicregionsandgenesassociatedwithmetabolicquantitativeloci(mQTL)orproductionofspecializedmetaboliteshasbeenperformedwithmajorcropstakingadvantageofavailablegeneticresources.
ExtensivemQTL69,249,phenotypic/genetic250,251analysesandgenomewideassociationstudy(GWAS)252ofricepopulationshavebeenreported,notingthatriceisaprimarycropfeedingthemajorityoftheworld'spopulation.
Inthesestudies,themQTLregions69,249andrelatedsingle-nucleotidepolymorphism(SNP)markers252foravarietyofflavonoidmoleculeshavebeenidentified.
Thesemetabolomicinvestigationswerebasedonthelarge-scaleidentificationofricemetabolitesusingasolidstrategyofstructuredeterminationbynaturalproductchemistry.
253Besidesthedirectrelationofgenomicregionsorgeneswithmetabolites,investigationofmetabolicsignaturesrepresentingtheresponsestoabioticstressesisalsoanimportantissuebeingaddressedthroughmetabolomics.
Forexample,metabolomicsstudiesrevealedcharacteristicmetabolicchangesassociatedwiththestressesofdraught,UVandnitrogendeficiencyinwheat254,255andmaize.
256,257Theapplicationtoherbalmedicinesandcrudedrugsisalsoexpectedtobenefitfrommetabolomicsanalyses.
258,259Metabolomicscandepictnotonlylargenumbersofchemicalcomponentsfoundinmixturesofherbalmedicines,260butcanalsocorrelatethosechemicalcomponentsfromplantswiththechemicalmarkersofpatientswhointaketheseherbalmedicines.
Ifonecansystematizeallcorrelationsofchemicalcomponentsbothfromherbalmedicinesandbodyfluidsofpatients,suchasbloodandurinewithdiagnosticindices,newprescriptionsofherbalmedicinemixturescouldbedevelopedtomaximizethetherapeuticeffects.
2616.
ConclusionsMetabolomicshassignificantlyadvancedourunderstandingofplantspecializedmetabolismandnaturalproductbiosynthesisatthemolecularandbiochemicallevelswithnumerousexamplesprovidedherewithin.
MetabolomicsisalsoenablingthebetterunderstandingofmedicinalplantsandtheidentificationofimportantmetabolicQTLsforenhancedbreeding.
Althoughmetabolomicshasprovenitsvalue,itstillfacessubstantialchallengesincludinglarge-scalemetaboliteidentifications.
AsimprovedtechnologiescontinuetheirPage10of15NaturalProductReportsJournalNameARTICLEThisjournalisTheRoyalSocietyofChemistry2012J.
Name.
,2012,00,1-3|11progressivemarchforward,thefieldofmetabolomicscanonlygetbetter.
Alargenumberofadvancingtechnologieswerereviewedwithinthisarticleandprovideaperspectiveontheexcitingandgrowingpotentialofmetabolomicsinthefuture!
7.
AcknowledgementsAlltheauthorswouldliketoacknowledgesupportfromtheMetabolomicsforaLowCarbonSocietyprogramfundedjointlybytheUSNationalScienceFoundationIOS(Award#1139489)andtheJapaneseScienceandTechnologyAgency(SICORP).
TheauthorswouldalsoliketothanktheNationalScienceFoundationIOSAward#1340058forfundingarelatedResearchCoordinationGrant:IntegratingandCoordinatingaNationalandInternationalPlant,Algae,andMicrobialMetabolomicsResearchCoordinationNetworkthatisenablinggreaterinteractionsbetweentheinternationalscientistsfundedthroughthejointNSF-JSTMetabolomicsforaLowCarbonSocietyProgram.
LWSwouldliketoacknowledgeboththeNationalScienceFoundation,MajorResearchInstrumentation,DBIAward#1126719andBrukerDaltonics/BioSpin;especiallyAikoBarschandUlrichBraumann,forUHPLC-MS-SPE-NMRinstrumentationsupport.
BJNacknowledgestheon-goingsupportbytheU.
S.
DepartmentofEnergy,OfficeofBasicEnergySciences,DivisionofChemicalSciences,Geosciences,andBiosciencesthroughtheAmesLaboratory.
TheAmesLaboratoryisoperatedfortheU.
S.
DepartmentofEnergybyIowaStateUniversityunderContractNo.
DE-AC02-07CH11358.
NotesandReferencesaTheSamuelRobertsNobleFoundation,PlantBiologyDivision,2510SamNobleParkway,Ardmore,OKUSA.
bDept.
ofBiochemistry,BiophysicsandMolecularBiology,IowaStateUniversity,Ames,IA50011.
cAmesLaboratory,U.
S.
DepartmentofEnergy,Ames,IA50011dRIKENCenterforSustainableResourceScience,Suehiro-cho,Tsurumi-ku,Yokohama230-0045,JapaneGraduateSchoolofPharmaceuticalSciences,ChibaUniversity,Inohana,Chuo-ku,Chiba260-8675,Japan.
1.
I.
F.
Stone,ThetrialofSocrates,Little,Brown,Boston,1988.
2.
B.
Robson,O.
K.
BaekandS.
Ekins,TheEnginesofHippocrates:FromtheDawnofMedicinetoMedicalandPharmaceuticalInformatics,Wiley,2009.
3.
E.
J.
DaSilva,E.
BaydounandA.
Badran,Biotechnologyandthedevelopingworld,2002.
4.
W.
Sneader,BMJ,2000,321,1591-1594.
5.
D.
Greenwood,JournalofAntimicrobialChemotherapy,1992,30,417-427.
6.
M.
C.
Wani,H.
L.
Taylor,M.
E.
Wall,P.
CoggonandA.
T.
McPhail,JournaloftheAmericanChemicalSociety,1971,93,2325-2327.
7.
J.
GoodmanandV.
Walsh,Thestoryoftaxol:natureandpoliticsinthepursuitofananti-cancerdrug,CambridgeUniversityPress,Cambridge;NewYork,2001.
8.
L.
H.
MillerandX.
Su,Cell,2011,146,855-858.
9.
S.
J.
CutlerandH.
G.
Cutler,Biologicallyactivenaturalproducts:pharmaceuticals,CRCPress,BocaRaton,FL,2000.
10.
J.
R.
Hanson,Naturalproducts:thesecondarymetabolites,RoyalSocietyofChemistry,Cambridge,2003.
11.
I.
A.
Graham,K.
Besser,S.
Blumer,C.
A.
Branigan,T.
Czechowski,L.
Elias,I.
Guterman,D.
Harvey,P.
G.
Isaac,A.
M.
Khan,T.
R.
Larson,Y.
Li,T.
Pawson,T.
Penfield,A.
M.
Rae,D.
A.
Rathbone,S.
Reid,J.
Ross,M.
F.
Smallwood,V.
Segura,T.
Townsend,D.
Vyas,T.
WinzerandD.
Bowles,Science,2010,327,328-331.
12.
T.
Townsend,V.
Segura,G.
Chigeza,T.
Penfield,A.
Rae,D.
Harvey,D.
BowlesandI.
A.
Graham,PloSone,2013,8,e61989.
13.
V.
Hale,J.
D.
Keasling,N.
RenningerandT.
T.
Diagana,AmJTropMedHyg,2007,77,198-202.
14.
A.
Ounaroon,G.
Decker,J.
Schmidt,F.
LottspeichandT.
M.
Kutchan,PlantJ.
,2003,36,808-819.
15.
J.
M.
GennityandP.
K.
Stumpf,ArchBiochemBiophys,1985,239,444-454.
16.
M.
Bafor,M.
A.
Smith,L.
Jonsson,K.
StobartandS.
Stymne,Biochem.
J.
,1991,280,507-514.
17.
M.
Lee,M.
Lenman,A.
