Download Multiomics of tomato glandular trichomes reveals

Plant Cell Advance Publication. Published on April 13, 2017, doi:10.1105/tpc.17.00060
LARGE-SCALEBIOLOGY
MultiomicsofTomatoGlandularTrichomesRevealsDistinctFeaturesof
CentralCarbonMetabolismSupportingHighProductivityofSpecialized
Metabolites
GerdU.Balcke1,StefanBennewitz1,NickBergau1,BenediktAthmer1,AnjaHenning1,Petra
Majovsky2,3,JoséM.Jiménez-Gómez4,5,WolfgangHoehenwarter2,AlainTissier1
1
LeibnizInstituteofPlantBiochemistry,Dept.ofCellandMetabolicBiology,Weinberg3,D-06120
Halle(Saale),Germany,
2
LeibnizInstituteofPlantBiochemistry,ProteomeAnalytics,Weinberg3,D-06120Halle/S.,Germany.
3
Currentaddress:CelerionSwitzerlandAG,Allmendstrasse32,8320Fehraltorf,Switzerland.
4
MaxPlanckInstituteforPlantBreedingResearch,Carl-von-Linné-Weg10,50829Cologne,Germany.
5
Currentaddress:InstitutJean-PierreBourgin,INRA,AgroParisTech,CNRS,UniversitéParis-Saclay,
RD10,78026VersaillesCedex,France.
Correspondingauthor:AlainTissier([email protected])
Shorttitle:Metabolicnetworkoftomatoglandulartrichomes
One-sentencesummary:Multi-omicsandstableisotopelabelingappliedtotomatoglandular
trichomesrevealuniquefeaturesforthesupplyofenergy,reducingpowerandcarboninthese
metaboliccellfactories.
Abstract
Glandulartrichomesaremetaboliccellfactorieswiththecapacitytoproducelargequantitiesof
secondarymetabolites.Littleisknownabouttheconnectionbetweencentralcarbonmetabolism
andmetabolicproductivityforsecondarymetabolitesinglandulartrichomes.Toaddressthisgapin
ourknowledge,weperformedcomparativemetabolomics,transcriptomics,proteomicsand13ClabelingoftypeVIglandulartrichomesandleavesfromacultivated(SolanumlycopersicumLA4024)
andawild(SolanumhabrochaitesLA1777)tomatoaccession.Specificfeaturesofglandulartrichomes
thatdrivetheformationofsecondarymetabolitescouldbeidentified.TomatotypeVItrichomesare
photosyntheticbutacquiretheircarbonessentiallyfromleafsucrose.Theenergyandreducing
powerfromphotosynthesisareusedtosupportthebiosynthesisofsecondarymetabolites,whilethe
comparativelyreducedCalvin-Benson-Basshamcycleactivitymaybeinvolvedinrecyclingmetabolic
CO2.Glandulartrichomescopewithoxidativestressbyproducinghighlevelsofpolyunsaturatedfatty
acids,oxylipins,andglutathione.Finally,distinctmechanismsarepresentinglandulartrichomesto
increasethesupplyofprecursorsfortheisoprenoidpathways.Particularly,thecitrate-malateshuttle
suppliescytosolicacetylCoAandplastidicglycolysisandmalicenzymesupporttheformationof
plastidicpyruvate.Amodelisproposedonhowglandulartrichomesachievehighmetabolic
productivity.
1
©2017 American Society of Plant Biologists. All Rights Reserved
Introduction
Glandulartrichomes(GTs)arespecializedsecretorycellsthatprotrudefromtheepidermisof
approximately30%ofallvascularplants(Fahn,2000).ThemorphologyofGTsisverydiverse
acrossplantspeciesasexemplifiedbythepeltatetrichomesoftheLamiaceae,thebiseriate
trichomes of the Asteraceae or the capitate trichomes of the Solanaceae. But they are all
multicellularandshareabasicplanwithbasal,stalk,andonetoafew(typically4–8)glandular
headcells(Croteauetal.,2005;Glasetal.,2012).Theglandularheadcellshaveincommon
thecapacitytoproducesubstancesofrelevancetothechemicalcommunicationoftheplant
with its environment, including short branched chain acyl sugars, flavonoids, phenolics,
alkaloids,andisoprenoids(Schilmilleretal.,2009;Schilmilleretal.,2010;Schmidtetal.,2011;
Kimetal.,2012;Brückneretal.,2014;Lietal.,2014).Someofthesesecondarymetabolites
wereshowntohaveantifeedant,ovipositiondeterrent,andinsomecasestoxicproperties
towardsinsects(Coatesetal.,1988;Nonomuraetal.,2009;Bleekeretal.,2011;Bleekeret
al.,2012;Glasetal.,2012).Furthermore,secondarymetabolitesproducedinGTshavehigh
commercial value in the cosmetics, food, and pharmaceutical industries. For instance,
artemisinin,asesquiterpenelactoneofArtemisiaannua,anditsderivativesincombination
therapy are currently regarded as the most effective treatment against malaria (Kokwaro,
2009).
OnenotablefeatureofGTsistheirmetabolicproductivity.Forexample,acylsugars(AS)in
Solanumpennelliicanrepresentupto20%oftheleafdryweight(Fobesetal.,1985)andin
certainSolanumhabrochaitesaccessionssesquiterpenecarboxylicacidsmayreachmorethan
12%oftheleafdryweight(FrelichowskiandJuvik,2005).Thesefiguressupportthenotion
thatGTscanbeconsideredashighlyactivemetaboliccellfactoriesbecausethecompounds
are produced exclusively there. For volatile compounds, this high productivity is often
associated with dedicated storage features in the GT. For example, in tomato and many
Lamiaceae species, the secreted metabolites are stored in an extracellular cavity whose
volumecanswelltoamultipleofthevolumeoftheglandularheadcells(Turneretal.,2000;
Bergauetal.,2015).
TodatethereisnoconceptualmodeltoexplainhowGTsachievesuchremarkablemetabolic
productivity.MoststudiesonGTsofarhavefocusedontheidentificationandcharacterization
ofenzymesdirectlyinvolvedintheformationofthemajortrichomesecondarymetabolites
2
(Croteauetal.,2005;Slocombeetal.,2008;Sallaudetal.,2009;Schmidtetal.,2011;Kimet
al.,2012;Schilmilleretal.,2012;Brückneretal.,2014),andnocomprehensivestudyhasbeen
conductedtolinkthedownstreambiosynthesisstepstocentralandenergymetabolismsin
these cells. Understanding how the core metabolic network in these specialized cells is
organizedtodeliversuchhighproductivitylevelswilloffernewopportunitiesinthebreeding
of plants with increased resistance to various aggressors and in metabolic engineering in
general.Tomato,duetoitsexcellentgeneticresourcesandextensivesequencedata,including
sequenced genomes from cultivated tomato and several closely related wild relatives, can
serveasagoodmodelforthestudyofthephysiologyanddevelopmentofGTs(Tissier,2012).
Forthisstudy,thecultivatedtomatoSolanumlycopersicumLA4024(hereafterreferredtoas
LA4024)andanaccessionfromawildrelative,S.habrochaitesLA1777(hereafterreferredto
asLA1777),werechosenasstudymaterial.ThemostabundantGTsinbothspeciesareoftype
VI,whichconferadistinctsecondarymetaboliteprofiletoeachspecies(Bergauetal.,2015).
InLA4024,themajormetabolitesproducedaretheflavonoidrutinandvariousmonoterpenes
(e.g.a-phellandrene),whereasLA1777producesmostlysesquiterpenecarboxylicacids(SCAs)
andacylsugars(McDowelletal.,2011;Glasetal.,2012).Ourrationalewastocomparethe
trichomesandtherespectivetrichome-freeleafinordertoidentifywhichfeaturesdistinguish
thesetwotissuesandtounderstandwhatmakesthetrichomesunique.Ourcomparisonis
basedonfoursetsofdata,respectivelyfromtranscriptomics,proteomics,metabolomics,and
13
C-labeling,whichwereprocessedbyuni-andmultivariateanalysesandusedtoaddresstwo
majorquestions:HowdoGTsgeneratetheenergyneededtoproducetheselargeamountsof
secondaryproductsandwhatarethecarbonsourcesthatfuelmetaboliteproduction?
Results
Morphology
Intomato,sevenclassesoftrichomeshavebeendescribed.Fourofthemareglandular,among
whichtypeI,IVandVIandVIIarepresentinLA1777,whiletypeI,VIandVIIoccurinLA4024
(McDowell et al., 2011; Glas et al., 2012). LA1777 is a wild tomato accession with roundshaped type VI trichomes as the dominant trichome type covering the leaf surface at high
density(Figure1).ThecultivatedtomatoLA4024iscloselyrelatedtoS.lycopersicumcv.Heinz
whosegenomewasrecentlypublished(Consortium,2012).Itsdominatingtrichometypeisa
3
four-leafclover-shapedtypeVI,whichoccursatalowerdensitythaninLA1777(Bergauetal.,
2015).
Inbothspecies,basedonmicroscopyanalysis,leaftrichomepreparationscontainedabout
80%typeVIglandularheadcellsand20%typeI/IVheadcellsinLA1777and20%typeIhead
cellsinLA4024,respectively.Furthermore,itwasnotpossibletofullyremovethestalkcells
ofbothtypesofglandularheads,whichaccountedfor37%ofallharvestedcells.IntypeVI
trichomes, four glandular head cells surround a storage cavity filled with secondary
metabolites,whichistypicallymuchlargerinthewildtomato(Bergauetal.,2015).Likeleaf
mesophyllcells,theglandularheadcellsoftypeVItrichomespossesstheirownchloroplasts
withintactthylakoidmembranes,suggestingtheyhavethecapacitytogeneratetheATPand
NADPHrequiredfortheassemblyofsecondarymetabolites.Notably,theouterenvelope(cell
wall+cuticle)oftypeVIGTcellsofbothtomatospeciesisslightlythickerthanthatofleaf
epidermalcells(about0.7µmversus0.5µm)and3–5timesthickerthanthedoublecellwall
betweenleafparenchymacells(Figure1).Thissuggeststhat,asinepidermalcells,gasand
waterexchangewiththeoutsidearelimited.
Metabolomics
Central metabolites are mostly polar. Therefore, to better understand the connection
betweenprimaryandsecondarymetabolisminglandulartrichomes,non-targetedprofiling
forpolarandsemi-polarmetaboliteswascarriedoutseparatelyusingtwoLC-MSmethods
and one GC-MS method (see Methods). Unless indicated, all metabolomics data discussed
belowshowedsignificantfoldchangesbasedonpairwiset-testingatasignificancelevelofp
<0.05.FordetailsseeSupplementalDatasets1–3.
Thetrichomesofwildandcultivatedtomatoproducedifferentsecondarymetabolites
As expected, metabolite profiling of secondary metabolites revealed significant differences
notonlybetweenbothspeciesbutalsobetweenthetrichomeandleafwithinaspecies(Figure
2, Supplemental Dataset 1). Venn diagrams show only 27% of the up- and 23% of the
downregulated mass features in trichomes versus leaves as shared patterns between both
tomato species (Supplemental Figure 1). Principal component analysis (PCA) of semi-polar
4
secondary metabolites measured by LC-MS separated both tomato lines on PC1 with 78%
coverageofvariance,indicatingverydifferentcompositioninthesecondarymetabolitesof
both species (Figure 2A). PC2 (15%) separated trichomes from leaves across the two lines,
demonstrating also joined latent similarities in the patterns of leaves and trichomes,
respectively.Here,signalsrelatingtotheglycoalkaloidstomatineanddehydrotomatine,which
areexclusivelyfoundintheleaves,stronglycontributedtothegroupseparation.Intrichomes,
the total ion chromatograms of LA4024 were dominated by monoterpenes (GC-MS) and
conjugated flavonols (mainly rutin, LC-MS), while sesquiterpenes (GC-MS), short branched
chain(C2-C12)acylsugars,andsesquiterpenecarboxylicacidsareprevalentinLC-MS-based
chromatogramsofLA1777(SupplementalFigure2).Theseresultsconfirmpreviousmetabolite
analysesoftomatotrichomesandunderlinethattrichomesofthesespeciesproducedifferent
terpenoidsecondarymetabolites(Slocombeetal.,2008;Besseretal.,2009;Schilmilleretal.,
2010;McDowelletal.,2011;Ekanayakaetal.,2014;Ghoshetal.,2014;Lietal.,2014).When
quantified,rutinalonecontributedto25±3%ofthecorrespondingtrichomedryweightin
LA4024,whereasthesumoftwomajorsesquiterpenecarboxylicacids((+)-(E)-α-santalene12-oic acid and (+)-(E)-endo-bergamotene-12-oic acid) added up to 23 ± 2% of the GT dry
weightinLA1777.BothvaluesillustratethecapabilityofGTtodirectmassivecarbonfluxes
towards distinct classes of secondary metabolites. To estimate the productivity of type VI
trichomes in the two species regarding terpenoids, we quantified the major sesquiterpene
carboxylicacids,i.e.,santalenoicandbergamotenoicacids,inLA1777andthemajorterpenes
(mostly monoterpenes and minor sesquiterpenes) in LA4024 in surface extracts of young
leafletsandinparallelestimatedthetrichomenumbersofleafletsofcomparablesize.We
found 6563 ± 618 (n=3) type VI trichomes per leaflet in LA1777 and 1804 ± 276 (n=5) in
LA4024.Thisgave65.82±7.85ng(n=3)ofSCAspertrichomeinLA1777versus0.68±0.20ng
(n=5)ofterpenespertrichomeinLA4024.Thus,theterpeneproductivityintypeVItrichomes
ofLA1777isaround97timeshigherthaninthoseofLA4024.
NewlyidentifiedmetabolitesincludeabundantoxylipinsderivedfromC18-andC20-PUFAs
Interestingly,wedetectedhighintensitiesoffreesaturated,mono-andpolyunsaturatedfatty
acids(PUFAs)thatwerefoundtobepreferentiallyenrichedinthetrichomesofbothtomatoes
(SupplementalFigure3A).Themostabundantwere18:2linoleicand18:3linolenicacidinboth
species and a C20:3-eicosatrienoic and C20:4-arachidonic acid in LA1777. The presence of
5
polyunsaturatedC20fattyacidsinLA1777isnoteworthy,sincethesearerareinplantsand
wereshowntomodulateplantresponsestostress(Savchenkoetal.,2010).Correspondingto
thechainlengthofthefattyacids,numerousoxylipinsthatstronglyaccumulatedinGTsas
comparedtothecorrespondingleaveswerealsodetected.Amongthese,dihydroxy-C18:1/
C18:2,hydroxy-C18:2/C18:3,trihydroxy-C18:3,hydroxy-C20:2/C20:3/C20:4anddihydroxy20:4/C20:5weremostabundant,withtheC20-oxylipinsagainaccumulatingpreferentiallyin
LA1777(SupplementalFigure3B).WecomparedLCretentiontimeandtheMS2spectraof
hydroxy-C18:2withauthenticstandardsoftheoxylipins13S-hydroxy-9Z,11E-octadecadienoic
acid (13(S)-HODE) and 9S-hydroxy-10E,12Z-octadecadienoic acid (9(S)-HODE). Both
substances with a precursor ion [M-H] of 295.227 amu co-eluted with a peak at 13.6 min,
which was also observed in GTs of both species. Yet, collision-induced dissociation (CID)spectraofthecompoundfromLA1777trichomesmatchedtheCID-spectraof(9(S)-HODE),
whereas those from LA4024 trichomes matched the spectra of 13(S)-HODE (Supplemental
Figure4).
Trichome-specificfeaturesofcentralcarbonandenergymetabolism
Tosurveythehydrophilicmetabolitesinvolvedincentralcarbonandenergymetabolism,we
compared the peak areas of 115 selected MS1 mass/ retention time features of known
identity.Thoseincludedintermediatesofthetricarboxylicacid(TCA)cycle,sugarphosphates,
freeaminoacids,redoxcouplesofglutathioneandNADcofactorsaswellasnucleotidesand
intermediatesofbothisoprenoidprecursorpathways.Vennanalysisshowedthatmorethan
athirdofallsignalsweresharedacrossbothtomatomodelsascommonfeaturesbetween
trichomesandleaves,respectively(SupplementalFigure1).
PCAidentifiedthreeprincipalcomponentswhichcover89.4%ofthevarianceinthedata.In
thiscase,PCAshowedastrongergroupseparationbetweentrichomesandleavesacrossthe
tomatolines(PC137.9%)thantheseparationbetweenbothtomatolinesonPC2(33.8%).This
indicates that, regardless of the tomato species, type VI-GTs have a highly distinct central
carbon and energy metabolism compared to leaves (Supplemental Figure 5). Also, cross
validationusingfiveoutofsixseriespertomatospeciestopredictthemissingdataseriesand
agoodpredictabilityofmetabolomedataofLA4024fromLA1777andviceversaunderline
6
this(SupplementalDataset4).Toidentifyfeaturesthatdistinguishtrichomesfromleavesas
theresponsevariables,partialleastsquareanalysis(PLS)wascarriedoutindividuallyforboth
species. This analysis showed a strong group separation based on Calvin-Benson-Bassham
(CBB)cycleintermediates,ADP-glucose,andglyceratewithlowerpeakintensitiesintrichome
samples as compared to leaves (e.g., ribulose-1,5-bisphosphate: 9.2-fold (LA1777), 8.6-fold
(LA4024); 3-phosphoglycerate: 5.3-fold (LA1777), 1.7-fold (LA4024); ADP-glucose: 10.6-fold
(LA1777), 8.6-fold (LA4024)) (Figure 3, Supplemental Dataset 3). Notably, the latter
metabolitesareinvolvedinphotosyntheticcarbonfixationandinstarchbiosynthesis,which
appeartobelessactiveintrichomesthaninleaves.Besidesthis,trichomesofLA1777and
LA4024accumulatedlargeamountsofinositolpolyphosphates(IP5andIP6)whichwerenot
detectedintheleaves(SupplementalDataset3).
Similarly,oxidizedglutathioneshowedincreasedlevelsinthetrichomesofbothspecies(i.e.,
in LA1777, GSSG: 1.2-fold (p=0.07); in LA4024, GSSG: 1.9-fold, Supplemental Dataset 3).
Althoughthereducedformofglutathionewas1.5-(LA1777)and3.7-fold(LA4024)higherin
theleaves,importantly,theratiobetweenoxidizedandreducedglutathioneisshiftedtomuch
highervaluesinGTsthaninleaves(inLA1777,Leaves:0.9,GTs:1.7;inLA4024,Leaves:0.4,
GT: 2.8). Thus, in trichomes a much larger fraction of the glutathione pool is oxidized.
Moreover, cystine levels were 4.6 and 5.3 times higher in GTs of LA1777 and LA4024,
respectively, implying increased replenishment of GSH in GTs. Ascorbate, another electron
scavenger,showeddecreasedlevelsintrichomesrelativetoleavesinLA1777(2.5-fold)and
wasstronglydepletedinthetrichomesofLA4024(Figure3,SupplementalDataset3).
TCAintermediatessuchasaconitate(ACT)andsuccinate(SUC)wereincreasedintrichomes
versusleaf(ACT:1.4-fold(LA1777),3.5-fold(LA4024);SUC:2.3-fold(LA4024)orshowedno
significantdifferencebetweentrichomeandleaf(LA1777).Finally,cyclicformsofAMPand
GMPshowedmuchhigherintensitiesinGTsthaninleaves(cAMP:4.0-fold,cGMP:3.5-fold
(LA1777);cAMP:9.2-fold,cGMP:18.4-fold(LA4024))(SupplementalDataset3).Interestingly,
the ATP levels in leaves and trichomes were comparable for both tomato species
(SupplementalDataset3).
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Intermediates of secondary metabolite precursor pathways are overrepresented in
trichomes
SinceGTsofLA4024andLA1777inparticularproduceterpenoidsfromboththemevalonate
(MEV) and the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathways, metabolites of these
pathways were measured, when possible. 3-hydroxy-3-methylglutaryl-coenzyme A (HMGCoA), an intermediate of the isoprenoid biosynthesis via the MEV pathway also showed
increased levels in the trichomes of both species (2.5-fold (LA1777) and 4.3-fold (LA4024))
versusleaves(SupplementalDataset3).InLA1777,intermediatesoftheMEPpathwayshowed
upto7-foldhigherlevelsintrichomesthaninleaves(SupplementalDataset3)andaremajor
contributorstothegroupseparationbetweenleavesandtrichomesofbothspecies(Figure
3A). Correspondingly, signals for cytidyl nucleotides, which are required for this pathway
(Caspietal.,2014),aresignificantlyupregulatedinthetrichomes(SupplementalDataset3).
Theseresultsareinaccordancewiththehighlevelsofsesquiterpenoidsthataresynthesized
intheplastidsofGTviatheMEPpathwayinLA1777(Besseretal.,2009;Sallaudetal.,2009;
Schilmilleretal.,2009).Bycontrast,metaboliclevelsoftheMEPpathwayintermediatesand
cytidylnucleotideswerenotormoderatelyincreasedinGTsofLA4024,withMEPshowinga
3.7-fold higher level than in the corresponding leaf as the metabolite with the largest
difference (Supplemental Dataset 3). Threonine and valine, which are involved in the
formationofbranchedshortchainacylresiduesofacylsugars(AS)weresignificantlyelevated
in the trichomes of LA1777 but not in LA4024, where AS were much less abundant
(Supplemental Dataset 3). In agreement with the increased lipid formation in GTs of both
species (see above) malonyl-CoA was strongly increased in GT (4.3-fold (LA1777), 2.5-fold
(LA4024))(SupplementalDataset3).
Together, the metabolite profiles of hydrophilic metabolites reflect that intermediates
requiredforthebiosynthesisoftrichome-specificsecondarymetabolitesarepresentathigh
levels,especiallyforthebiosynthesisofterpenoids.Inaddition,glandulartrichomesofLA1777
appearedtobemetabolicallymoreactivethanthoseofLA4024.
8
Transcriptomics,Proteomics,and13C-Labeling
Tocorrelateourmetabolomicsdatawithgeneexpression,weperformedtranscriptomicand
proteomic analysis on the same samples. The transcriptomics data were generated with a
custom-designedmicroarraybasedonRNA-Seqdatathatwereproducedforthisproject(see
Methods).
Wereportquantile-normalizedtranscriptomedataandproteomedataofbiologicaltriplicates
ofisolatedtrichomesandtrichome-freeleaves(SupplementalDataset5).Unlessindicated,all
datadiscussedbelowshowedsignificantfold-changesatasignificancelevelp<0.05.Aglobal
overviewoftherelativetranscriptandproteinlevelsintrichomesandtrichome-freeleaves
withregardstocellularmetabolismisprovidedinFigure4(transcriptomicsofLA1777)and
SupplementalFigures6and7(transcriptomicsandproteomicsofLA4024andproteomicsof
LA1777).Venndiagramsshowingcommonpatternsofleafandtrichome-specificomicsdata
across both tomato models (ca. 26–30%) as well as species-specific data are shown in
SupplementalFigure1.
GlandulartrichomesconstituteastrongsucrosesinkwithlowCalvin-Benson-BasshamCycle
(CBB)activitybuthighexpressionofphotosystemgenes
OneoutstandingquestionconcernstheoriginofthecarbonsupplytoGTs.Unlikemintpeltate
GTs, tomato GTs possess chloroplasts. This raises the question of whether carbon fixation
takesplaceinthesecellsand,ifso,towhatextentitcontributestothecarbonsupplyofthe
GTsoriftheydependonleavesastheirmaincarbonsupplier.Toanswerthesequestions,we
examinedourtranscriptomicsandproteomicsdata.First,analysisofthetranscriptomedata
of genes involved in sucrose degradation showed that several cell walls and cytosolic
invertasesaswellassucrosesynthasesweresignificantly(p<0.05witht-teststatistics)overrepresentedintrichomescomparedtoleaves(Figure5).Thisisparticularlyclearforsucrose
synthaseswherethecumulativeexpressionofthetwomajorisoformsis7.5and7.4times
higherinGTsinLA1777andLA4024,respectively.Conversely,thecumulatedexpressionof
invertaseinhibitorsis10.5-and19.3-foldstrongerinleavesversustrichomesofLA1777and
LA4024, respectively. Additionally, genes encoding sucrose symporters, which are typically
involvedinsucroseexport,areexpressedatlevelsthatare6.4and6.9timeshigherinleaves
than in trichomes of LA1777 and LA4024, respectively (Supplemental Dataset 5). These
9
expressionprofileswereconfirmedbyRT-qPCR(SupplementalTable1).Enrichmentanalysis
demonstrated overrepresentation of genes and proteins involved in plastidic and cytosolic
glycolysisinGTs,butthiseffectwassignificantonlyinLA1777(SupplementalDataset6).
Next,wefoundthattranscriptsencodingenzymesoftheCBBcyclewereamongthetop2000
highest expressed genes only in the leaf samples of both tomato species but not in their
trichomes (Figure 6A and B). Here, the difference in transcript levels between leaves and
trichomes was significant (Figure 7), with average fold-changes within this ontology group
being6.3(LA1777)and8.4(LA4024).Theseresultsareconsistentwiththeproteinabundances
obtainedfromshotgunproteomics(SupplementalDataset5),RT-qPCRofselectedCBBcycle
genes(SupplementalTable1),andthemetaboliteprofilesofCBBintermediates(Figure3).On
average,genesencodingphotorespiration,whichiscloselycoupledtotheactivityoftheCBB
cycle,showedupto22-fold(LA1777)and34-fold(LA4024)lowerexpressionandupto4-fold
(LA1777) and 46-fold (LA4024) lower protein levels in GTs as compared to leaves of the
correspondingspecies(Figure4,SupplementalFigures6and7,SupplementalDatasets5and
SupplementalTable1).Bothatthetranscriptandproteinlevels,enrichmentanalysesrevealed
strongleaf-specificoverrepresentationofsignalswithintheMapManontologygroups1.2PSphotorespirationand1.3PS.CBB-cycle(SupplementalDataset6).Furthermore,transcriptsof
carbonic anhydrase (CA) showed leaf-specific expression and higher protein abundance in
leavesthanintrichomes(LA17772.8-fold;LA40248.5-fold,p<0.05).
Paralleling lower ADP-glucose levels in GTs, glucose-1-phosphate adenylyltransferase, a
unidirectional starch biosynthesis enzyme, showed significantly lower transcript (2- and 3fold)andprotein(13-and57-fold)levelsinGTsofbothspeciesascomparedtotheirleaves
(Figure7,SupplementalDataset5).Likewise,starchmetabolismwasoverrepresentedatthe
transcriptlevelintheleavesofbothtomatospecies(SupplementalDataset6).
By contrast, genes of photosystems (PS) I and II represented the ontology class with the
highest expression levels in all samples (leaves and trichomes of both species). Although
expressionlevelswerehigherinleavesformostofthehighlyabundantPSIandPSIIgenes,
theselevelsdifferedbylessthantwo-foldwhencomparingtrichomesandleaves(Figure6C,
Figure7,SupplementalDataset5).SincemanyPSproteinsareintegratedinmembranes,they
couldnotbereliablydetectedbytheproteomicsmethodused.
10
13
C-CO2andglucoselabelingconfirmglandulartrichomesassinkorgans
13
C-labelingofLA1777with13C-CO2showedsignificantlyslowerincorporationof13C-isotopes
into3-phosphoglycerate(3-PGA)andribulose-1,5-bisphosphate(RU-1,5-BP)despitesmaller
poolsizesintrichomesversusleaves(Figure8A).Forexample,after10minthepercentageof
labeledCinRU-1,5-BPisonly8%intrichomes,versus68%inleaves.Becausethepoolsizeis
smallerinGT,inabsoluteamountofC,thisrepresentsa32-foldhigherincorporationofCO2
in leaves versus trichomes. In contrast to the CBB intermediates, the fraction of label in
sucroseinthetrichomesisnotsignificantlydifferenttothatofleavesafter10min,andof31%
versus50%intheleavesafter30min(Figure8A).Also,inabsoluteamounts(basedonpeak
area),theincorporationoflabelinsucroseintrichomesismuchhigherthanthatinRU-1,5BP, indicating that in GT sucrose must be largely imported from the leaves. The pattern
distribution of heavy isotopologs in sucrose was similar for leaf and trichome but the 13C
enrichment in trichomes lagged behind the
13
C-enrichment observed in the leaf
(SupplementalFigure8A;SupplementalDataset7(standarddeviationisotopologs)).Similar
enrichmentof3-PGA(68%after10minand84%after30min)andRU-1,5-BP(68%after10
minand81%after30min)with13CdemonstratesthattheCBBcycleinleavesislargelyfedby
atmosphericCO2(Figure8A).Bycontrast,inGTs,strongerenrichmentwith13Cof3-PGA(22%
after10minand44%after30min)thanofRU-1,5-BP(8%after10minand32%after30min)
upon 13C-CO2labelingisobserved.Thisindicatesthatthelabelin3-PGAinpartcomesfrom
thebreakdownoflabeledsucroseimportedfromtheleaf.