Banas,M.
Bafor,S.
Singh,M.
Schweizer,R.
Nilsson,C.
Liljenberg,A.
Dahlqvist,P.
O.
Gummeson,S.
Sjodahl,A.
GreenandS.
Stymne,Science,1998,280,915-918.
18.
M.
Bafor,M.
A.
Smith,L.
Jonsson,K.
StobartandS.
Stymne,Biochem.
Biophys.
,1993,303,145-151.
19.
E.
B.
Cahoon,K.
G.
Ripp,S.
E.
HallandA.
J.
Kinney,J.
Biol.
Chem.
,2001,276,2637-2643.
20.
J.
M.
Dyer,D.
C.
Chapital,J.
C.
Kuan,R.
T.
Mullen,C.
Turner,T.
A.
McKeonandA.
B.
Pepperman,PlantPhysiol.
,2002,130,2027-2038.
21.
P.
Sperling,M.
Lee,T.
Girke,U.
Zahringer,S.
StymneandE.
Heinz,Eur.
J.
Biochem.
,2000,267,3801-3811.
22.
T.
Cakir,K.
R.
Patil,Z.
Onsan,K.
O.
Ulgen,B.
KirdarandJ.
Nielsen,Molecularsystemsbiology,2006,2,50.
23.
D.
B.
Murray,M.
BeckmannandH.
Kitano,Proc.
Natl.
Acad.
Sci.
USA,2007,104,2241-2246.
24.
M.
T.
Kresnowati,W.
A.
vanWinden,M.
J.
Almering,A.
tenPierick,C.
Ras,T.
A.
Knijnenburg,P.
Daran-Lapujade,J.
T.
Pronk,J.
J.
HeijnenandJ.
M.
Daran,Molecularsystemsbiology,2006,2,49.
25.
M.
A.
Smith,L.
Jonsson,S.
StymneandK.
Stobart,Biochem.
J.
,1992,287,141-144.
26.
N.
Amiour,S.
Imbaud,G.
Clement,N.
Agier,M.
Zivy,B.
Valot,T.
Balliau,P.
Armengaud,I.
Quillere,R.
Canas,T.
Tercet-LaforgueandB.
Hirel,J.
Exp.
Bot.
,2012,63,5017-5033.
27.
T.
T.
DangandP.
J.
Facchini,PlantPhysiol.
,2012,159,618-631.
28.
A.
Jauhiainen,O.
Nerman,G.
MichailidisandR.
Jornsten,Biostatistics,2012,13,748-761.
29.
S.
Osorio,R.
Alba,Z.
Nikoloski,A.
Kochevenko,A.
R.
FernieandJ.
J.
Giovannoni,PlantPhysiol.
,2012,159,1713-1729.
30.
S.
P.
Singh,X.
R.
Zhou,Q.
Liu,S.
StymneandA.
G.
Green,Curr.
Opin.
PlantBiol.
,2005,8,197-203.
31.
Y.
Gibon,B.
Usadel,O.
E.
Blaesing,B.
Kamlage,M.
Hoehne,R.
TretheweyandM.
Stitt,Genomebiology,2006,7,R76.
32.
P.
Bais,S.
M.
Moon,K.
He,R.
Leitao,K.
Dreher,T.
Walk,Y.
Sucaet,L.
Barkan,G.
Wohlgemuth,M.
R.
Roth,E.
S.
Wurtele,P.
Dixon,O.
Fiehn,B.
M.
Lange,V.
Shulaev,L.
W.
Sumner,R.
Welti,B.
J.
Nikolau,S.
Y.
RheeandJ.
A.
Dickerson,PlantPhysiol.
,2010,152,1807-1816.
33.
S.
M.
Quanbeck,L.
Brachova,A.
A.
Campbell,X.
Guan,A.
Perera,K.
He,S.
Y.
Rhee,P.
Bais,J.
A.
Dickerson,P.
Dixon,G.
Wohlgemuth,O.
Fiehn,L.
Barkan,I.
Lange,B.
M.
Lange,I.
Lee,D.
Cortes,C.
Salazar,J.
Shuman,V.
Shulaev,D.
V.
Huhman,L.
W.
Sumner,M.
R.
Roth,R.
Welti,H.
Ilarslan,E.
S.
WurteleandB.
J.
Nikolau,Front.
PlantSci.
,2012,3,15.
34.
D.
W.
J.
James,E.
Lim,J.
Keller,I.
Plooy,E.
RalstonandH.
K.
Dooner,PlantCell,1995,7,309-319.
35.
C.
S.
Oh,D.
A.
Toke,S.
MandalaandC.
E.
Martin,J.
Biol.
Chem.
,1997,272,17376-17384.
36.
M.
N.
Ngaki,G.
V.
Louie,R.
N.
Philippe,G.
Manning,F.
Pojer,M.
E.
Bowman,L.
Li,E.
Larsen,E.
S.
WurteleandJ.
P.
Noel,Nature,2012,485,530-533.
37.
H.
Goda,E.
Sasaki,K.
Akiyama,A.
Maruyama-Nakashita,K.
Nakabayashi,W.
Li,M.
Ogawa,Y.
Yamauchi,J.
Preston,K.
Aoki,T.
Kiba,S.
Takatsuto,S.
Fujioka,T.
Asami,T.
Nakano,H.
Page11of15NaturalProductReportsARTICLEJournalName12|J.
Name.
,2012,00,1-3ThisjournalisTheRoyalSocietyofChemistry2012Kato,T.
Mizuno,H.
Sakakibara,S.
Yamaguchi,E.
Nambara,Y.
Kamiya,H.
Takahashi,M.
Y.
Hirai,T.
Sakurai,K.
Shinozaki,K.
Saito,S.
YoshidaandY.
Shimada,PlantJ.
,2008,55,526-542.
38.
E.
S.
Wurtele,J.
Chappell,A.
D.
Jones,M.
D.
Celiz,N.
Ransom,M.
Hur,L.
Rizshsky,P.
Dixon,J.
Liu,M.
P.
WidrlechnerandB.
J.
Nikolau,Metabolites,2012,InPress.
39.
M.
C.
CrispinandE.
S.
Wurtele,inBiotechnologyforMedicinalPlants,eds.
S.
Chandra,H.
LataandA.
Varma,SpringerBerlinHeidelberg,,2013,pp.
395-411.
40.
H.
Goda,E.
Sasaki,K.
Akiyama,A.
Maruyama-Nakashita,K.
Nakabayashi,W.
Li,M.
Ogawa,Y.
Yamauchi,J.
Preston,K.
Aoki,T.
Kiba,S.
Takatsuto,S.
Fujioka,T.
Asami,T.
Nakano,H.
Kato,T.
Mizuno,H.
Sakakibara,S.
Yamaguchi,E.
Nambara,Y.
Kamiya,H.
Takahashi,M.
Y.
Hirai,T.
Sakurai,K.
Shinozaki,K.
Saito,S.
YoshidaandY.
Shimada,ThePlantjournal:forcellandmolecularbiology,2008,55,526-542.
41.
H.
Jenkins,N.
Hardy,M.
Beckmann,J.
Draper,A.
R.
Smith,J.
Taylor,O.
Fiehn,R.
Goodacre,R.
J.
Bino,R.
Hall,J.
Kopka,G.
A.
Lane,B.
M.
Lange,J.
R.
Liu,P.
Mendes,B.
J.
Nikolau,S.
G.
Oliver,N.
W.
Paton,S.
Rhee,U.
Roessner-Tunali,K.
Saito,J.
Smedsgaard,L.
W.
Sumner,T.
Wang,S.
Walsh,E.
S.
WurteleandD.
B.
Kell,Nat.
Biotechnol.
,2004,22,1601-1606.
42.
E.
Wurtele,J.
Chappell,A.
Jones,M.