LabelingwithU13C-glucoseinambientCO2atmosphereresultedinintensiveincorporationof
13
C-label in sucrose (Figure 8B). Here, 13C enrichment in the trichomes lagged behind that
observedinleaves.Interestingly,thelabelingpatternof13C-isotopologswassimilarbetween
leavesandtrichomes,particularlyatthelatertimepoints(SupplementalFigure8).Amongthe
heavier isotopologs, the signatures of 6x13C and 12x13C most strongly contributed to the
labelingpatternsinleavesandtrichomes,whichcanbeinterpretedasdirectincorporationof
U13C-glucoseintosucrosebysucrose-phosphatesynthaseintheleavesandtransportofsuch
labeledsucroseintothetrichomes.Thefractionoflabelintheleavesin3-PGAandRU-1,5-BP
is lower than with 13C-CO2, reflecting the incorporation of ambient 12C-CO2 (Figure 8B).
Nonetheless, significant incorporation of label could be observed in CBB intermediates,
indicatingreplenishmentof 13Cfromglucosecatabolism.Intrichomes,thisphenomenonis
11
exacerbated,resultingforexamplein30%labeledCinRU-1,5-BPafter24hversus17%inthe
leaves(Figure8B).Thus,despitealowerfixationrateviatheCBBintrichomescomparedto
leaves(Figure8A),refixationofCO2appearstoplayproportionallyastrongerpartintrichomes
than in leaves. This is further supported by the pattern of labeling, showing preferential
enrichment of heavier isotopologs in 3-PGA and RU-1,5-BP in trichomes versus leaves
(SupplementalFigure8).
DifferentialgeneexpressionisconsistentwiththemetabolitespectraobservedinGTsof
wild-typeandcultivatedtomato
Isoprenoid biosynthesis is highly active in GTs of both tomato species. Besides protein
turnoverandRNAregulation,isoprenoidsynthesisrepresentedthecategorywithsomeofthe
mosthighlyexpressedgenesinthetrichomesofLA1777andLA4024(Figure6).Bothspecies
producehighlevelsofterpenesthataresynthesizedinthecytosol(sesquiterpenesinboth
species)(vanDerHoevenetal.,2000)andintheplastids(monoterpenesforS.lycopersicum
(Schilmilleretal.,2009)andsesquiterpenesforLA1777(Sallaudetal.,2009)).Therefore,one
should expect high level expression of both the mevalonate and the methyl-erythritol
phosphatepathwaysinbothspecies.Genesencodingenzymesoftheisoprenoidprecursor
pathways and the corresponding proteins were strongly enriched in the trichomes
(Supplemental Dataset 6) and showed significant trichome-specific expression (Figure 4,
SupplementalFigure9)inbothaccessions.Accordingly,highexpressionofgenesandproteins
of the MEV and MEP pathways as well as high levels of metabolic intermediates of these
pathways could be measured (Supplemental Dataset 3). In LA1777 GTs, we note the
particularlyhighlevelsofmethylerythritol-cyclodiphosphate(MEcPP),anintermediateofthe
MEP pathway, which supplies precursors for the highly abundant sesquiterpene carboxylic
acidsproducedinthisspecies(Figures3andSupplementalDataset3).Sincethegenesofboth
theMEVandMEPpathwaysareover-expressedinGTs,exchangeofisoprenyldiphosphate
precursorsbetweencompartmentsisunlikelybutcannotbeexcluded.
Amino acid metabolism. Although significant in both lines, stronger differences in LA1777
thaninLA4024betweentrichomesandleavescouldbeobservedwithregardtoaminoacid
metabolism(Figure4,SupplementalFigure6and7).InLA1777inparticular,themetabolism
12
ofbranchedchainaminoacidswasupregulated,illustratedbytheenrichmentanalysisofall
transcripts (bin 13.4.2.1 in Supplemental Dataset 6) and genes such as those encoding 3isopropylmalate
dehydratase
(Solyc03g005730)
or
2-isopropylmalate
synthase
(Solyc08g014230), which is consistent with the higher metabolite levels of valine, and
leucine/isoleucine (Supplemental Datasets 3 and 5). Interestingly, genes involved in the
degradation of branched chain amino acids, such as 3-hydroxyisobutyryl-CoA hydrolase
(Solyc07g044710), 3-methyl-2-oxobutanoate dehydrogenase (Solyc04g063350), IsovalerylCoA dehydrogenase (Solyc06g073560) or Enoyl-CoA-hydratase (Solyc07g043680), are also
over-expressedinGTs(SupplementalDataset5).Branchedchainaminoacidsareprecursors
of acyl sugars, which are preferentially formed in LA1777 (Slocombe et al., 2008). We also
observed high levels of aromatic amino acid decarboxylases in GTs (e.g., Solyc08g066250,
SupplementalDataset5),whosefunctionisunclear.
Flavonoidmetabolism.Severalgenesimplicatedinflavonoidmetabolismwereupregulated
in trichomes with a strong contribution to the biosynthesis of chalcones, flavonols and
dihydroflavonols(Figures4,SupplementalFigure6and7,SupplementalDataset5).Acommon
patterninbothtomatospeciesisthesignificantenrichmentoftranscriptsandproteinsofthe
MapManbin16.8(SupplementalDataset6),includingtranscriptsencodingproteinssuchas
chalcone synthase (Solyc05g053550) or chalcone-flavonone isomerase (Solyc05g010320).
Highest transcript and protein levels were observed in the trichomes of LA4024 as shown
earlierindetailforS.lycopersicumM-82(Schilmilleretal.,2010)andcorrelatewellwiththe
large amount of rutin found in trichomes of LA4024, but also for the presence of various
methylatedmyricetinderivativesinbothspecies(Schmidtetal.,2011;Kimetal.,2014).
Lipidmetabolism.InLA1777,25%(p=0.05)ofalltranscriptsand56%(p=0.01)ofallproteins
detectedinMapManbin11weresignificantlyupregulatedinGTs.InLA4024,21%(p=0.05)
ofalltranscriptsand49%(p=0.01)ofallproteinsdetectedinMapManbin11weresignificantly
upregulatedinGTs.Furthermore,enrichmentanalysisshowedamarkedcontributionofgenes
encodinglongfattyacidmodificationandlipiddegradationspecifictotheGTsofbothspecies
(SupplementalDataset6,SupplementalDataset5).Lipidformationinplantsnormallytakes
placeintheplastids.Therefore,weexpectedtofindtranscriptsfortheplastidicacetyl-CoA
carboxylaseandmalonyl-CoAacylcarrierproteintransacylase,theinitiatingenzymesoffatty
acidbiosynthesis,bothofwhicharestronglyupregulated.However,differentsubclustersof
13
the acetyl-CoA carboxylase protein complex showed only up to two-fold higher levels of
transcript(e.g.,Solyc12g056940)inbothspecies.Thetransacylasewasexpressedatrelatively
lowlevelsinbothspeciesanddidnotshowasignificantdifferencebetweentrichomesand
leaves(Solyc01g006980).Afterplastidicbiosynthesisoffattyacidswithchainlengthsofupto
C16-C18, extension to longer fatty acids requires their export out of the plastid to the
endoplasmicreticulum(Samuelsetal.,2008;KunstandSamuels,2009).Theextensionoffatty
acids from long (C16, C18) to very long chains is catalyzed by β-ketoacyl-CoA synthase, βketoacyl-CoAreductase,β-hydroxyacyl-CoAdehydratase,andenoyl-CoAreductase(Kunstand
Samuels, 2009). Inspection of highly abundant but differentially expressed genes revealed
strongupregulationoffattyacidelongasesandacyl-CoAligasesinGTs(e.g.,Solyc07g043630,
Solyc03g031940, Solyc09g083050, and Solyc08g067410; Supplemental Dataset 5).
Furthermore, in agreement with the accumulation of polyunsaturated fatty acids in GTs in
bothspecies,massiveexpressionwasalsoobservedforseveraltrichome-specificdesaturases.
Solyc11g008680 (log2-fold change of 4.1 in LA1777; log2-fold change of 0.53 in LA4024),
Solyc01g009960(log2-foldchangeof3.7inLA1777;log2-foldchangeof2.83inLA4024)and
Solyc06g059710(log2-foldchangeof1.5inLA1777;log2-foldchangeof4.79inLA4024)encode
acyl-carrier protein desaturases. Solyc01g006430 (log2-fold change of 1.31 in LA1777; log2foldchangeof1.87inLA4024)encodesadesaturaseoftheendoplasmicreticulumandisalso
upregulatedinGTsofbothspecies.Highexpressionoftheselipidbiosynthesisgenesisingood
agreementwiththemeasurementoflargeamountsofpolyunsaturatedC18(inLA4024)and
C20(inLA1777)fattyacids(seeabove).
VariousROSdetoxificationsystemsareover-expressedinGTs
Photosynthetic oxygenesis, the activity of oxidoreductases and high metabolic activity in
generalresultintheformationofreactiveoxygenspecies(ROS)suchassingletoxygen.ROS
staininginLA1777indicatedmuchstrongerROSformationinGTscomparedwithleafmatter
(Supplemental Figure 10). Enzymes that are involved in the detoxification of ROS include
superoxidedismutase,catalases,andperoxidasesfortheoxidationofglutathione,ascorbate,
orlipids.Veryhighlevelsofsuperoxidedismutasegeneexpressioncouldbedetectedinthe
GTs of both species, with, e.g., Solyc01g067740 belonging to the top 15 most abundant
14
enzymesinthetrichomesofbothspecieswithproteinlevelsbeing31-fold(LA1777)and39fold(LA4024)higherthanintheleaves(SupplementalDataset5).
The high levels of polyunsaturated fatty acids and of oxylipins derived thereof (see above)
suggestedthatthesecouldplayaroleinthequenchingofROS,ashasbeenshowninother
plantsystems(Schmid-Siegertetal.,2016).Lipoxygenasesrepresentanessentialcomponent
ofthisdetoxificationpathway,sincetheyconsumeH2O2andgeneratelipidperoxidesthatcan
be further metabolized. The cumulative gene expression of all 21 lipoxygenases was
comparable between leaves and trichomes in both species. Yet, closer inspection of the
subcellularlocalizationshowedmuchhighertranscriptandproteinlevelsformembersofthe
cytosolicLOX1/LOX5familywith9S-lipoxygenaseactivity.ThehighestlevelswerefoundinGTs
of LA1777 (Supplemental Figure 11). By contrast, homologs of the LOX2/LOX3 family in
Arabidopsisthalianawith13S-lipoxygenaseactivity,whicharelocalizedintheplastids,were
enriched in leaves with one exception (Solyc01g006540). This gene appeared to also be
trichome-specificinLA4024andshowedoneofthehighestproteinabundancesmeasuredof
all (Supplemental Figure 11D). These expression results were confirmed by RT-qPCR
(SupplementalTable1).
Furthermore, Solyc07g049690, encoding the fatty acid hydroperoxide lyase, was highly
expressedintrichomesofLA1777andLA4024withtranscriptlevelsbeingsixtimesandthree
times higher and protein abundances being 50 and 150 times higher than in leaves
(Supplemental Dataset 5). This finding supports the contribution of lipid oxidation as one
important mechanism involved in ROS detoxification. Whereas in leaves expression of
hydroperoxide lyase is inducible, in GTs it appears to be constitutively expressed, since no
extraenvironmentalstresswasappliedduringourexperiments.Altogether,theseresultsare
inagreementwiththemeasurementsof9S-hydroxy-10E,12Z-octadecadienoicacid(9-HODE)
inLA1777and13S-hydroxy-9Z,11E-octadecadienoicacid(13-HODE)inLA4024,respectively.
Lipid hydroperoxides can be recycled by glutathione peroxidases via the oxidation of
glutathione(GSH)toglutathionedisulfide(GSSG).Inbothspecies,mostgenesencodingGSH
peroxidases showed higher transcript levels and higher cumulative protein abundances in
trichomes than in leaves and were significantly enriched in the GT protein of both species
(Supplemental Figure 12, Supplemental Dataset 6). The higher GSSG/GSH oxidation state
15
mentionedaboveisfurthercorroboratedbythehighexpressioninGTsofcysteinesynthase,
which is required for GSH replenishment. Five out of six cysteine synthase-coding genes
showedhigherexpressionintrichomesofbothtomatolines.Particularly,Solyc01g097940,
whichdominatedtheexpressionofthisenzymefamily,showed15-17-foldhighertranscript
levelsascomparedtothecorrespondingleaves(SupplementalDataset5).
In contrast to GSH peroxidases, other enzyme families contributing to the cell redox
homeostasisdidnotshowamajorupregulationinGTs(SupplementalFigure12).Forinstance,
glutaredoxins showed similar transcript levels in GTs and leaves of both species, with one
isoform-encoding gene (Solyc09g074570) being outstandingly high in the leaves
(SupplementalDataset5).Amongthethioredoxins,withoneexception(Solyc05g018700with
transcript levels being 4-5 higher in GTs and moderately abundant protein present only in
GTs), similar or lower transcript levels were detected in GTs relative to the leaves of both
species (e.g., Solyc07g063190 or Solyc04g081970). With one exception (Solyc04g082460),
catalase-codingtranscriptswerecomparableorhigherintomatoleavesascomparedtoGTs
(SupplementalDataset5).SimilarorlowertranscriptandproteinlevelsinGTscomparedto
leaves in both species were found also for the most abundant ascorbate peroxidases (i.e.,
Solyc06g005160);thustheglutathione-ascorbatecycleappearstoequallycontributetoH2O2
removal in GTs and leaves despite the depletion observed for ascorbate in trichomes of
LA4024 (Supplemental Dataset 3). Other peroxidases included polyphenol oxidases, which
showed GT-specific expression in LA4024 (Solyc08g074620, Solyc02g078650, Supplemental
Dataset5).