Celiz,N.
Ransom,M.
Hur,L.
Rizshsky,M.
Crispin,P.
Dixon,J.
Liu,M.
P.
WidrlechnerandB.
Nikolau,Metabolites,2012,2,1031-1059.
43.
M.
Hur,A.
A.
Campbell,M.
Almeida-de-Macedo,L.
Li,N.
Ransom,A.
Jose,M.
Crispin,B.
J.
NikolauandE.
S.
Wurtele,Naturalproductreports,2013,30,565-583.
44.
L.
A.
Giddings,D.
K.
Liscombe,J.
P.
Hamilton,K.
L.
Childs,D.
DellaPenna,C.
R.
BuellandS.
E.
O'Connor,J.
Biol.
Chem.
,2011,286,16751-16757.
45.
Y.
S.
Yeo,S.
E.
Nybo,A.
G.
Chittiboyina,A.
D.
Weerasooriya,Y.
H.
Wang,E.
Góngora‐Castillo,B.
Vaillancourt,C.
R.
Buell,D.
DellaPenna,M.
D.
Celiz,A.
D.
Jones,E.
S.
Wurtele,N.
Ransom,N.
Dudareva,K.
A.
Shabaan,N.
Tibrewal,S.
Chandra,T.
Smillie,I.
A.
Khan,R.
M.
Coates,D.
S.
WattandJ.
Chappell,J.
Biol.
Chem.
,2012,InReview.
46.
S.
Takahashi,Y.
Yeo,B.
T.
Greenhagen,T.
McMullin,L.
Song,J.
Maurina-Brunker,R.
Rosson,J.
P.
NoelandJ.
Chappell,Biotechnologyandbioengineering,2007,97,170-181.
47.
M.
CrispinandE.
Wurtele,inBiotechnologyforMedicinalPlants,eds.
S.
Chandra,H.
LataandA.
Varma,SpringerBerlinHeidelberg,2013,pp.
395-411.
48.
M.
C.
Crispin,M.
Hur,T.
Park,Y.
H.
KimandE.
S.
Wurtele,Physiologiaplantarum,2013,148,354-370.
49.
K.
Saito,Currentopinioninplantbiology,2013,16,373-380.
50.
A.
R.
Fernie,Phytochemistry,2007,68,2861-2880.
51.
K.
SaitoandF.
Matsuda,AnnuRevPlantBiol,2010,61,463-489.
52.
K.
Yonekura-SakakibaraandK.
Saito,Naturalproductreports,2009,26,1466-1487.
53.
K.
Saito,K.
Yonekura-Sakakibara,R.
Nakabayashi,Y.
Higashi,M.
Yamazaki,T.
TohgeandA.
R.
Fernie,PlantPhysiologyandBiochemistry,2013,72,21-34.
54.
B.
Winkel-Shirley,inAdvancesinBotanicalResearch,2002,pp.
75-94.
55.
L.
Lepiniec,I.
Debeaujon,J.
-M.
Routaboul,A.
Baudry,L.
Pourcel,N.
NesiandM.
Caboche,AnnualReviewofPlantBiology,2006,57,405-430.
56.
P.
Jones,B.
Messner,J.
Nakajima,A.
R.
SchaffnerandK.
Saito,TheJournalofbiologicalchemistry,2003,278,43910-43918.
57.
T.
Tohge,Yonekura-Sakakibara,K.
,Niida,R.
,Watanabe-Takahashi,A.
,Saito,K.
,PureApplChem,2007,79,811-823.
58.
T.
Tohge,Y.
Nishiyama,M.
Y.
Hirai,M.
Yano,J.
Nakajima,M.
Awazuhara,E.
Inoue,H.
Takahashi,D.
B.
Goodenowe,M.
Kitayama,M.
Noji,M.
YamazakiandK.
Saito,ThePlantjournal:forcellandmolecularbiology,2005,42,218-235.
59.
K.
Yonekura-Sakakibara,A.
Fukushima,R.
Nakabayashi,K.
Hanada,F.
Matsuda,S.
Sugawara,E.
Inoue,T.
Kuromori,T.
Itoandetal.
,PlantJournal,2012,69,154-167.
60.
K.
Yonekura-Sakakibara,T.
Tohge,F.
Matsuda,R.
Nakabayashi,H.
Takayama,R.
Niida,A.
Watanabe-Takahashi,E.
InoueandK.
Saito,PlantCell,2008,20,2160-2176.
61.
K.
Yonekura-Sakakibara,T.
Tohge,R.
NiidaandK.
Saito,TheJournalofbiologicalchemistry,2007,282,14932-14941.
62.
K.
Yonekura-Sakakibara,R.
Nakabayashi,S.
Sugawara,T.
Tohge,T.
Ito,M.
Koyanagi,M.
Kitajima,H.
TakayamaandK.
Saito,ThePlantjournal:forcellandmolecularbiology,2014,79,769-782.
63.
J.
Luo,Y.
Nishiyama,C.
Fuell,G.
Taguchi,K.
Elliott,L.
Hill,Y.
Tanaka,M.
Kitayama,M.
Yamazaki,P.
Bailey,A.
Parr,A.
J.
Michael,K.
SaitoandC.
Martin,ThePlantjournal:forcellandmolecularbiology,2007,50,678-695.
64.
J.
C.
D'Auria,M.
Reichelt,K.
Luck,A.
SvatosandJ.
Gershenzon,FEBSLetters,2007,581,872-878.
65.
I.
Muzac,J.
Wang,D.
Anzellotti,H.
ZhangandR.
K.
Ibrahim,Archivesofbiochemistryandbiophysics,2000,375,385-388.
66.
F.
Matsuda,M.
Y.
Hirai,E.
Sasaki,K.
Akiyama,K.
Yonekura-Sakakibara,N.
J.
Provart,T.
Sakurai,Y.
ShimadaandK.
Saito,Plantphysiology,2010,152,566-578.
67.
F.
Matsuda,R.
Nakabayashi,Y.
Sawada,M.
Suzuki,M.
Y.
Hirai,S.
KanayaandK.
Saito,Frontiersinplantscience,2011,2,40.
68.
A.
Fukushima,M.
Kusano,R.
F.
Mejia,M.
Iwasa,M.
Kobayashi,N.
Hayashi,A.
Watanabe-Takahashi,T.
Narisawa,T.
Tohge,M.
Hur,E.
S.
Wurtele,B.
J.
NikolauandK.
Saito,Plantphysiology,2014,165,948-961.
69.
F.
Matsuda,Y.
Okazaki,A.
Oikawa,M.
Kusano,R.
Nakabayashi,J.
Kikuchi,J.
-I.
Yonemaru,K.
Ebana,M.
Yanoandetal.
,PlantJournal,2012,70,624-636.
70.
L.
Gong,W.
Chen,Y.
Gao,X.
Liu,H.
Zhang,C.
Xu,S.
Yu,Q.
ZhangandJ.
Luo,ProceedingsoftheNationalAcademyofSciences,2013.
71.
M.
Brazier-Hicks,K.
M.
Evans,M.
C.
Gershater,H.
Puschmann,P.
G.
SteelandR.
Edwards,JournalofBiologicalChemistry,2009,284,17926-17934.
72.
R.
A.
Dixon,D.
-Y.
XieandS.
B.
Sharma,NewPhytologist,2005,165,9-28.
73.
D.
-Y.
Xie,S.
B.
Sharma,N.
L.
Paiva,D.
FerreiraandR.
A.
Dixon,Science,2003,299,396-399.
74.
Y.
Pang,X.
Cheng,D.
Huhman,J.
Ma,G.
Peel,K.
Yonekura-Sakakibara,K.
Saito,G.
Shen,L.
Sumner,Y.
Tang,J.
Wen,J.
YunandR.
Dixon,Planta,2013,238,139-154.
75.
S.
Kitamura,F.
Matsuda,T.
Tohge,K.
Yonekura-Sakakibara,M.
Yamazaki,K.
SaitoandI.
Narumi,ThePlantjournal:forcellandmolecularbiology,2010,62,549-559.
76.
L.
Pourcel,J.
-M.
Routaboul,L.
Kerhoas,M.
Caboche,L.
LepiniecandI.
Debeaujon,ThePlantCellOnline,2005,17,2966-2980.
77.
J.
Zhao,Y.
PangandR.
A.
Dixon,Plantphysiology,2010,153,437-443.
78.
M.
Y.
Hirai,M.
Yano,D.
B.
Goodenowe,S.
Kanaya,T.
Kimura,M.
Awazuhara,M.
Arita,T.
FujiwaraandK.
Saito,ProcNatlAcadSciUSA,2004,101,10205-10210.
79.
M.
Y.
Hirai,M.
Klein,Y.
Fujikawa,M.
Yano,D.
B.
Goodenowe,Y.
Yamazaki,S.
Kanaya,Y.
Nakamura,M.
Kitayamaandetal.
,JournalofBiologicalChemistry,2005,280,25590-25595.
80.
M.
Y.
Hirai,K.
Sugiyama,Y.
Sawada,T.
Tohge,T.
Obayashi,A.
Suzuki,R.
Araki,N.
Sakurai,H.
Suzuki,K.
Aoki,H.
Goda,O.
I.
Nishizawa,D.
ShibataandK.
Saito,ProcNatlAcadSciUSA,2007,104,6478-6483.
81.
T.
Obayashi,K.
Kinoshita,K.
Nakai,M.
Shibaoka,S.
Hayashi,M.
Saeki,D.
Shibata,K.
SaitoandH.
Ohta,NucleicAcidsResearch,2007,35,D863-D869.
82.
E.
K.
F.
Chan,H.
C.
Rowe,J.
A.
Corwin,B.
JosephandD.
J.
Kliebenstein,PLoSBiol,2011,9,e1001125.
83.
D.
J.
Kliebenstein,J.
Kroymann,P.
Brown,A.
Figuth,D.
Pedersen,J.
GershenzonandT.
Mitchell-Olds,Plantphysiology,2001,126,811-825.
84.
B.
G.
Hansen,D.
J.
KliebensteinandB.
A.
Halkier,ThePlantJournal,2007,50,902-910.
85.
F.
Geu-Flores,M.
E.
Mldrup,C.
Bttcher,C.
E.
Olsen,D.
ScheelandB.
A.
Halkier,PlantCell,2011,23,2456-2469.
86.
J.
M.
Augustin,V.
Kuzina,S.
B.
AndersenandS.
Bak,Phytochemistry,2011,72,435-457.
87.
S.
SawaiandK.
Saito,Frontiersinplantscience,2011,2.
88.
A.
Gholami,N.
DeGeyter,J.
Pollier,S.
GoormachtigandA.
Goossens,Naturalproductreports,2014,31,356-380.
Page12of15NaturalProductReportsJournalNameARTICLEThisjournalisTheRoyalSocietyofChemistry2012J.
Name.
,2012,00,1-3|1389.
H.
Seki,K.
Ohyama,S.
Sawai,M.
Mizutani,T.
Ohnishi,H.
Sudo,T.
Akashi,T.
Aoki,K.
Saitoandetal.
,ProceedingsoftheNationalAcademyofSciencesoftheUnitedStatesofAmerica,2008,105,14204-14209.
90.
H.
Seki,S.
Sawai,K.
Ohyama,M.
Mizutani,T.
Ohnishi,H.
Sudo,E.
O.
Fukushima,T.
Akashi,T.
Aoki,K.
SaitoandT.
Muranaka,PlantCell,2011,23,4112-4123.
91.
M.
Carelli,E.
Biazzi,F.
Panara,A.
Tava,L.
Scaramelli,A.
Porceddu,N.
Graham,M.
Odoardi,E.
Piano,S.
Arcioni,S.
May,C.
ScottiandO.
Calderini,ThePlantCellOnline,2011,23,3070-3081.
92.
E.
O.
Fukushima,H.
Seki,K.
Ohyama,E.
Ono,N.
Umemoto,M.
Mizutani,K.
SaitoandT.
Muranaka,PlantandCellPhysiology,2011,52,2050-2061.
93.
M.
A.
Naoumkina,L.
V.
Modolo,D.
V.
Huhman,E.
Urbanczyk-Wochniak,Y.
Tang,L.
W.
SumnerandR.
A.
Dixon,PlantCell,2010,22,850-866.
94.
J.
M.
Augustin,S.
Drok,T.
Shinoda,K.
Sanmiya,J.
K.
Nielsen,B.
Khakimov,C.
E.
Olsen,E.
H.
Hansen,V.
Kuzina,C.
T.
Ekstrm,T.
HauserandS.
Bak,Plantphysiology,2012,160,1881-1895.
95.
T.
Sayama,E.
Ono,K.
Takagi,Y.
Takada,M.
Horikawa,Y.
Nakamoto,A.
Hirose,H.
Sasama,M.
Ohashi,H.
Hasegawa,T.
Terakawa,A.
Kikuchi,S.
Kato,N.
Tatsuzaki,C.
TsukamotoandM.
Ishimoto,PlantCell,2012,24,2123-2138.
96.
A.
Osbourn,Plantphysiology,2010,154,531-535.
97.
S.
Boycheva,L.
Daviet,J.
L.
WolfenderandT.
B.
Fitzpatrick,Trendsinplantscience,2014.
98.
X.
Qi,S.
Bakht,M.
Leggett,C.
Maxwell,R.
MeltonandA.
Osbourn,ProceedingsoftheNationalAcademyofSciencesoftheUnitedStatesofAmerica,2004,101,8233-8238.
99.
B.
FieldandA.
E.
Osbourn,Science,2008,320,543-547.
100.
B.
Field,A.
-S.
Fiston-Lavier,A.
Kemen,K.
Geisler,H.
QuesnevilleandA.
E.
Osbourn,ProceedingsoftheNationalAcademyofSciences,2011,108,16116-16121.
101.
M.
Itkin,U.
Heinig,O.
Tzfadia,A.
J.
Bhide,B.
Shinde,P.
D.
Cardenas,S.
E.
Bocobza,T.
Unger,S.
Malitsky,R.
Finkers,Y.
Tikunov,A.
Bovy,Y.
Chikate,P.
Singh,I.
Rogachev,J.
Beekwilder,A.
P.
GiriandA.
Aharoni,Science,2013,341,175-179.
102.
S.
Sawai,K.
Ohyama,S.
Yasumoto,H.
Seki,T.
Sakuma,T.
Yamamoto,Y.
Takebayashi,M.
Kojima,H.
Sakakibara,T.
Aoki,T.
Muranaka,K.
SaitoandN.
Umemoto,PlantCell,2014.
103.
A.
Gesell,M.
Rolf,J.
Ziegler,M.
L.
DiazChavez,F.
C.
HuangandT.
M.
Kutchan,TheJournalofbiologicalchemistry,2009,284,24432-24442.
104.
J.
M.
HagelandP.
J.
Facchini,Naturechemicalbiology,2010,6,273-275.
105.
T.
Winzer,V.
Gazda,Z.
He,F.
Kaminski,M.
Kern,T.
R.
Larson,Y.
Li,F.
Meade,R.
Teodor,F.
E.
Vaistij,C.
Walker,T.
A.
BowserandI.
A.
Graham,Science,2012,336,1704-1708.
106.
F.
Geu-Flores,N.
H.
Sherden,V.
Courdavault,V.
Burlat,W.
S.
Glenn,C.
Wu,E.
Nims,Y.
CuiandS.