Altogether,ourresultsbasedonmetabolomics,transcriptomicsandproteomics,indicatethat
specific ROS-detoxification pathways show increased activity in GTs. In particular, the
oxidation of unsaturated lipids and glutathione-based detoxification of reactive peroxides
appeartoplayimportantroles.
16
ThemetabolismofGTsisdirectedtowardsthesupplyofprecursorsandcofactorsforthe
majormetabolitepathways
The abundant biosynthesis of secondary metabolites by GTs requires the supply of carbon
fromprecursorsaswellastheprovisionoflargequantitiesofATPandNAD(P)H.Sincethe
major metabolites in GTs are terpenoids produced from the MEP (plastidic) and the
mevalonate (cytosolic) pathways, it was thus expected that processes delivering triose
phosphates and pyruvic acid (for the MEP pathway) or acetyl CoA (for the mevalonate
pathway)areupregulatedinGTsoftomato.
Supply of acetyl CoA in the cytosol. Upon glycolysis, acetyl CoA is produced in the
mitochondria. Acetyl CoA, however, cannot be transported across the mitochondrial
membrane. The supply of acetyl CoA in the cytosol can be achieved via the citrate shuttle
(Oliver et al., 2009; Xing et al., 2014). Citrate is transported from the mitochondria or the
peroxisomes into the cytosol and cleavage of citrate by the cytosolic ATP-citrate lyase
produces oxaloacetate and acetyl CoA. Microarray expression data showed that citrate
synthases (CS; Solyc01g073740 - mitochondrial, Solyc12g011000 - peroxisomal) were
moderately,butsignificantly(p<0.05witht-teststatistics)upregulatedinthetrichomesof
bothspecies(SupplementalDataset5)andupto23-foldhigherproteinlevelswerefoundin
GTs relative to the corresponding leaves (Supplemental Dataset 5). Genes encoding ATPcitrate-lyasesshowedhighertranscriptlevelsinLA1777trichomesandhighercorresponding
proteinlevelsinGTsrelativetoleafmatter,supportingcitrateexportfrommitochondriato
thecytosolinGTs(Figure9,SupplementalDataset5).Despitethelargerpoolsizeofcitrate
comparedtoisocitrate,isotopicpatternsofcitrateafter24hU13C-glucoselabelingshoweda
muchhigherincorporationof13Cintocitratethanwasobservedforisocitrate(Supplemental
Figure13).InaccordancewiththesmallercitratepoolsizeinGTsversusinleaves(p<0.05
witht-teststatistics),the 13CenrichmentincitratewasmoreintenseinGTsthaninleaves.
Thus,supportingthetranscriptomics/proteomicsdata,labelingindicatesthatcitrateislargely
exported from mitochondria rather than turned into isocitrate via mitochondrial TCA
enzymes.Moreover,incontrasttoCS,mostotherTCAenzymesshowedcomparableoronly
slightlyincreasedtranscriptandproteinlevels(<3-fold)fortrichomesversusleavesofthe
correspondingline(SupplementalDataset5).OurdatathereforesupportthefactthatinGTs
thecitrate-malate-pyruvateshuttleisusedtosupplyacetylCoAinthecytosol,neededtofuel
17
fattyacidelongation,thebiosynthesisofisoprenyldiphosphatesviathemevalonatepathway
or other key metabolites produced in the cytosol. Higher transcript and protein levels for
cytosolic phosphoenolpyruvate carboxykinase (PEPCK), cytosolic pyruvate kinase (cPK),
cytosolic malic enzyme (cME), and cytosolic and mitochondrial pyruvate dehydrogenase
(cPDH, mPDH) support that carbon withdrawn from the TCA cycle by the ATP-citrate lyase
complex is replenished by malate and pyruvate (Figure 9, Supplemental Dataset 5,
Supplemental Table 1). Upregulated transcripts and elevated protein levels of
phosphoenolpyruvatecarboxylase(PEPC)andPEPCKinGTsindicatethatanapleroticroutes
to produce oxaloacetate may additionally facilitate the production of cytosolic acetyl-CoA
(Tcherkez et al., 2011). For PEPCK, a bidirectional enzyme, preferred carboxylation was
demonstrated in C4 plants for high physiological ATP/ADP ratios (Chen et al., 2002). On
average,theATP/ADPratio(basedonpeakarea)was1.5xand2.7xhigherinGTsofLA1777
andLA4024,respectively,ascomparedtothecorrespondingleaves(SupplementalDataset3).
This,andincreasedproteinlevelsofPEPCinGTsversusleavesinbothspecies,alsoinfersthat
CO2isincorporatedintooxoaloacetate.
Supplyofpyruvateandglyceraldehyde-3-phosphateintheplastids.Thelargequantitiesof
plastidicterpenoidsproducedinGTsofbothtomatospecies(monoterpenoidsforLA4024and
sesquiterpenoidsforLA1777),indicatethatfluxthroughtheMEPpathwayisimportant.This
is supported by high transcript and protein levels of MEP pathway enzymes in GTs
(Supplemental Figure 9, Supplemental Dataset 5, Supplemental Table 2). Such high MEP
pathwayfluxwouldrequireanappropriatesupplyofitsprecursors,namelyglyceraldehyde3-phosphate(GAP)andpyruvate(PYR).PlastidicPYRcanbeproducedviaplastidicglycolysis,
byplastidicisoformsofmalicenzymeorimportedfromthecytosol(Oliveretal.,2009;Weber
andBrautigam,2013;Eisenhutetal.,2015;Shtaidaetal.,2015).GAPintheplastidscanbe
supplied by the non-oxidative pentose phosphate pathway, the CBB cycle, by plastidic
glycolysisorbytranslocationfromthecytosol(Flüggeetal.,2011).
Plastidicandcytosolicglycolysis.Sincethesubcellularlocalizationofcytosolicandplastidic
poolsofglycolyticmetabolitescannotbeunambiguouslyassignedbymetabolomics,itisnot
possibletoconcludewhetherplastidicorcytosolicglycolysissuppliesthecarbonforGAPor
PYRinGTsbasedonmetabolomicsdata.Thus,transcriptomicsandproteomicsresultswere
mined to address this question. Two genes encoding plastidic pyruvate kinases (pPK)
18
(Solyc01g106780 (LA1777: log2-fold change 3.15; LA4024: log2-fold change 1.84) and
Solyc03g007810 (LA1777: log2-fold change 1.80; LA4024: log2-fold change 2.0) show
significantlyincreasedexpressionintrichomesaswellasageneencodingaplastidicenolase
(Solyc03g114500), although in this case differential expression is more pronounced for
LA1777(log2-foldchange2.3)thanforLA4024(log2-foldchange0.4)(SupplementalFigure14,
Supplemental Dataset 5, Supplemental Table 1) in particular at the protein level (log2-fold
change3.38inLA1777;nosignificantchangeinLA4024).Inbothspecies,hightranscriptlevels
and protein abundances of several isoforms of fructose-bisphosphate aldolase (FBA) were
predicted to be plastidic. Those with the highest expression levels were leaf-specific (e.g.,
Solyc02g062340) and are associated with aldolase activity in the CBB cycle. However, one
plastidic FBA (Solyc05g008600) and two cytosolic FBAs (Solyc09g009260, Solyc10g083570)
showedhighertranscriptandproteinlevelsintrichomesthaninleaveswithabsolutevalues
beinghighinGTsofbothspecies(Figure14,SupplementalDataset5).Additionally,hexose
breakdown in GTs appears to proceed via cytosolic diphosphate-dependent
phosphofructokinases,whichshowedhigherexpressioninGTsascomparedtoleaves(e.g.,
Solyc04g082880, Solyc12g095760, Solyc02g081160, Supplemental Figure 14, Supplemental
Dataset5).Thus,inGTs,provisionoftriosephosphatesseemstobeassistedviaacombination
of cytosolic and plastidic glycolysis. This implies the transport of various sugar and triose
phosphatesbetweenthecytosolandtheplastids.
Otherroutes.Alternatively,pyruvatecanbeproducedviadecarboxylationofmalatebythe
plastidic NADP-dependent malic enzyme (pNADP-ME). Genes encoding pNADP-ME,
particularly Solyc12g044600, were most strongly expressed in GTs of LA1777 (Log2-fold
change=1.2)andLA4024(Log2-foldchange=0.3)andalsoexhibitedhigherproteinlevelsinGTs
versus leaves but only in LA1777 (Log2-fold change=3.94) (Supplemental Dataset 5).
Transketolase,aGAP-producingenzymeinvolvedinthenon-oxidativebranchofthepentose
phosphate pathway showed high expression of two plastidic isoforms (Solyc10g018300,
Solyc05g050970, Supplemental Dataset 5) of which the latter showed increased transcript
levelsandproteinabundanceinGTsofbothspeciesascomparedtothecorrespondingleaves.
Contributionoftheplastidicoxidativepentosephosphatepathway.Intheoxidativepentose
phosphate pathway glucose-6-phosphate is converted to a pentose phosphate with the
release of NADPH. One plastidic isoform of glucose-6-phosphate 1-dehydrogenase
19
(Solyc05g015950)showedelevatedtranscriptlevelsinGTs(Log2-foldchange=1.7inLA1777,
1.09 in LA4024; Supplemental Dataset 5), which was also confirmed by RT-qPCR
(SupplementalTable1)andproteinlevelsweremarkedlyelevated inGTsversusleavesbut
only in LA1777 (Log2-fold change=3.89, Supplemental Dataset 5). Furthermore,
Solyc12g056120 which encodes a plastidic 6-phosphogluconate dehydrogenase shows
elevatedtranscriptandremarkablyhighproteinlevelsintrichomes(Log2-foldchange=2.01in
LA1777,2.13inLA4024; SupplementalDataset5-SI).Highactivityoftheoxidativepentose
phosphate pathway would serve a dual function: to supply NADPH as well as ribulose-5phosphateforthereplenishmentofRU-1,5-BPintheCBBcycle.
Transport. The compartmentalization of metabolism implies exchange between
compartments,whichplayanessentialroleinensuringappropriatesupplyofprecursorsfor
compartment-specificmetabolicpathways.Particularlyrelevantarethetransportofsugar,
sugar-phosphates,triose-phosphatesandcarboxylicacidstoandfromthechloroplasts.The
moststronglyexpressedisoformoftheglucose-6-phosphatetranslocator(Solyc07g064270)is
slightly over-expressed in trichomes (log2-fold change = 1.32 in LA1777; 0.59 in LA4024),
whereas the most strongly expressed isoform of the phosphoenolpyruvate translocator
(Solyc03g112870) shows no differential expression between leaves and trichomes in both
species.TriosephosphatetranslocatorSolyc10g008980showedhightranscriptlevelsinGTs
ofbothtomatolineseventhoughtheexpressionlevelwasthreetimeshigherintheleaves
thaninthetrichomes(log2-foldchange=1.27inLA1777;1.53inLA4024).Triosephosphate
translocationinleavesisutilizedtoshuttletriosephosphatesproducedbytheCBBcyclefrom
thechloroplaststothecytosol(LudewigandFlügge,2013).However,asglycolyticbreakdown
ofhexoseinthecytosolofGTsisindicatedbyhighexpressionofgenesofthetoppartofthe
glycolysis pathway, reverse transport of triose phosphate from the cytosol to the plastids
wouldalsobeplausible.Ashexosephosphateistranslocatedfromthecytosolintotheplastids
ofGTs,itcanbeconvertedtoGAPviaplastidicglycolysisortheplastidicpentosephosphate
pathway.
Among the putative transmembrane transporters for pyruvate, mitochondrial
Solyc10g051120, encoding a putative mitochondrial isoform, was particularly strongly
overexpressedinGTs,2.6-foldinLA1777(q-value=1.42x10-6)and24-foldinLA4024(q-value
=1.06x10-11),supportingthefactthatpyruvateisconvertedtoacetyl-CoAandcitrateinthe
20
mitochondria,thelatterbeingthenexportedtothecytosoltobeconvertedtoacetyl-CoAby
ATP-citratelyase(seeabove).
The tomato genome contains several homologs of the bile acid sodium symporter (BASS)
family.InArabidopsisthaliana,theBASS2protein(At2g26900)wasshowntofunctionasa
plastidial pyruvate sodium-dependent transporter (Furumoto et al., 2011). A phylogenetic
analysis of the BASS homologs from Arabidopsis and tomato (Supplemental Figure 15;
Supplemental Dataset 8) indicates a strong conservation of the respective BASS proteins
betweenthespecies,ratherthanwithinspecies.Thissuggeststhattherespectivefunctions
of the BASS proteins between these two species are well conserved. The BASS2 putative
ortholog of tomato (Solyc05g017950) shows comparable expression levels in leaves and
trichomes in both species, indicating that pyruvate import via BASS2 is not induced in
trichomes compared to leaves. By contrast, in LA1777, Solyc08g007590 (BASS6) is overexpressed in trichomes (Log2-fold change=2.05). Arabidopsis has two putative orthologs of
Solyc08g007590,BASS5andBASS6,althoughithasahigherpercentageofsequenceidentity
withBASS6thanwithBASS5.Interestingly,BASS5(At4g12030)isinvolvedinthetransportof
methionine-derived alpha-keto acids required for the biosynthesis of the corresponding
glucosinolates(Gigolashvilietal.,2009),butthefunctionofBASS6isunknown.Sincetomato
isnotknowntoproduceglucosinolates,Solyc08g007590couldbeinvolvedintheimportof
other carboxylic acids with a functional group in the alpha position, for example C4dicarboxylicacidssuchasmalateoroxaloacetate.InLA4024,Solyc09g055940(putativeBASS3
ortholog)isoverexpressedintrichomes(Log2-foldchange=1.5;p=0.05).However,thefunction
ofBASS3isunknown.ItssimilaritytoBASS5/6couldsuggestitisalsoinvolvedinthetransport
ofcarboxylicacidswithafunctionalgroupinthealphaposition.Inaddition,ahomologofthe
Arabidopsisdicarboxylicacidtransporter(DIT1),Solyc11g065830,showsslightoverexpression
intrichomesofLA1777(Log2-foldchange=0.69)butalowerexpressionintrichomesofLA4024
(Log2-foldchange=2.19).