E.
O'Connor,Nature,2012,492,138-142.
107.
K.
Asada,V.
Salim,S.
Masada-Atsumi,E.
Edmunds,M.
Nagatoshi,K.
Terasaka,H.
MizukamiandV.
DeLuca,ThePlantCellOnline,2013,25,4123-4134.
108.
K.
Miettinen,L.
Dong,N.
Navrot,T.
Schneider,V.
Burlat,J.
Pollier,L.
Woittiez,S.
vanderKrol,R.
Lugan,T.
Ilc,R.
Verpoorte,K.
M.
Oksman-Caldentey,E.
Martinoia,H.
Bouwmeester,A.
Goossens,J.
MemelinkandD.
Werck-Reichhart,Naturecommunications,2014,5,3606.
109.
V.
Salim,B.
Wiens,S.
Masada-Atsumi,F.
YuandV.
DeLuca,Phytochemistry,2014,101,23-31.
110.
M.
Yamazaki,K.
Mochida,T.
Asano,R.
Nakabayashi,M.
Chiba,N.
Udomson,Y.
Yamazaki,D.
Goodenowe,U.
Sankawa,T.
Yoshida,A.
Toyoda,Y.
Totoki,Y.
Sakaki,E.
Góngora-Castillo,C.
Buell,T.
SakuraiandK.
Saito,Plant&cellphysiology,2013,54,686-696.
111.
T.
Asano,K.
Kobayashi,E.
Kashihara,H.
Sudo,R.
Sasaki,Y.
Iijima,K.
Aoki,D.
Shibata,K.
SaitoandM.
Yamazaki,Phytochemistry,2013,91,128-139.
112.
R.
NakabayashiandK.
Saito,AnalyticalandBioanalyticalChemistry,2013,405,5005-5011.
113.
S.
Bunsupa,M.
YamazakiandK.
Saito,Frontiersinplantscience,2012,3,239.
114.
S.
Bunsupa,K.
Katayama,E.
Ikeura,A.
Oikawa,K.
Toyooka,K.
SaitoandM.
Yamazaki,PlantCell,2012,24,1202-1216.
115.
R.
N.
Stewart,K.
H.
NorrisandS.
Asen,Phytochemistry,1975,14,937-942.
116.
G.
HrazdinaandG.
J.
Wagner,inTheBiochemistryofPlantPhenols,eds.
C.
F.
VanSumereandP.
J.
Lea,OxfordUniversityPress,Oxford,England,1985,pp.
119-133.
117.
A.
M.
ZobelandG.
Hrazdina,BiotechHistochem,1995,70,1-6.
118.
M.
E.
Galway,J.
D.
Masucci,A.
M.
Lloyd,V.
Walbot,R.
W.
DavisandJ.
W.
Schiefelbein,Developmentalbiology,1994,166,740-754.
119.
K.
Noda,B.
J.
Glover,P.
LinsteadandC.
Martin,Nature,1994,369,661-664.
120.
W.
H.
OutlawandS.
Zhang,Journalofexperimentalbotany,2001,52,605-614.
121.
W.
Wirtz,M.
StittandH.
W.
Heldt,Plantphysiology,1980,66,187-193.
122.
M.
Stitt,W.
WirtzandH.
W.
Heldt,Biochimicaetbiophysicaacta,1980,593,85-102.
123.
S.
Klie,S.
Krueger,L.
Krall,P.
Giavalisco,U.
I.
Flugge,L.
WillmitzerandD.
Steinhauser,Frontiersinplantscience,2011,2,55.
124.
J.
Kehr,Currentopinioninplantbiology,2003,6,617-621.
125.
M.
L.
Reyzer,Y.
S.
Hsieh,K.
Ng,W.
A.
KorfmacherandR.
M.
Caprioli,JournalofMassSpectrometry,2003,38,1081-1092.
126.
R.
L.
CaldwellandR.
M.
Caprioli,Molecular&CellularProteomics,2005,4,394-401.
127.
Y.
J.
Lee,D.
C.
Perdian,Z.
H.
Song,E.
S.
YeungandB.
J.
Nikolau,PlantJournal,2012,70,81-95.
128.
P.
Chaurand,M.
StoeckliandR.
Caprioli,AnalChem,1999,71,5263-5270.
129.
N.
Ishida,M.
KoizumiandH.
Kano,Ann.
Bot.
,2000,86,259-278.
130.
W.
Kockenberger,Trendsinplantscience,2001,6,286-292.
131.
L.
Borisjuk,H.
RolletschekandT.
Neuberger,PlantJournal,2012,70,129-146.
132.
H.
VanAsandJ.
vanDuynhoven,Journalofmagneticresonance(SanDiego,Calif.
:1997),2013,229,25-34.
133.
J.
S.
Fletcher,J.
C.
VickermanandN.
Winograd,Currentopinioninchemicalbiology,2011,15,733-740.
134.
E.
H.
SeeleyandR.
M.
Caprioli,AnalChem,2012,84,2105-2110.
135.
J.
S.
Fletcher,N.
P.
LockyerandJ.
C.
Vickerman,Massspectrometryreviews,2011,30,142-174.
136.
S.
Cha,H.
Zhang,H.
I.
Ilarslan,E.
S.
Wurtele,L.
Brachova,B.
J.
NikolauandE.
S.
Yeung,ThePlantjournal:forcellandmolecularbiology,2008,55,348-360.
137.
Y.
J.
Lee,D.
C.
Perdian,Z.
Song,E.
S.
YeungandB.
J.
Nikolau,ThePlantjournal:forcellandmolecularbiology,2012,70,81-95.
138.
M.
M.
Burrell,C.
J.
EarnshawandM.
R.
Clench,JournalofExperimentalBotany,2007,58,757-763.
139.
A.
Benninghoven,F.
G.
RudenauerandH.
W.
Werner,Secondaryionmassspectrometry:basicconcepts,instrumentalaspects,applicationsandtrends,JohnWiley&Sons,NewYork,1987.
140.
M.
PacholskiandN.
Winograd,Chemicalreviews,1999,99,2977-3006.
141.
D.
B.
Lazof,J.
G.
Goldsmith,T.
W.
RuftyandR.
W.
Linton,Plantphysiology,1996,112,1289-1300.
142.
R.
Metzner,H.
U.
Schneider,U.
BreuerandW.
H.
Schroeder,Plantphysiology,2008,147,1774-1787.
143.
K.
L.
Moore,M.
Schroder,Z.
Wu,B.
G.
H.
Martin,C.
R.
Hawes,S.
P.
McGrath,M.
J.
Hawkesford,J.
FengMa,F.
-J.
ZhaoandC.
R.
M.
Grovenor,Plantphysiology,2011,156,913-924.
144.
A.
Seyer,J.
Einhorn,A.
BrunelleandO.
Laprevote,AnalChem,2010,82,2326-2333.
145.
K.
L.
Moore,E.
Lombi,F.
J.
ZhaoandC.
R.
Grovenor,AnalBioanalChem,2012,402,3263-3273.
Page13of15NaturalProductReportsARTICLEJournalName14|J.
Name.
,2012,00,1-3ThisjournalisTheRoyalSocietyofChemistry2012146.
D.
Hlscher,R.
Shroff,K.
Knop,M.
Gottschaldt,A.
Crecelius,B.
Schneider,D.
G.
Heckel,U.
S.
SchubertandA.
Svato,ThePlantjournal:forcellandmolecularbiology,2009,60,907-918.
147.
S.
Cha,Z.
Song,B.
J.
NikolauandE.
S.
Yeung,AnalChem,2009,81,2991-3000.
148.
P.
Nemes,A.
A.
Barton,Y.
LiandA.
Vertes,AnalyticalChemistry,2008,80,4575-4582.
149.
P.
Nemes,A.
A.