Supply of ATP and reducing power. As for the oxidative pentose phosphate pathway,
transcriptlevelsofgenesandproteinsencodingNADPH-producingenzymefamiliesotherthan
PSIwereincreasedinGTsascomparedtoleaftissue(SupplementalFigure16).Thus,intomato
GTs,inadditiontoNADPHproductionviaphotosynthesis,thecentralcarbonmetabolismis
directedintotheproductionofthereducingcofactorNADPH.Onaverage,theexpressionof
21
genes in the MapMan ontology group 1.1.4 PS.lightreaction.ATP synthase resulted in
transcriptlevelsbeing3.4-fold(LA1777)and3.1-fold(LA14024)higherinleavesthaninthe
correspondingGTs.Ontheotherhand,mitochondrialATPasetranscripts(MapMangroup9.9
mitochondrial electron transport/ATP synthesis.F1-ATPase) were comparable (0.8-fold
(LA1777)and0.8-fold(LA4024))betweenleavesandGTsofbothspecies.Thus,photosynthetic
ATPandNADPHproductionintomatoGTsisbasedonpronouncedexpressionlevelsofboth
photosystems(Figure7).However,itscontributiontotheenergetichomeostasisappearsto
belowerthaninleaves.
Discussion
Toidentifywhichmetabolicroutesdeliverenergy,reductionequivalentsandcarbonintomato
GT,weusedamulti-omicsapproachand13C-labelingexperiments.Wetookadvantageofthe
recentsequencingofthetomatogenomeforacomprehensivesurveyofgeneandprotein
expression.ThehighdegreeofsequencesimilaritybetweenLA4024andLA1777allowedus
to use a single microarray that was designed to hybridize to the RNA of both species. By
comparing trichome samples (dominated by type VI trichomes) with nearly trichome-free
leavesintwotomatospecies,weidentifiedmetabolicprocessesthatdrivethehighproduction
ofsecondarymetabolitesinthistypeofGT.AsthesecondarymetabolismintypeVIGTsof
LA1777andLA4024widelyoverlaps,weonlybrieflydescribedprocessesthatarespecificto
eitherofthelineswhenevertheycontributedtotheformationofhighlyabundantsecondary
metabolites.Themainaim,however,wastoemphasizeregulatoryprinciplesthatarecommon
tobothtomatomodels.
Trichome-specificcentralcarbonmetabolismdespitedifferentsecondarymetabolites
Metabolomicsdataofsecondarymetabolitesgenerallyconfirmsearlierworks(Besseretal.,
2009;Schilmilleretal.,2010;Ghoshetal.,2014).InLA1777trichomes,themetaboliteprofile
isdominatedbyshortbranchedchainacylsugarswithfattyacidchainlengthsbetweenC2C12,flavonoids,freelong-chainfattyacidsandsesquiterpenecarboxylicacids.InLA4024,the
flavanol conjugates dominated the metabolic profile in LC/MS. Beyond these confirmative
measurements,tothebestofourknowledge,acomprehensiveanalysisofthecentralcarbon
and energy metabolism of GT has not been reported to date. PCA analysis of hydrophilic
22
metabolomesshowedthatGTsofLA1777andLA4024havemoreincommonwitheachother
than with leaves from the same species. This indicates that GTs share unique metabolic
processesthatrelatetotheirhighmetabolicactivitydespitethedifferentmetabolitesthey
produce. The combination of these data with transcriptomics, proteomics and 13C-labeling
dataallowedustoextractthesalientfeaturesofthesemetaboliccellfactories,whichwillbe
discussedbelow,andtointegratethemintoametabolicmodel.
SucroseisamajorcarbonsourceforGTs
Severalargumentssupportthefactthatsucroseisthemajorcarbonsource.Firstly,thepool
size of sucrose in the leaves is about twice as large as that of trichomes, implying sucrose
provisionfromtheleavesviaaconcentrationgradient(Figure8).Secondly,thepatternsof
labeledformsofsucroseweresimilarfortrichomesandleafmatter,especiallyatlatertime
points. Thirdly, the percentage of labeled isotopologs in GT lagged behind the enrichment
observed in leaves during earlier stages. Signals of labeled glucose were low in leaf and
trichomes, implying a rapid conversion of glucose into sucrose in the leaf. Instead,
incorporationoflabelwasfoundinsucroseinall12positionswiththen+12,n+11,n+10and
n+6isotopologsdominatingthesignalafter 13C-labelingwith 13C-CO2andthen+6andn+12
13
C- isotopologs dominating the signal after U13C-glucose labeling (Figure 8, Supplemental
Figure8).Inaddition,sucrose-degradingenzymeswereoverexpressedintrichomesinalmost
all compartments as shown in Figure 5 for cell wall invertases, cytosolic invertases and
cytosolicsucrosesynthase.Inparallel,invertaseinhibitorswerestronglyreducedintrichomes,
supportingstrongerdegradationofsucroseintheapoplasm.Sucrosedecompositioninnonphotosynthetic GTs of other plant families displayed relatively high enrichment in EST
libraries, for instance in peltate trichomes of basil (Gang et al., 2001). From this, it was
proposedthatcarbonofabundantphenylpropenesoriginatesfromsucroseimportedfrom
theunderlyingleaves(Gangetal.,2001).Inspearmint(Menthaspicata),severaltranscripts
encodingenzymesforsucrosecatabolismwereexpressedmoreinnon-photosyntheticpeltate
GTsthanintrichome-freeleafmatter(Jinetal.,2014).Intobacco(Nicotianatabacum),which
liketomatohasphotosyntheticglandulartrichomes,highRuBisCOactivitywasindicatedby
representationinESTlibrariesandexpressionlevelsmeasuredbyRT-qPCR(Cuietal.,2011).
Besides RuBisCO, carbohydrate metabolism represented more than 10% of all proteins
definedbythegeneontology(GO)inatrichome-specificcDNAlibraryoftobaccotrichomes.
23
BecauseGTsoftomatoalsoexpressphotosynthesisgenes(forbothphotosystemsandcarbon
fixation) at high levels, the fact that sucrose constitutes a major carbon source raises the
questionofthecontributionofphotosynthesistoCsupplyintrichomes.
Uncouplinglightphotosynthesisfromthedarkreactions
In GTs, genes encoding proteins of both photosystems were expressed at roughly half the
valuesobservedforleavesbuthadstillthemostabundanttranscriptsinGTs,implyingthat
both photosystems are highly active in GTs in delivering ATP, NADPH and O2. By contrast,
genesfortheCBBcycleandphotorespirationwere4to5timeslowerintrichomescompared
to leaves. This indicates that there is an uncoupling of gene expression between light
photosynthesisandcarbonfixationmediatedbytheCBBcycleinGTs.Consistentwiththis,
genesforstarchbiosynthesisanddegradationarealsodown-regulatedinGTs,showingthat
carbonthatisincorporatedinGTsisnotstoredasstarch.TheloweractivityoftheCBBcycle
inGTswasconfirmedbyour13Clabelingexperiments,whichindicatedthatsomeCO2isfixed
viatheRuBisCOcomplex,butalargepartofthe13C-labelwasacquiredindirectlyaftersupply
andbreakdownofsucroseandnotdirectlyfromtheatmosphere.ThatlittleatmosphericCO2
isfixedinGTsisfurthercorroboratedbythepresenceofanenvelope(cellwallandcuticle),
which is even thicker than that of epidermal cells and likely to significantly restrict gas
exchange(Figure1).
Thehighmetabolicactivityoftheglandularcellsrequireshighlevelsofchemicalenergyand
reducingpower.Forexample,theplastidicMEPpathwayrequires1xATP,1xCTPand2x
NADPHandthecytosolicmevalonatepathwayrequires3xATPand2xNADPHtogenerate
oneC5body.Onecanthereforeassumethattheenergyandreducingpowerresultingfrom
theactivityofthephotosystemsinGTssupportthishighmetabolicactivity.Theuncouplingof
lightphotosynthesisfromthedarkreactionsraisestheissueofthefunctionoftheCBBcycle
inGTs.DespitethelowerexpressionofCBBcyclegenesinGTscomparedtotheleaf,their
expressionisfarfromnegligible.ThemoststronglyexpressedRuBisCOsmallsubunitisoform
(Solyc02g085950)stillranksamongthetop350mosthighlyexpressedgenesinGTsofLA1777
andLA4024.CO2producedinternallyinthetrichomesispotentiallyrecycledviatheactivityof
RuBisCO without a full CBB cycle, as previously shown in developing green Brassica seeds
(Schwender et al., 2004). In our 13C-U-glucose labeling experiments, RU-1,5-BP and 3-PGA
24
display stronger 13C enrichment in trichomes than in leaves, particularly with heavier
isotoplogs(e.g.,n+4andn+5forRU-1,5-BP,andn+3for3-PGA)(Figure8andSupplemental
Figure8).ThisimpliesthatintrichomestheCBBcycleisreplenishedviasucrosebreakdown
products. This could be accomplished through the supply of C5 units from the oxidative
pentose phosphate pathway or through glycolytic formation of triose phosphates, both of
whichareoverexpressedintrichomes.
ElevatedlipidmetabolismandlipidoxidationarecommonfeaturesinGT–Hydroperoxide
lyaseiscoexpressedwithgenesoftheMEPpathway
Tightlyconnectedtoactivephotosystemsandtheactivityofoxidoreductasesistheformation
ofreactiveoxygenspecies(ROS),thepresenceofwhichwassubstantiatedbyROSstaining
experiments(SupplementalFigure10).Ourdata(bothmetaboliteandgeneexpression)point
totheactivationofmechanismsthatprovideincreasedprotectionagainstoxidativestressin
GTs. Firstly, we see high levels of unsaturated fatty acids (C20:4 in LA1777 and C18:3 in
LA4024) and also high levels of oxidized derivatives of these PUFAs. Next, we see high
expression of genes encoding lipoxygenases and hydroperoxide lyase, indicating lipid
peroxidation activity. Interestingly, hydroperoxide lyase was shown to be induced by high
levels of 2-C-Methyl-D-erythritol-2,4-cyclopyrophosphate (MEcPP) via retrograde signaling
from plastid to nucleus (Xiao et al., 2012). Moreover, abiotic stresses robustly modulated
MEcPP levels and thus induced hydroperoxide lyase expression. As in hydroxy-methylbutenyl-diphosphate-synthase(HDS,EC1.17.7.1)mutantlinesofA.thaliana,highlevelsof
MEcPPalsocoincidedwithhighhydroperoxidelyasetranscriptioninGTs.However,whether
highhydroperoxidelyaseexpressioninGTsistheresultofhighMEcPPlevelsremainstobe
demonstrated. The strong upregulation of GSH peroxidase and other peroxidases was also
observed in tomato GTs contributing to the detoxification of ROS. The induction of similar
processesthatcopewithROShasalsobeenobservedintrichomesfromotherspecies.For
example,strongupregulationoflipidandglutathionemetabolismwasshowninArtemisiaand
minttrichomes(Soetaertetal.,2013;Jinetal.,2014).Soetaertetal.alsoshowedthat—as
intomato—non-plastidicelongasesanddesaturasesaswellasglutathionemetabolismwere
strongly elevated in GTs of Artemisia annua. Thus, detoxification of ROS by high levels of
peroxidasesandlipoxygenationseemtobeatrichome-specificfeature.
25
IncreasedsupplyofprecursorsforsecondarymetabolitepathwaysinGTs
Tomato GTs produce large quantities of terpenoids both from the cytosolic MEV and the
plastidicMEPpathways.Thereforeitistobeexpectedthatthemetabolitesthatareatthe
originofthesepathwaysshouldalsobesuppliedinadequateamounts.Theprecursorofthe
MEV pathway is acetyl-CoA. Our data support a strong induction of the citrate-malatepyruvateshuttlesystem,whichallowsthesynthesisofacetylCoAinthecytosol.Akeyelement
of this shuttle is the cytosolic ATP-citrate lyase, which cleaves citrate exported from the
mitochondria to acetyl-CoA and oxalo-acetate. ATP-citrate lyase is strongly enriched in
trichomes(Figure9),asareanumberofenzymestypicallyassociatedwiththisshuttlesystem.
Recently,Xingetal.highlightedthecentralroleofATP-citrate-lyaseintheproductionofcutin
andacetyl-CoA-derivedpolyhydroxybutyrateintransgenicA.thaliana(Xingetal.,2014).Thus
the overexpression ATP-citrate lyase together with a number of enzymes involved in this
process constitutes a potential engineering target for increasing flux through the MEV
pathway.
The precursors of the MEP pathway are pyruvate (PYR) and glyceraldehyde-3-phosphate
(GAP).Bothareintermediatesofglycolysis,whichcantakeplaceeitherinthecytosolorinthe
plastids. Several genes encoding plastidic isoforms of enzymes of the lower part of the
glycolysispathway,suchaspyruvatekinaseandenolase,wereoverexpressedinGTs.Since
some enzymes of glycolysis are also shared by the CBB cycle, which is overall significantly
down-regulatedinGTs,itisofinteresttoseehowspecificisoformsareexpressedinGTs.Thus,
Solyc05g008600,whichencodesapredictedplastidicfructose-bisphosphatealdolase(FBA),is
overexpressedintrichomesandshowstheoppositetrendasothergenesencodingplastidic
FBAs.ThereisthereforeastrongindicationthatplastidicglycolysisisfullyexpressedinGTs.