BartonandA.
Vertes,AnalyticalChemistry,2009,81,6668-6675.
150.
P.
NemesandA.
Vertes,AnalyticalChemistry,2007,79,8098-8106.
151.
P.
Nemes,A.
S.
WoodsandA.
Vertes,Analyticalchemistry,2010,82,982-988.
152.
B.
Shrestha,P.
Nemes,J.
Nazarian,Y.
Hathout,E.
P.
HoffmanandA.
Vertes,TheAnalyst,2010,135,751-758.
153.
B.
Shrestha,P.
NemesandA.
Vertes,AppliedPhysicsA,2010,101,121-126.
154.
B.
Shrestha,J.
M.
PattandA.
Vertes,AnalyticalChemistry,2011,83,2947-2955.
155.
A.
Svatos,TrendsBiotechnol,2010,28,425-434.
156.
M.
Peukert,A.
Matros,G.
Lattanzio,S.
Kaspar,J.
AbadiaandH.
P.
Mock,TheNewphytologist,2012,193,806-815.
157.
T.
Mueller,S.
Oradu,D.
R.
Ifa,R.
G.
CooksandB.
Kraeutler,AnalyticalChemistry,2011,83,5754-5761.
158.
L.
W.
Sumner,D.
S.
Yang,B.
J.
Bench,B.
S.
Watson,C.
LiandA.
D.
Jones,AnnualPlantReviews,2011,43,343-366.
159.
D.
C.
PerdianandY.
J.
Lee,AnalChem,2010,82,9393-9400.
160.
A.
K.
Mullen,M.
R.
Clench,S.
CroslandandK.
R.
Sharples,Rapidcommunicationsinmassspectrometry:RCM,2005,19,2507-2516.
161.
S.
Robinson,K.
Warburton,M.
Seymour,M.
ClenchandJ.
Thomas-Oates,TheNewphytologist,2007,173,438-444.
162.
V.
Vrkoslav,A.
Muck,J.
CvackaandA.
Svatos,JournaloftheAmericanSocietyforMassSpectrometry,2010,21,220-231.
163.
P.
Franceschi,Y.
Dong,K.
Strupat,U.
VrhovsekandF.
Mattivi,JExpBot,2012,63,1123-1133.
164.
M.
Ha,J.
H.
Kwak,Y.
KimandO.
P.
Zee,FoodChemistry,2012,133,1155-1162.
165.
J.
Sarsby,M.
W.
Towers,C.
Stain,R.
CramerandO.
A.
Koroleva,Phytochemistry,2012,77,110-118.
166.
Y.
Yoshimura,H.
Enomoto,T.
Moriyama,Y.
Kawamura,M.
SetouandN.
Zaima,AnalBioanalChem,2012,403,1885-1895.
167.
Y.
Yoshimura,N.
Zaima,T.
MoriyamaandY.
Kawamura,PloSone,2012,7,e31285.
168.
H.
Ye,E.
Gemperline,M.
Venkateshwaran,R.
Chen,P.
M.
Delaux,M.
Howes-Podoll,J.
M.
AneandL.
Li,ThePlantjournal:forcellandmolecularbiology,2013,75,130-145.
169.
J.
H.
Jun,Z.
H.
Song,Z.
J.
Liu,B.
J.
Nikolau,E.
S.
YeungandY.
J.
Lee,AnalyticalChemistry,2010,82,3255-3265.
170.
S.
Cha,Z.
Song,B.
J.
NikolauandE.
S.
Yeung,AnalyticalChemistry,2009,81,2991-3000.
171.
C.
Sluszny,E.
S.
YeungandB.
J.
Nikolau,J.
Am.
Soc.
MassSpectrom.
,2005,16,107-115.
172.
Y.
J.
Lee,D.
C.
Perdian,Z.
Song,E.
S.
YeungandB.
J.
Nikolau,ThePlantjournal:forcellandmolecularbiology,2012,70,81-95.
173.
R.
G.
Cooks,Z.
Ouyang,Z.
TakatsandJ.
M.
Wiseman,Science,2006,311,1566-1570.
174.
A.
B.
CostaandR.
GrahamCooks,ChemicalPhysicsLetters,2008,464,1-8.
175.
I.
Cotte-Rodriguez,C.
C.
MulliganandR.
G.
Cooks,AnalChem,2007,79,7069-7077.
176.
Q.
Hu,N.
Talaty,R.
J.
NollandR.
G.
Cooks,Rapidcommunicationsinmassspectrometry,2006,20,3403-3408.
177.
R.
G.
Cooks,N.
E.
Manicke,A.
L.
Dill,D.
R.
Ifa,L.
S.
Eberlin,A.
B.
Costa,H.
Wang,G.
HuangandZ.
Ouyang,Faradaydiscussions,2011,149,247-267.
178.
A.
L.
Dill,D.
R.
Ifa,N.
E.
Manicke,A.
B.
Costa,J.
A.
Ramos-Vara,D.
W.
KnappandR.
G.
Cooks,Analyticalchemistry,2009,81,8758-8764.
179.
L.
S.
Eberlin,A.
L.
Dill,A.
B.
Costa,D.
R.
Ifa,L.
Cheng,T.
Masterson,M.
Koch,T.
L.
RatliffandR.
G.
Cooks,Analyticalchemistry,2010,82,3430-3434.
180.
S.
R.
Ellis,C.
Wu,J.
M.
Deeley,X.
Zhu,R.
J.
Truscott,R.
G.
Cooks,T.
W.
MitchellandS.
J.
Blanksby,JournaloftheAmericanSocietyforMassSpectrometry,2010,21,2095-2104.
181.
M.
Girod,Y.
Shi,J.
-X.
ChengandR.
G.
Cooks,Analyticalchemistry,2010,83,207-215.
182.
D.
R.
Ifa,J.
M.
Wiseman,Q.
SongandR.
G.
Cooks,InternationalJournalofMassSpectrometry,2007,259,8-15.
183.
N.
E.
Manicke,M.
Nefliu,C.
Wu,J.
W.
Woods,V.
Reiser,R.
C.
HendricksonandR.
G.
Cooks,Analyticalchemistry,2009,81,8702-8707.
184.
J.
M.
Wiseman,D.
R.
Ifa,Y.
Zhu,C.
B.
Kissinger,N.
E.
Manicke,P.
T.
KissingerandR.
G.
Cooks,ProceedingsoftheNationalAcademyofSciences,2008,105,18120-18125.
185.
A.
U.
Jackson,A.
Tata,C.
Wu,R.
H.
Perry,G.
Haas,L.
WestandR.
G.
Cooks,TheAnalyst,2009,134,867-874.
186.
N.
Talaty,Z.
TakatsandR.
G.
Cooks,TheAnalyst,2005,130,1624-1633.
187.
P.
J.
Roach,J.
LaskinandA.
Laskin,TheAnalyst,2010,135,2233-2236.
188.
A.
R.
Korte,Z.
H.
Song,B.
J.
NikolauandY.
J.
Lee,AnalyticalMethods,2012,4,474-481.
189.
M.
Koestler,D.
Kirsch,A.
Hester,A.
Leisner,S.
GuentherandB.
Spengler,Rapidcommunicationsinmassspectrometry:RCM,2008,22,3275-3285.
190.
A.
Rmpp,S.
Guenther,Y.
Schober,O.
Schulz,Z.
Takats,W.
KummerandB.
Spengler,Angewandtechemieinternationaledition,2010,49,3834-3838.
191.
P.
C.
Lauterbur,Nature,1973,242,190-191.
192.
M.
Rokitta,A.
Peuke,U.
ZimmermannandA.
Haase,Protoplasma,1999,209,126-131.
193.
T.
W.
J.
Scheenen,D.
vanDusschoten,P.
A.
deJagerandH.
VanAs,J.