MEP pathway precursors could also be provided by other means, for example via the CBB
cycle,whichalthoughdownregulatedintrichomes,isstillactiveandcansupplyGAP.Further,
wealsonotetheincreasedexpressionofglucose-6-phosphateandtriosephosphateplastidic
transportersinGTs,indicatingthatG6Pandtriosephosphatesrepresentimportantcarbon
supplies to the chloroplasts of GTs. Another potential source of precursors for the MEP
pathway comes from C4 dicarboxylic acids. Particularly in LA1777, we noted a significant
overexpression of a plastidic isoform of NADP-dependent malic enzyme (NADP-ME,
Solyc12g044600).MEconvertsmalatetopyruvate,therebyreleasingCO2andNADPH.High
26
levels of malate in the plastids would require transport of malate from the cytosol to the
plastids. Of the characterized dicarboxylic acid transporters, only one (Solyc11g065830)
showedmoderatelyincreasedexpressionlevelsintrichomes,butseveralgenesannotatedas
encodingmitochondrialcarrierproteins(e.g.Solyc05g052640),whichhavehomologytoyeast
mitochondrialdicarboxylicacidtransporters,showstrongexpressioninGT.Theexactfunction
ofthesetransportersinplantsisnotknown,anditistemptingtospeculatethattheymight
beinvolvedintheimportofdicarboxylicacidsintotheplastidsinthecontextofcellswithlow
CBBactivity.Alsoofinterest,istheoverexpressionofgenescodingfortransportersofthe
BASSfamily,Solyc08g007590inLA1777andSolyc09g055940inLA4024,whichareputative
orthologsofBASS6andBASS3,respectively.ThefunctionofthesetransportersinArabidopsis
isnotyetknown,butBASS5,whichiscloselyrelatedtoBASS6,isinvolvedinthetransportof
methionine-deriveda-ketoacids,suggestingBASS6andBASS3arealsopotentialcandidates
forthetransportofC4-dicarboxylicacids,suchasmalate.InconnectiontoC4metabolism,
high level expression of PEPC in LA1777 trichomes and of PEPCK in the trichomes of both
specieswereobserved.AlthoughPEPCiswellestablishedasaCO2fixatingenzyme,PEPCKis
typicallyregardedasadecarboxylatingenzyme.However,PEPCKisabidirectionalenzymeand
preferredcarboxylationwasdemonstratedinC4plantsforhighphysiologicalATP/ADPratios
(Chenetal.,2002),whichisthecaseintomatoGTs.Thus,highexpressionofPEPCandPEPCK
could indicate a transient CO2 fixation in the form of C4 dicarboxylic acids, which when
transported to the plastids would be further converted to pyruvate. CO2 released by this
reactioncouldthenberecycledbyRuBisCo,therebyincreasingthecarbonefficiencyofthe
glandularcells.Thusitappearsthatthesupplyofpyruvateandglyceraldehyde-3-phosphate
fortheMEPpathwayissupportedbyseveralprocessesinvolvingplastidsandthecytosol.GTs
ofLA1777andLA4024produceterpenesthataresynthesizedinthecytosol(sesquiterpenes
in LA4024 and LA1777) and in plastids (monoterpenes in LA4024 and sesquiterpenes in
LA1777).ThisraisesthequestionofthecrosstalkbetweentheMEVandMEPpathwaysinGTs,
aphenomenonthathasbeenobservedpreviouslyinseveralplantsystems(Dudarevaetal.,
2005;LipkoandSwiezewska,2016).Ourtranscriptomicsandproteomicsdata(Supplemental
Figure9)indicatethatboththeMEVandMEPpathwaysarehighlyactiveinGTs,particularly
inLA1777,inagreementwiththehigherterpeneproductivityinthisspecies.Fromthis,itcan
beconcludedthateachoftheisoprenoidprecursorpathwayslikelysuppliesprecursorsfor
27
theterpenesthatareproducedintheirrespectivecompartment.However,itisnotpossible
toexcludethetransferofprecursorsfromonecompartmenttotheotherwiththeavailable
data. This would require labeling with, for example, deuterated deoxyxylulose or
mevalonolactone.
ThecomparisonbetweenLA4024andLA1777indicatesmanysimilaritiesinGTsbutalsosome
differences.SomenotablefeaturesareforexamplethehigherexpressionofgenesintheMEP
pathway,oftheplastidicglycolysis(enolaseandpyruvatekinase)ortheplastidicNADP-malic
enzymeinLA1777,whichlikelycontributetoahigherfluxintheplastidicisoprenoidpathway
inthatspecies.ThisisconsistentwiththesignificantlyhigherproductivityofGTsinLA1777
(97timesmoreterpenoidsproducedthaninLA4024).
Amodelforglandulartrichomemetabolicefficiency
Integratingtheseobservations,weproposeamodelonhowprimarymetabolismintomato
typeVIGTsisorganizedtosupplyadequateamountsofprecursorsforthemajormetabolic
output,whichconsistsessentiallyofterpenoidsinLA1777(Figure10).Sucroseimportedfrom
the leaves constitutes the major carbon source. The light-dependent reactions of
photosynthesissupplyenergyandreducingpowerforthemetabolicactivity,althoughamajor
differencewithmesophyllcellsisthattheenergyandreducingpowerarenotusedprimarily
forcarbonfixationviatheCBBcycle,butforthesecondarymetabolitepathways,principally
terpenoidandlipidbiosynthesis.ApossiblecontributionoftheCBBcycleisre-fixationofCO2
generated by the metabolic activity, thereby increasing the carbon efficiency. The
photosyntheticactivitygeneratesROS,whicharedealtwithbyoxidationofPUFAsandother
anti-oxidantmechanismssuchassuperoxidedismutaseorglutathioneperoxidases.Thedirect
supplyofprecursorsforisoprenoidbiosynthesisissupportedbytheincreasedexpressionof
specificenzymes.Foracetyl-CoAinthecytosol,thecitrate-malate-pyruvateshuttle,including
ATP-citrate-lyase,appearstoplayakeyrole,whileforGAPandPYRintheplastidsseveral
sources are likely. Plastidic glycolysis probably plays a major role, but the recycling of
metabolicCO2throughRuBisCOandreplenishmentofcarbonunitsfromtheoxidativepentose
phosphate pathway could contribute significantly to the CBB cycle. Additionally, PYR and
reducing power can be produced by the activity of the plastidic ME from malate. This,
28
however,alsorequiresthepresenceofplastidicmalateaswellasarefluxofC4-unitsfromthe
cytosol into the plastids. Since the low expression of known C4 translocators does not
correspondwiththeobservationofhighlyexpressedplastidicME,theexistenceofnovel,yet
undiscoveredC4transportersisanticipated.
Our model provides an initial framework for understanding these metabolic cell factories.
Evaluatingthecontributionsofindividualenzymestotheglobalcarbonandenergybalanceof
thesecellswillbethefocusofsubsequentwork.Thismodelalsoprovidessomenovelclues
for applications in metabolic engineering of isoprenoids by transposing the knowledge to
othersystems,forexampletoyeastormicroalgae.
Methods
Plantgrowth
LA4024 and LA1777 seeds were obtained from the Tomato Genetics Resource Center, UC
Davis, USA. Plants were grown in the greenhouse on soil in the following conditions: 65%
humidity, in addition to sunlight artificial light tgenerated by MT250DL metal halide lamps
withanintensityof165µmols-1mm2(16hlightperiod),25°C,)andwateredwithtapwater
everytwodays.Onceaweektheplantsweretreatedwithafertilizersolution(0.1%Kamasol
BrilliantBlau,CompoExpertGmbH,Germany).Plantmaterialwasharvested12and14weeks
aftersowingduringthelightphaseintheearlyafternoon.
Harvestoftrichomeandleafmaterial
Fortrichomeharvest,whichalwaystookplaceataroundnoon,20–30cmlongtomatoleaves
cutonebyonewereplacedinthepalmofoneglovedhandandexposedtothelight,and
trichomeswerequickly(inlessthan5s)brushedofftheleaflets(around5cmeach)witha2
cmbroadpaintbrushthathadbeendippedinliquidnitrogenasdescribedinBalckeetal.
(2014).Eachofthethreebiologicalreplicateswerecomposedofleavesof15plantsforLA1777
and 50 plants for LA4024. For each biological replicate, the leaves were cryo-brushed as
describedabove,resultinginapooloftrichomes.Asubsetofthebrushedleavesdevoidof
trichomeswerelikewiseusedtomakeapoolofleaves.Bothpooledtrichomesandpooled
29
trichome-freeleaveswereimmediatelycollectedinmortarsfilledwithliquidnitrogen.The
crudequenchedtrichomefractionwasfurtherenrichedunderliquidnitrogenconditionsby
sieving through steel sieves of 150 µm mesh width (Retsch, Haan, Germany)(Balcke et al.,
2014).Trichome-freeleavesweregentlygroundinamortarlikewisefilledwithliquidnitrogen.
After removal of all liquid nitrogen during storage at –80°C, leaves and trichomes were
lyophilizedovernightforsubsequentmetabolomicsorremainedfrozeninadeepfreezeruntil
use.
Metaboliteextraction
Using wall-reinforced cryo-tubes of 1.6 mL volume (Precellys Steel Kit 2.8mm, Peqlab
BiotechnologieGmbH,Erlangen,Germany)filledwith5steelbeads(3mm),25mgaliquotsof
dry leaf or trichome powder from three independent pools were resuspended in 900 µL
dichloromethane/ethanol(–80°C).Then,200µLof50mMaqueousammoniumacetatebuffer
(0°C,pH6.7)wasaddedtoeachvialandcellrupture/metaboliteextractionwasachievedby
FastPrepbeadbeating(3x20s,speed6.5m/s,1stround–80°C,2ndroundroomtemperature;
FastPrep24 instrument with cryo adapter, MP Biomedicals LLC, Santa Ana, CA, USA). After
phaseseparationbycentrifugationat20,000xg(2min,4°C),150µLoftheaqueousphase
was collected and the Fastprep beating was repeated after adding another 150 µL of
ammonium acetate. After the second centrifugation, another 200 µL aqueous phase was
combinedwiththefirstextractand600µLoftheorganicphasewascollected.Then,500µL
fresh tetrahydrofuran (THF) was added twice to the extraction residue and the Fastprep
beating was repeated. Each time, 450 µL THF extract was combined with the first organic
phaseextract.Theaqueousphasewasthenlyophilizedandtheorganicphasewasdriedina
streamofnitrogengas.
ForanalysisofhydrophilicintermediatesbyionpairingLC-(-)ESI-MS,lyophilizedsamplesof
theaqueousphaseweredissolvedin150µLofdeionizedwaterandfiltratedover0.2µmPVDF
(96well,Corning,450xg,5min,4°C).Foranalysisofsemi-polarintermediatesintheorganic
phase, dried samples were dissolved in 180 µL buffer of 75% methanol and 25% water,
filtratedover0.2µmPVDF(96well,Corning,450xg,5min,0°C),andprocessedbyreverse
phase LC-(+)ESI-MS and reverse phase LC-(-)ESI-MS. Detailed chromatography and mass
30
spectrometryparametersareprovidedinSupplementalTable3,SupplementalTable4and
SupplementalDataset9.
Foranalysisofvolatilemetabolites,surfaceextractswerecollectedfrom6leafletsofcirca5
cmlengthbyadding2mln-Hexaneandshakingfor1minute.Thehexanewastransferredto
acleantubeandcentrifugedat16,000xgfor90secondstoremovedebris.Thehexaneextract
wasthendirectlyinjectedintheGC.
Metaboliteprofiling.
Detectionapproach,datamatrix,datanormalization
Non-targeted ion detection in LC-MS was achieved using an Acquity UPLC (Waters) and a
TripleTOF5600massspectrometerusingthesoftwareAnalyst1.6TF(Sciex,Toronto,Canada).
Hydrophilicmetaboliteswereseparatedbyionpairingchromatography(SupplementalTable
3)andsemi-polarmetaboliteswereseparatedbyreversedphaseUPLC(Balckeetal.,2014).
TOF-MS1-massfeatureswereassayedbetween65–1250Dasimultaneouslywithanarrayof
non-targetedQToF-MS2scanexperimentsbysequentialwindowacquisitionofalltheoretical
fragment-ionspectra(SWATH)-QToF-MS/MS(Hopfgartneretal.,2012).Duringthelatter,the
transmissionrangeforprecursorionsintheQ1-quadrupolwassetfor20mstomasswindows
of33Daltonandwasincrementedfromm/z=65-1250.Simultaneously,CID-fragmentation
wasacquiredinrelationtoeachSWATHtransmissionwindow.Thus,compromisingbetween
Q1 dwell times and MS2 spectra purity, LC-SWATH-MS/MS measurements allow for rapid
assessmentofMS1andMS2spectrawithcycletimesofbelow1s.Intotal,LC-SWATH-MS/MS
resultedin8308MS1distinctmass/retentiontimefeatureswithreliableCIDfragmentation
(3833aqueousphase,3475fromtheorganicphase).OutofallLC-MSfeatures,weassigned
thestructureof243metabolitesbycomparisontotheretentiontime,exactmass(MS1),and
CIDfragmentationpattern(MS2)ofauthenticstandards(SupplementalDatasets1,2and3).
All data presented were normalized to dry weight. Since secondary metabolites of GT
contributesignificantlytothedryweightofGTs,whichisnotthecaseinleaves,normalization
based on dry weight to compare different plant organs may bias the conclusions made.
Therefore,othernormalizationapproachesweretestedincludingnormalizationtothetotal
nitrogencontentthoughttorepresentthetotalpoolofproteinsandnucleicacids.Basedon
31
thisnormalizationhowever,similartrendswereobservedandwechosetopresentthedata
normalizedtodryweight.
Ifauthenticstandardswerenotavailable,wepredictedthemetaboliteidentityoraffiliation
to a class of secondary metabolites by exact mass, isotopic distribution of carbon in MS1
spectra,CIDfragmentationpatterns,andtheplausibilityofthemetaboliteappearanceand
retention time in the chromatogram of aqueous or organic extracts. All LC- and MSparametersandalistofusedauthenticstandardsaregiveninSupplementalDatasets1,2,3,
and9,andSupplementalTables3and4.
Volatilemetabolitesweremeasuredfrom1µlofthehexanesupernatantbyGC–MSonaTrace
GCUltragaschromatographcoupledtoanISQmassspectrometer(ThermoScientific).GC-MS
analyseswereperformedaccordingtoBrückneretal.(2014).
Estimationoftherutin,sesquiterpenecarboxylicacidandterpenecontentinGT
Twenty-five milligrams of dry trichome material were exhaustively extracted by 10
consecutive extractions (short vortexing) with a mixture of 75% methanol and 25% water.
AliquotsoftheextractsweredilutedtomeetthelinearrangeofthefollowingLC-MSanalysis.
Rutin(Sigma)and(E,E)-farnesoicacid(EchelonInc.)purestandardswereusedforexternal
calibration of Rutin in LA4024 and of two major sesquiterpene carboxylic acids ((+)-(E)-αsantalene12-oicacidand(+)-(E)-endo-bergamotene-12-oicacid)inLA1777trichomeextracts.