Exp.
Bot.
,2000,51,1751-1759.
194.
T.
W.
J.
Scheenen,F.
J.
Vergeldt,A.
M.
HeemskerkandH.
VanAs,Plantphysiology,2007,144,1157-1165.
195.
H.
VanAs,JExpBot,2007,58,743-756.
196.
C.
W.
Windt,E.
GerkemaandH.
VanAs,Plantphysiology,2009,151,830-842.
197.
L.
Ciobanu,D.
A.
SeeberandC.
H.
Pennington,Journalofmagneticresonance(SanDiego,Calif.
:1997),2002,158,178-182.
198.
G.
Melkus,H.
Rolletschek,J.
Fuchs,V.
Radchuk,E.
Grafahrend-Belau,N.
Sreenivasulu,T.
Rutten,D.
Weier,N.
Heinzel,F.
Schreiber,T.
Altmann,P.
M.
JakobandL.
Borisjuk,Plantbiotechnologyjournal,2011,9,1022-1037.
199.
G.
Melkus,H.
Rolletschek,R.
Radchuk,J.
Fuchs,T.
Rutten,U.
Wobus,T.
Altmann,P.
JakobandL.
Borisjuk,Plantphysiology,2009,151,1139-1154.
200.
J.
Lambert,P.
Lampen,A.
vonBohlenandR.
Hergenroder,AnalBioanalChem,2006,384,231-236.
201.
A.
Rollins,J.
Barber,R.
ElliottandB.
Wood,Plantphysiology,1989,91,1243-1246.
202.
L.
Borisjuk,H.
RolletschekandT.
Neuberger,Progressinlipidresearch,2013,52,465-487.
203.
J.
Fuchs,T.
Neuberger,H.
Rolletschek,S.
Schiebold,T.
H.
Nguyen,N.
Borisjuk,A.
Borner,G.
Melkus,P.
JakobandL.
Borisjuk,Plantphysiology,2013,161,583-593.
204.
R.
J.
Bino,R.
D.
Hall,O.
Fiehn,J.
Kopka,K.
Saito,J.
Draper,B.
J.
Nikolau,P.
Mendes,U.
Roessner-Tunali,M.
H.
Beale,R.
N.
Trethewey,B.
M.
Lange,E.
S.
WurteleandL.
W.
Sumner,TrendsPlantSci.
,2004,9,418-425.
205.
R.
M.
Salek,C.
Steinbeck,M.
R.
Viant,R.
GoodacreandW.
B.
Dunn,GigaScience,2013,2,13.
206.
W.
B.
Dunn,A.
Erban,R.
J.
M.
Weber,D.
J.
Creek,M.
Brown,R.
Breitling,T.
Hankemeier,R.
Goodacre,S.
NeumannandJ.
Kopka,Metabolomics:OfficialjournaloftheMetabolomicSociety,2013,9,S44-S66.
207.
L.
Sumner,A.
Amberg,D.
Barrett,M.
Beale,R.
Beger,C.
Daykin,T.
Fan,O.
Fiehn,R.
Goodacre,J.
Griffin,T.
Hankemeier,N.
Hardy,J.
Harnly,R.
Higashi,J.
Kopka,A.
Lane,J.
Lindon,P.
Marriott,A.
Nicholls,M.
Reily,J.
ThadenandM.
Viant,Metabolomics,2007,3,211-221.
208.
J.
L.
Wolfender,E.
F.
QueirozandK.
Hostettmann,MagnResonChem,2005,43,697-709.
Page14of15NaturalProductReportsJournalNameARTICLEThisjournalisTheRoyalSocietyofChemistry2012J.
Name.
,2012,00,1-3|15209.
M.
Lambert,J.
L.
Wolfender,D.
Staerk,S.
B.
Christensen,K.
HostettmannandJ.
W.
Jaroszewski,AnalChem,2007,79,727-735.
210.
A.
Castro,S.
Moco,J.
CollandJ.
Vervoort,JNatProd,2010,73,962-965.
211.
J.
vanderHooft,V.
Mihaleva,R.
deVos,R.
BinoandJ.
Vervoort,MagnResonChem,2011,49Suppl1,S55-60.
212.
H.
Horai,M.
Arita,S.
Kanaya,Y.
Nihei,T.
Ikeda,K.
Suwa,Y.
Ojima,K.
Tanaka,S.
Tanaka,K.
Aoshima,Y.
Oda,Y.
Kakazu,M.
Kusano,T.
Tohge,F.
Matsuda,Y.
Sawada,M.
Y.
Hirai,H.
Nakanishi,K.
Ikeda,N.
Akimoto,T.
Maoka,H.
Takahashi,T.
Ara,N.
Sakurai,H.
Suzuki,D.
Shibata,S.
Neumann,T.
Iida,K.
Funatsu,F.
Matsuura,T.
Soga,R.
Taguchi,K.
SaitoandT.
Nishioka,Journalofmassspectrometry:JMS,2010,45,703-714.
213.
D.
S.
Wishart,T.
Jewison,A.
C.
Guo,M.
Wilson,C.
Knox,Y.
Liu,Y.
Djoumbou,R.
Mandal,F.
Aziat,E.
Dong,S.
Bouatra,I.
Sinelnikov,D.
Arndt,J.
Xia,P.
Liu,F.
Yallou,T.
Bjorndahl,R.
Perez-Pineiro,R.
Eisner,F.
Allen,V.
Neveu,R.
GreinerandA.
Scalbert,Nucleicacidsresearch,2013,41,D801-807.
214.
C.
A.
Smith,G.
O'Maille,E.
J.
Want,C.
Qin,S.
A.
Trauger,T.
R.
Brandon,D.
E.
Custodio,R.
AbagyanandG.
Siuzdak,TherDrugMonit,2005,27,747-751.
215.
R.
Tautenhahn,K.
Cho,W.
Uritboonthai,Z.
Zhu,G.
J.
PattiandG.
Siuzdak,Naturebiotechnology,2012,30,826-828.
216.
J.
Kopka,N.
Schauer,S.
Krueger,C.
Birkemeyer,B.
Usadel,E.
Bergmuller,P.
Dormann,W.
Weckwerth,Y.
Gibon,M.
Stitt,L.
Willmitzer,A.
R.
FernieandD.
Steinhauser,Bioinformatics(Oxford,England),2005,21,1635-1638.
217.
Y.
Sawada,R.
Nakabayashi,Y.
Yamada,M.
Suzuki,M.
Sato,A.
Sakata,K.
Akiyama,T.
Sakurai,F.
Matsuda,T.
Aoki,M.
Y.
HiraiandK.
Saito,Phytochemistry,2012,82,38-45.
218.
F.
Matsuda,K.
Yonekura-Sakakibara,R.
Niida,T.
Kuromori,K.
ShinozakiandK.
Saito,ThePlantjournal:forcellandmolecularbiology,2009,57,555-577.
219.
H.
Neuweger,S.
P.
Albaum,M.
Dondrup,M.
Persicke,T.
Watt,K.
Niehaus,J.
StoyeandA.
Goesmann,Bioinformatics(Oxford,England),2008,24,2726-2732.
220.
Q.
Cui,I.
A.
Lewis,A.
D.
Hegeman,M.
E.
Anderson,J.
Li,C.
F.
Schulte,W.
M.
Westler,H.
R.
Eghbalnia,M.
R.
SussmanandJ.
L.
Markley,NatBiotechnol,2008,26,162-164.
221.
E.
ChamparnaudandC.
Hopley,Rapidcommunicationsinmassspectrometry:RCM,2011,25,1001-1007.
222.
K.
Scheubert,F.
HufskyandS.
Bocker,JCheminform,2013,5,12.
223.
M.
GerlichandS.
Neumann,Journalofmassspectrometry:JMS,2013,48,291-298.