Sincethelattercompoundswerebothnotcommerciallyavailable,weassumedthesameMS
responseaswithfarnesoicacid.FortheestimationofterpenecontentinLA4024,leafsurface
extractswerepreparedfromabout1.5cmlongleafletsinthreeconsecutiveextractionswith
hexane containing dodecane at a concentration of 0.15 mg l-1 and measured by GC-MS as
describedabove.Theconcentrationoftheterpeneswasestimatedbyaddingthepeakarea
ofthefivemajormonoterpenesdetectedandusingthepeakareaofdodecaneasareference.
Shot-gunproteomics
ProteinExtractionandTrypsinDigestion
One-hundredmilligramsoffrozen,powderedtomatotrichomesorleaves(threeindependent
poolseach)werewashedwith10%TCAinacetonefollowedbyacetonebothat–20°C.The
tissuepelletwassuspendedin400µlofextractionbuffer(100mMTris-HCl(pH8.5),1%(w/v)
32
SDS,25%(w/v)sucrose,5mMEDTA,0.5%(v/v)ß-mercaptoethanoland1%plantprotease
inhibitorcocktail(Roche)),bothaddedfreshandthesuspensionwasmixedvigorouslyfor30
minutesatroomtemperature(RT).Anequalvolumeofwatersaturatedphenolwasadded
andthehomogenatewasfurthermixedfor30minutes.Thehomogenatewascentrifugedat
10,000gfor10minutesatRT.Anequalvolumeofreextractionbuffer(100mMTris-HCl(pH
8.5),20mMKCl,25%(w/v)sucrose,10mMEDTA,0.5%(v/v)ß-mercaptoethanoladdedfresh)
wasaddedtothetopphenolphase.Thehomogenatewasmixedvigorouslyfor15minutesat
RT and centrifuged as described above. Proteins were precipitated from the phenol phase
with5volumesof–20°C100mMammoniumacetateinmethanolovernight.Proteinswere
collectedbycentrifugation,washedtwotimeswith20%50mMammoniumbicarbonate,80%
acetoneanddissolvedin8Murea,50mMammoniumbicarbonate.Theconcentrationofthe
protein solution was measured using the 2-D Quant Kit (GE Healthcare) following the
manufacturer’sinstructions.Disulfidebondswerereducedwith200mMdithiothreitol(DTT),
100mMTris-HClandalkylatedwithanexcessof200mMiodoacetamide(IAA),100mMTrisHCl.Proteinsweredigestedwithtrypsin(enzymetoproteinratio1:50)at37°Covernight.The
peptidesolutionwasdesaltedwithC18reversephasebatchchromatographyinSTAGEtips.
TheC18matrixwasconditionedwith80%ACN,0.1%FAinddH2Oandequilibratedwith0.1%
FAinddH2O,andretainedpeptideswerewashedwith0.1%FAinddH2Oandelutedwith80%
ACN,0.1%FAinddH2O.Peptidesweredriedinavacuumconcentratoranddissolvedin5%
ACN,0.1%TFA.
LiquidChromatographyandMassSpectrometry
Four micrograms of peptides were injected into an EASY nLC1000 liquid chromatography
system from Thermo Fisher Scientific. Peptides were separated using C18 reverse phase
chemistryonanEASY-columnSC001pre-column(length2cm,innerdiameter100µm,particle
diameter5µm)in-linewithanEASY-SprayES803column(length50cm,innerdiameter75
µm,particlediameter2µm)bothfromThermoFisherScientificwithagradientfrom5%to
40%acetonitrilein9handaflowrateof250nl/min.Thecolumntemperaturewassetto40°C.
Peptideionswereelectrosprayedon-lineintoanLTQOrbitrapVelosPromassspectrometer
viaanEASYSprayionsourcebothfromThermoFisherScientific.Thesprayvoltagewas1.9kV,
thecapillarytemperaturewas275°C,theS-lensRFlevelwas50%,themultipoleoffsetwas-7
V.FullMSsurveyscansofthetotalionpopulationwerecarriedoutwitharesolutionof60,000
33
in the Orbitrap mass analyzer. The automatic gain control (AGC) target value was 106; the
maximuminjectiontime(maxIT)was500ms,and1microscanwasacquired.Fullscanswere
followedbyupto20data-dependent(DDA)MS/MSscansofthemostabundantprecursor
ionswithaminimumsignalthresholdof500fragmentedusingcollision-induceddissociation
(CID)intheLTQmassanalyzer.TheAGCtargetvaluewas104;themaxITwas200ms,and1
microscanwasacquired.Thedynamicexclusionrepeatcountwas1andtherepeatduration
was30s,theexclusiondurationwas240s,andtheexclusionmasswidthwas+/-10ppm.
Massspectrawerecalibratedinternallyinrealtimeusingthem/z445.120024.
PeptideandProteinIdentificationandQuantification
Peptidespectralmatches(PSMs)weregeneratedwithMascotsoftwarev2.4.0fromMatrix
Science linked to Proteome Discoverer v1.4 (PD) from Thermo Fisher Scientific. A signal to
noisethresholdof1.5wasusedtofilterionsignalpeaksfromMSfullscans.TheInternational
TomatoAnnotationGroup(ITAG)release2.3ofthetomatoproteomedatabaseamendedwith
commoncontaminants(34727sequences,11956401residues)wassearchedwithaprecursor
iontoleranceof7ppm,afragmentiontoleranceof0.8Da,enzymesettingtrypsin,2tolerated
missed cleavages and carbamidomethylation of cystein as a fixed modification. The family
wise PSM error rate was controlled via the false discovery rate (FDR/q-values) using the
target/decoydatabasemodelforestimatingfalsepositivePSMswiththetargetdecoyPSM
validatormoduleinPD.PeptidesandproteinswereidentifiedusingthePeptideandProtein
ProphetalgorithmsinScaffoldv.4.0.5.A95%peptidethresholdandan80%proteinthreshold
wereequivalenttoa0.01%peptideFDRanda0.2%proteinFDR.Proteinswerequantified
usingthe“QuantitativeValue(NormalizedTotalSpectra)”optioninScaffold.
Transcriptomics
RNApreparation
TotalRNAwasisolatedfromthreeindependentpoolsoftrichome-freeleavesandtrichome
preparationsusingtheQiagenRNeasy™Kit.TheRNAwasDNAsetreatedwiththeAmbion
DNA-free™ Kit, quantified via NanoDrop™ and quality assessed using the Qiagen Qiaxcel™
capillaryelectrophoresissystem.OnlysampleswithanestimatedRINnumberof8orgreater
wereusedfordownstreamapplications.
34
Microarray
Seventeen mRNA samples from six S. habrochaites accessions (LA1731, LA1753, LA1777,
LA2158, LA2167, LA2650) and two S. lycopersicum accessions (LA4024, LA4005) were
sequenced to obtain an average of 59,080,335 single end, 101 bp reads per library
(SupplementalTable5).Readswerealignedtothetomatoreferencegenomeversion2.4using
TopHat2v2.0.6withthefollowingparameters:--max-insertion-length9--max-deletion-length
9-p8-g1--library-typefr-unstranded-m1--read-gap-length12--read-edit-dist12--readmismatches 8 --read-realign-edit-dist 0 --no-coverage-search --segment-mismatches 3. An
averageof82%ofallreadsmappeduniquelytothereference,withslightlymorereadsaligned
intheS.lycopersicumsamples(SupplementalTable5).Foreachalignment,duplicatedreads
werethenremovedandindelswererealignedusingPicardv1.65andGATKv2.2.8withdefault
parameters.Alignmentsforalllibrariesfromthesameaccessionweremerged,resultinginan
averageof103millionreadsperaccession(rangefrom74to166million,SupplementalTable
5).VariantsinallalignmentswerecalledsimultaneouslyusingGATKandtheresultingfilewas
usedtodefineasetofexonicregionscoveredbyRNA-seqreadsbutfreeofanypolymorphism
between LA4024 and LA1777. This analysis resulted in 403,328 regions larger than 25 bp
distributedacross25,892outofthe34,727cDNAspresentintheITAGannotationv2.30.These
sequenceswereprovidedtoAffymetrix,where398,006ofthesesequenceswereselectedto
beincludedinthearray.Inaddition,anumberofprobestogenotypeLA1777andLA4024
were added to the chip. From the 363,286 SNPs found between these two accessions, we
filteredforthosethathadnoothervariantin20bponeachsideandwereatleast20ntaway
fromanexonboundary.Thisresultedin14,557probescontainingasingleSNPthatwerealso
includedinthearray.
Triplicate microarray hybridizations of leaf and trichome RNA preparations of LA1777 and
LA4024 were performed on an Affymetrix GeneAtlas™ System with the custom designed
microarraychips(seedescriptionabove).OnehundredfiftynanogramsoftotalRNAofeach
preparationwasusedasstartingtemplateutilizingtheAmbionWTExpressionKit,theGene
Chip Eukaryotic Poly A RNA Control Kit and the Gene Chip WT Terminal Sequencing Kit
according to manufacturer’s protocols. Throughout the preparation, quality controls were
conducted using the Qiagen Qiaxcel™ capillary electrophoresis system. Hybridization was
performedfor20hat48°C.
35
QuantitativeReal-TimePCR
FirststrandcDNAfromthreebiologicalreplicateswassynthesizedfrom150ngDNAsetreated
totalRNAusingamixofoligo(dT)18andrandomhexamerprimerswiththeProtoScriptIIFirst
Strand cDNA Synthesis Kit (New England Biolabs). Samples were then prepared for the
FluidigmFLEXsix™chipsaccordingtothemanufacturer’smanualandRT-qPCRwasperformed
intechnicaltriplicatesonaFluidigmBiomarkHDsystem.Quantitative(q)PCRprimersforthe
target genes were designed using the Primer3 program (Koressaar and Remm, 2007;
Untergasseretal.,2012)andsequencescanbefoundinSupplementalTable1.Datawere
analyzedusingtheFluidigmRealTimePCRAnalysisSoftwareV.4.1.2andrelativeexpression
tothecontrolgeneSerine/threonine-proteinphosphatase2(Solyc05g006590)wascalculated
usingthe2(ΔCt)method(LivakandSchmittgen,2001).
13
C-Labeling
LabelingwithU13C-glucose:10leaveswith7leafletseachwerecutfromLA1777plantsand
immediatelyputin50mLtubesfilledwith35mLU-13C-glucose(99%purity,EUROISO-TOP,
Saint-AubinCedex,France)(10gL-1intapwater).Theleaveswereexposedtopermanentlight
(165µmols-1mm2)forupto24h.
Labeling with 13C-CO2: Six-week-old LA1777 plants grown in hydroponics on expanded clay
were placed in a labeling cabinet (PhytolabelBox, developed together with Elektrochemie
HalleGmbH)(dimensions:60x60x60cm). 13C-CO2labeling(99%purity,EUROISO-TOP)inthe
light(165µmols-1mm2)forupto180minwasconductedat380ppmpartialpressureof13CCO2, 70% humidity and 20% oxygen, which was supplied from synthetic air (Linde AG,
Germany). Residual atmospheric CO2 was removed by flushing the chamber’s atmosphere
overdiverchalk(Spherasorb,Wokingham,UK)for20minbeforetheonsetof13C-CO2-labeling.
Forharvest,theleaveswerecutatthestalkandwereimmediatelyquenchedinliquidnitrogen
(LN2).AftergentlecrushingofleafmatterinamortarfilledwithLN2,leafpiecesandtrichomes
wereseparatedbyshakingfor20swithcrusheddryiceinSchottbottlesof500mL.During
thisprocess,thelidmustnotbetightlyclosedtoallowpressurerelease.Thefrozenleafpieces
andtrichomeheadsobtainedweresubsequentlysievedinliquidnitrogenover150µmsteel
36
sieves,withthesievebeingrepeatedlyliftedandsettledinLN2.Thetrichome-freeleafpieces
remained on the 150 µm sieve, and the GT heads were collected on a 45 µm sieve. Two
biological replicates were made from pools of three plants each (both for leaves and
trichomes).Allplantmatterwaslyophilizedovernight.Thereafterthesebiologicalreplicates
werefurtherdividedintothreealiquotsof25mgdryweighteach,whichwereextractedand
measured independently as technical replicates. The plant material was kept frozen
throughouttheentireprocedureandstoredovernightat-25°Ctoletthedryiceandliquid
nitrogenevaporate.Furtherstorageoccurredat-80°C.Metaboliteextractionwasperformed
as described above. Each pool was extracted in triplicate, resulting in hexuplicate
determinations. For LC-MS/MS analysis of the aqueous extraction phase by ion pairing
chromatography,aspecialmultiplereactionmonitoringmethod,wasusedwhereisotopolog
isoformsofselectedmetaboliteswererecordedusingaSciex6500QTRAPandthesoftware
Analyst1.7(Sciex,Toronto,Canada)(SupplementalTable4).
Dataanalysis
Metabolomics data. Peak quantification of known compounds by area was done using the
softwareMultiquant3.0(Sciex).Retrospectiveextractionofm/zandretentiontimefeatures
fromuntargetedLC-TOF-MS1runswasperformedusingthesoftwareMarkerView1(Sciex).
PCA and PLS analysis on dry weight-normalized data were conducted using SIMCA 13.0.3
(Umetrics, Umea, Sweden). For this, each individual metabolite signal of interest was first
dividedbytheaverageofthecorrespondingsignalintheleafsamplesofLA1777.Then,the
dataratiowaslog10-transformedandfinallyPareto-scaled.Thisway,up-anddown-regulated
signalsofanyintensityarenormallydistributedbutrelativechangesbetweentrichomeand
leafareemphasized.WearbitrarilychoseGTofLA1777tonormalizealldata.
R2X[1] is the cumulative sum of squares of the entire X that can be explained by principal
component1.(X=log-normalizedpeakheightsrelativetoLA1777leafmatter).Q2X(cum)The
cumulative fraction of the total variation of X and Y that can be predicted by principal
component1forallofitsx-variablesandy-variables(X=log-normalizedpeakheightsrelative
toLA1777leafmatter;Y-variables1=GT;2=leaves).