224.
S.
Wolf,S.
Schmidt,M.
Muller-HannemannandS.
Neumann,BMCbioinformatics,2010,11,148.
225.
M.
Heinonen,A.
Rantanen,T.
Mielikainen,J.
Kokkonen,J.
Kiuru,R.
A.
KetolaandJ.
Rousu,Rapidcommunicationsinmassspectrometry:RCM,2008,22,3043-3052.
226.
T.
Kind,K.
H.
Liu,Y.
Leedo,B.
DeFelice,J.
K.
MeissenandO.
Fiehn,NatMethods,2013,10,755-758.
227.
F.
Rasche,K.
Scheubert,F.
Hufsky,T.
Zichner,M.
Kai,A.
SvatosandS.
Bocker,Analyticalchemistry,2012,84,3417-3426.
228.
S.
E.
Stein,JournaloftheAmericanSocietyforMassSpectrometry,1995,6,644-655.
229.
M.
L.
Bandu,K.
R.
Watkins,M.
L.
Bretthauer,C.
A.
MooreandH.
Desaire,Analyticalchemistry,2004,76,1746-1753.
230.
K.
Klagkou,F.
Pullen,M.
Harrison,A.
Organ,A.
FirthandG.
J.
Langley,Rapidcommunicationsinmassspectrometry:RCM,2003,17,1163-1168.
231.
J.
A.
TaylorandR.
S.
Johnson,Rapidcommunicationsinmassspectrometry:RCM,1997,11,1067-1075.
232.
L.
J.
Kangas,T.
O.
Metz,G.
Isaac,B.
T.
Schrom,B.
Ginovska-Pangovska,L.
Wang,L.
Tan,R.
R.
LewisandJ.
H.
Miller,Bioinformatics(Oxford,England),2012,28,1705-1713.
233.
H.
Zhang,S.
SinghandV.
N.
Reinhold,Analyticalchemistry,2005,77,6263-6270.
234.
A.
Kameyama,S.
Nakaya,H.
Ito,N.
Kikuchi,T.
Angata,M.
Nakamura,H.
K.
IshidaandH.
Narimatsu,JProteomeRes,2006,5,808-814.
235.
R.
Wei,G.
LiandA.
B.
Seymour,Analyticalchemistry,2010,82,5527-5533.
236.
Y.
Sawada,K.
Akiyama,A.
Sakata,A.
Kuwahara,H.
Otsuki,T.
Sakurai,K.
SaitoandM.
Y.
Hirai,Plant&cellphysiology,2009,50,37-47.
237.
U.
Vrhovsek,D.
Masuero,M.
Gasperotti,P.
Franceschi,L.
Caputi,R.
ViolaandF.
Mattivi,Journalofagriculturalandfoodchemistry,2012,60,8831-8840.
238.
E.
Carvalho,P.
Franceschi,A.
Feller,L.
Palmieri,R.
WehrensandS.
Martens,PlantPhysiolBiochem,2013,72,79-86.
239.
A.
Schurmann,V.
Dvorak,C.
Cruzer,P.
ButcherandA.
Kaufmann,RapidCommunMassSpectrom,2009,23,1196-1200.
240.
D.
Hurtaud-Pessel,E.
Verdon,J.
BlotandP.
Sanders,FoodAdditContam,2006,23,569-578.
241.
B.
J.
Berendsen,L.
A.
StolkerandM.
W.
Nielen,JournaloftheAmericanSocietyforMassSpectrometry,2013,24,154-163.
242.
.
J.
Pozo,J.
V.
Sancho,M.
Ibáez,F.
HernándezandW.
M.
A.
Niessen,TrACTrendsinAnalyticalChemistry,2006,25,1030-1042.
243.
H.
Gao,O.
L.
Materne,D.
L.
HoweandC.
L.
Brummel,Rapidcommunicationsinmassspectrometry:RCM,2007,21,3683-3693.
244.
R.
G.
DickinsonandA.
L.
Raymond,J.
Amer.
Chem.
Soc.
,1923,45,22-29.
245.
J.
P.
Glusker,ProteinScience,1994,3,2465-2469.
246.
Y.
Inokuma,S.
Yoshioka,J.
Ariyoshi,T.
Arai,Y.
Hitora,K.
Takada,S.
Matsunaga,K.
RissanenandM.
Fujita,Nature,2013,495,461-466.
247.
Y.
Inokuma,S.
Yoshioka,J.
Ariyoshi,T.
AraiandM.
Fujita,Natureprotocols,2014,9,246-252.
248.
N.
Carreno-Quintero,H.
J.
BouwmeesterandJ.
J.
B.
Keurentjes,TrendsinGenetics,2013,29,41-50.
249.
L.
Gong,W.
Chen,Y.
Gao,X.
Liu,H.
Zhang,C.
Xu,S.
Yu,Q.
ZhangandJ.
Luo,ProceedingsoftheNationalAcademyofSciences,2013,110,20320-20325.
250.
H.
Redestig,M.
Kusano,K.
Ebana,M.
Kobayashi,A.
Oikawa,Y.
Okazaki,F.
Matsuda,M.
Arita,N.
Fujitaandetal.
,BMCSystemsBiology,2011,5,176251.
M.
N.
Calingacion,C.
Boualaphanh,V.
D.
Daygon,R.
Anacleto,R.
SackvilleHamilton,B.
Biais,C.
Deborde,M.
Maucourt,A.
Moing,R.
Mumm,R.
C.
H.
Vos,A.
Erban,J.
Kopka,T.
H.
Hansen,K.
H.
Laursen,J.
K.
Schjoerring,R.
D.
HallandM.
A.
Fitzgerald,Metabolomics:OfficialjournaloftheMetabolomicSociety,2012,8,771-783.
252.
F.
Matsuda,R.
Nakabayashi,Z.
Yang,Y.
Okazaki,J.
Yonemaru,K.
Ebana,M.
YanoandK.
Saito,PlantJournal,2014,accepted.
253.
Z.
Yang,R.
Nakabayashi,Y.
Okazaki,T.
Mori,S.
Takamatsu,S.
Kitanaka,J.
KikuchiandK.
Saito,Metabolomics:OfficialjournaloftheMetabolomicSociety,2013,1-13.
254.
J.
B.
Bowne,T.
A.
Erwin,J.
Juttner,T.
Schnurbusch,P.
Langridge,A.
BacicandU.
Roessner,MolecularPlant,2012,5,418-429.
255.
C.
B.
Hill,J.
D.
Taylor,J.
Edwards,D.
Mather,A.
Bacic,P.
LangridgeandU.
Roessner,Plantphysiology,2013,162,1266-1281.
256.
N.
Amiour,S.
Imbaud,G.
Clément,N.
Agier,M.
Zivy,B.
Valot,T.
Balliau,P.
Armengaud,I.
Quilleré,R.
Caas,T.
Tercet-LaforgueandB.
Hirel,JournalofExperimentalBotany,2012,63,5017-5033.
257.
P.
Casati,M.
Campi,D.
Morrow,J.
FernandesandV.
Walbot,BMCGenomics,2011,12,321.
258.
J.
Qiu,Nature,2007,448,126-128.
259.
M.
Wang,R.
-J.
A.
N.
Lamers,H.
A.
A.
J.
Korthout,J.
H.
J.
vanNesselrooij,R.
F.
Witkamp,R.
vanderHeijden,P.
J.
Voshol,L.
M.
Havekes,R.
VerpoorteandJ.
vanderGreef,PhytotherapyResearch,2005,19,173-182.
260.
A.
Zhang,H.
Sun,Z.
Wang,W.
Sun,P.
WangandX.
Wang,PlantaMed,2010,76,2026-2035.
261.
T.
Okadaande.
al.
,Submitted,2014.
Page15of15NaturalProductReports