37
Other omics data processing and statistical analysis. Affymetrix exon microarrays were
hybridized and imaged using the Affymetrix GeneAtlas System and pre-processed by
Affymetrix Power Tools (v. 1.15.1). Multiple probes of each probeset were summarized by
median polish. Raw datasets were normalized by robust multi-array averaging (RMA). To
remove background noise, data were filtered for undetected probesets as described in
(Lockstone, 2011) using Detection Above Background (DABG) tests at the exon-level
implemented in APT. Undetected probesets were excluded prior to differential gene
expressionanalysis.
Linear models were fitted with Bioconductor's limma package (Smyth, 2005) and p-values
were adjusted using Benjamini and Hochberg false discovery rate (< 0.05) procedure
(Benjamini and Hochberg, 1995). Differentially expressed genes were identified by a
significancethresholdof0.05andaminimallog2-fold-changeof±1.
Low expressed proteins detected by quantitative shotgun proteomics were filtered by an
independentfilteringapproachusingthegenefilterpackage(Gentleman,2009)andexcluded
from further analyses. The PLGEM package (Pavelka et al., 2004) was used for differential
expressionanalyseswithasignificancethresholdof0.01.Thesuitabilityofthispackagefor
analyzingdatasetsderivedfromshotgunproteomicswasprovenearlier(Pavelkaetal.,2008).
Afterdataprocessingandnormalization,20445genesand4390proteinsannotatedwitha
Solyc tomato ID formed the basis for gene expression and protein abundance analysis
between trichomes and trichome-free leaves. Individual values as well as the averages of
triplicates,log2-foldchangebetweengroupsandthefalsediscoveryrate(FDR)adjustedpvaluesfromamoderatedt-test(Smyth,2005)areshowninSupplementalDataset5.Principal
componentanalysisandhierarchicalclusteringdemonstratingthatreplicatesgrouptogether
asexpectedareshowninSupplementalFigure17.
Correlation between transcriptomics and proteomics within tissues was estimated by
calculatingthePearsoncoefficient,whichproducedslightlypositivervaluesof0.341(LA1777
leaf), 0.367 (LA1777 trichome) and 0.418 (LA4024-leaf), 0.414 (LA4024 – trichome).
Moderatelypositivecorrelationsbetweentranscriptomeandproteomedataareusualand
havebeenreportedpreviously(Maieretal.,2009).InarecentstudyinZeamays(maize),
similarcorrelationsvalueswereobserved,butenrichmentcategorieswerefoundtobehighly
38
similar between transcriptome and proteome data (Walley et al., 2016). The enrichment
analysis (see Supplemental Dataset 6) points to a similar trend. Functional categories that
showastrongenrichmentatthetranscriptomelevelarealsoenrichedattheproteomelevel
(e.g.,photosynthesis,Calvincycle,isoprenoidmetabolism,lipidmetabolism).
The MapMan hierarchical ontology was used for functional annotation of the studied
transcriptomes and proteomes of both Solanum species (Thimm et al., 2004). The official
mappingfilefortheITAG2.3releasewasusedinallsubsequentanalyses.Enrichmentanalyses
wereperformedusingPageManintheMapManprogram.
PhylogeneticAnalysis
AphylogenetictreeoftheBileAcidSodiumSymporter(BASS)proteinfamilyfromArabidopsis
and tomato (S. lycopersicum) was generated with the Geneious software v 6.1.8
(www.geneious.com).FirsttheBASSproteinsequencesfromthesetwospecieswerealigned
using the MUSCLE alignment algorithm with the following options: maximum number of
iterations=10;distancemeasure=kmer6_6forthefirsttwoiterationsthenpctid_kimurafor
thesubsequentones,clusteringmethod=UPGMB;treerootingmethod=pseudo;sequence
weighingscheme=CLUSTALW;terminalgaps=halfpenalty;objectivescore=spm;anchor
spacing=32;gapopenscore=-1.ThealignmentwasthenfedintotheGeneiousTreeBuilder
with the following options: genetic distance model = Jukes-Cantor; tree build method =
neighbor-joiningwithoutoutgroup;numberofreplicatesforthebootstrapresampling=1000;
supportthresholdof45%.ThetreewasthenexportedintheunrootedlayouttoCorelDraw
formanualeditingandannotatingofBASSfunctions.
Predictionofthesubcellularlocalization
TheproteinsequenceoftheindividualSolGeneIDwasloadedintoWolfPSort(accessiblevia
http://www.genscript.com/wolf-psort.html),
Sherloc2
(http://abi.inf.uni-
tuebingen.de/Services/SherLoc2) and iPSort (http://ipsort.hgc.jp). In some cases, the
respectiveproteinsequenceswereblastedagainsttheclosesthomologinArabidopsisandthe
SUBApredictiontoolwasalsoused(accessibleviahttp://www.arabidopsis.org).
39
Lightmicroscopy
Microscopyanalysisofleavesandtrichomeswasperformedaspreviouslypublished(Bergau
et al., 2015). Trichomes on the leaves were observed in brightfield with an AZ100 (Nikon,
Japan). Fluorescence was observed with a LSM 710 microscope (Zeiss, Jena, Germany).
Autofluorescencewasexcitedat405nmandrecordedat420–545nm(cellwall)and645–735
nm(chloroplasts).
Transmissionelectronmicroscopy
Leaves and trichomes were fixed with 3% glutaraldehyde (Sigma Aldrich,Taufkirchen,
Germany)insodiumcacodylatebuffer(SCB)pH7.2for4hatroomtemperature,washedwith
SCB,postfixedwith1%osmiumtetroxide(CarlRoth,Karlsruhe,Germany)inSCB,dehydrated
inagradedethanolseries,andembeddedinepoxyresin.Afterpolymerization,thematerial
wassectionedwithanUltramicrotomeS(Leica;Wetzlar,Germany).Ultrathinsections(80nm)
were transferred to formvar-coated grids and post-stained with uranyl acetate and lead
citrate.ThesectionswereobservedwithaZeissLibra120transmissionelectronmicroscope
operating at 120 kV (Carl Zeiss Microscopy, Oberkochen, Germany). Images were taken
applyingaDual-SpeedonaxisSSCCDcamera(BM-2k-120;TRS,Moorenweis,Germany).
ROSstaining
Staining with dihydroethidium (Sigma) was performed according to (Owusu-Ansah et al.,
2008).Insteadofthedescribedbuffer,asorbitolbuffer(200mMsorbitol,50mMTris-Cl,20
mM Suc, 10 mM KCl, 5 mM MgCl2, 5 mM succinic acid, 1 mM EGTA, 0.5 mM K2HPO4 and
0,015%TritonX-100)wasused.Stainingoccurredafter3x3minvacuumfiltrationofasingle
tomatoleaflet.Thelaseremissionwavelengthwas514nm.Thefluorescencereadoutwindow
wassetto540–620nm.
AccessionNumbers
ThemetabolomicsrawdataandmetaboliteabundancesareavailableatMetaboLightsunder
the accession number MTBLS297. The mass spectrometry proteomics data have been
deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the
40
datasetidentifierPXD003154.TranscriptomicsrawdataareavailableinArrayExpressunder
theaccessionnumberE-MTAB-4482.
SupplementalData
SupplementalFigure1.Venndiagramsofdifferentiallyregulatedomicssignalswithalog2foldchange>1.
Supplemental Figure 2. Total GC-MS and LC-MS ion chromatograms from S. habrochaites
LA1777andS.lycopersicumLA4024extracts.
SupplementalFigure3.Fattyacidandoxylipinpeakareasfromtrichomeandtrichome-free
leafextracts.
SupplementalFigure4.MS/MSspectraofapeakelutingat13.8minwith[M-H]:m/z295.228.
Supplemental Figure 5. Scores of a principle component analysis comparing 115 selected
signalsfromhydrophilicextractsofleaves(LVS)andtrichomes(TRI)ofLA1777andLA4024.
Supplemental Figure 6. MapMan transcriptomics and proteomics overview of the cellular
metabolisminLA4024.
SupplementalFigure7.MapManproteomicsoverviewofthecellularmetabolisminLA1777.
SupplementalFigure8.TimecourseafterpulselabelingofLA1777with13C-CO2andU-13C-Dglucose.
Supplemental Figure 9. Biosynthesis of isoprenoid precursors in leaves and trichomes of
LA4024andLA1777.
Supplemental Figure 10. ROS staining of LA1777 using dihydroethidium (DHE) and laser
scanningmicroscopy.
Supplemental Figure 11. Transcript levels and protein abundances of lipoxygenases of
cytosolicandplastidiclocalization.
SupplementalFigure12.DifferentialtranscriptandproteinlevelsofREDOXenzymefamilies.
Supplemental Figure 13. 13C-incorporation into citrate and isocitrate after 24 h labeling of
LA1777withU-13C-D-glucoseinpermanentlight.
SupplementalFigure14.MapManoverviewofcytosolicandplastidicglycolysisandplastid
transporters(triosephosphate,putativepyruvateandmalate)inLA1777.
Supplemental Figure 15. Phylogenetic tree of proteins of the bile acid sodium symporter
(BASS)familyfromArabidopsisthalianaandSolanumlycopersicum.
41
Supplemental Figure 16. Expression of NADPH-producing enzyme families in GT versus
trichome-freeleaves.
Supplemental Figure 17. Principle component and hierarchical cluster analyses of
transcriptomeandproteomedata.
SupplementalTable1.GeneswhoseexpressionwasverifiedbyqRT-PCRandcorresponding
primers.
SupplementalTable2.ListofgenesoftheMEVandMEPpathways.
SupplementalTable3.Chromatographyconditions.
SupplementalTable4.MSparametersfortheuntargetedanalysisofhydrophilicandsemipolarmetabolitesby(-)ESI-SWATH-MS/MS.
SupplementalTable5.SummaryofRNAseqsamplesanddata.
SupplementalDataset1.PeakHeightsofsemi-polarmetabolitesasextractedfromTOF-MS1
byMarkerView(ESInegative).
SupplementalDataset2.PeakheightsofhydrophilicmetabolitesasextractedfromTOF-MS1
byMarkerView(ESInegative).
SupplementalDataset3.Peakareasofhydrophilicmetabolites.
Supplemental Dataset 4. Prediction of loadings of the principal component analysis of
hydrophilicmetabolites.
SupplementalDataset5.Transcriptomicsandproteomicsdata.
SupplementalDataset6.Enrichmentanalysis.
SupplementalDataset7.Standarddeviation(n=6measurements)ofisotopologsfrom13Clabelingexperiments.
SupplementalDataset8.MultiplealignmentofBASSproteinsfromArabidopsisthalianaand
Solanumlycopersicum.
SupplementalDataset9.MSparametersforthetargetedanalysisofselectedmetabolitesand
their13C-isotopologsvia(-)ESIandscheduledmultiplereactionmonitoring.
Acknowledgments
WewouldliketoacknowledgetheassistanceofGerdHausewiththeelectronmicroscopy,
(Biozentrum,Martin-LutherUniversitätHalle-Wittenberg)andMarioBauerattheHelmholtzCentreforEnvironmentalResearchforprovidingaccesstotheBiomarkHDsystem.Weare
gratefultoMichaelHahnandcoworkersofElektrochemieHalleGmbHforthecollaborative
42
development of a prototype for 13C-phytolabeling. This work was funded in part by the
DeutscheForschungsgemeinschaft(grantnumberTI800/1)toAT.
AuthorContributions
GBdevelopedthemetaboliteprofilingapproachandthePhytolabelBoxincollaborationwith
ElektrochemieHalleGmbH,performedtheomicsdataanalysis,andwrotethemanuscript.SB
extracted RNA, conducted hybridizations, and performed RT-qPCR and GC-MS runs. NB
prepared the plant material and extracts, conducted LC-MS runs, contributed to the data
analysis,performedROSstaining,andgeneratedmicrographs.AHassistedGBandNBwith
sample preparation. BA performed bioinformatics data analysis of transcriptome and
proteomedata.PMandWHperformedshot-gunproteomicsmeasurementsandanalysis.
JJGanalyzedtheRNAseqdataoftomatoanddesignedthegeneexpressionmicroarraybased
on these data. AT conceived and supervised the project, performed transcriptome and
proteome data analysis, and wrote the manuscript. All authors read and revised the
manuscript.
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Figure 1. Bright field, fluorescence and electron microscopy of type VI
glandular trichomes. Light microscopy: leaflets of S. lycopersicum LA4024 (A)
and wild-type S. habrochaites LA1777 (C) showing different trichome density. (B
and D) Left side: section of leaves from LA4024 (B) and LA1777 (D) showing the
epidermis and mesophyll cells; right side: fluorescence microscopy of trichome
head cells from LA4024 (B) and LA1777 (D) viewed from above (top) or from the
side (bottom). Transmission electron microscopy: (E) section of a leaf from
LA1777 highlighting the thickness of the outer cell wall and cuticle of the
epidermal cells and the cell wall of internal cells; (F) head of a type VI trichome
from LA1777 illustrating the size of the intercellular storage cavity and the
thickness of the outer cell wall.
A
B
Component R2X
1
2
3
Q2(cum)
0.775 0.743
0.152 0.906
0.047 0.962
LA1777TRI
LA4024TRI LA1777LVS
LA4024LVS Figure 2. Principle component analysis of semi-polar metabolites measured in
negative ESI-mode. The data consist of 3476 signals, each corresponding to a unique m/z
and retention time combination. A) Scores and B) Loadings. In the loading plot, the colors of
the circles correspond to metabolite classes of interest, and the size of the circle reflects the
average pool size of the respective metabolite over all of the samples. For simplicity, only
signals that could be associated with a metabolite group are shown (in total 129). The entire
data set, i.e. including unknowns, is presented in Supplemental Dataset 1. All data are
weight-normalized and Pareto-scaled. Abbreviations: FA – fatty acids, PUFA –
polyunsaturated fatty acid, AS – acyl sugar, SQTCA - sesquiterpene carboxylic acid; R2X Cumulative sum of squares of the entire X explained by principal components 1-3. (X = lognormalized peak heights relative to LA1777 leaf matter); Q2X(cum) - Cumulative fraction of
the total variation of X and Y that can be predicted by principal components.
Figure 3 . Loadings of PLS-analyses of 115 selected MS signals from
hydrophilic extracts. Red: related to photosynthesis and starch formation,
black: related to isoprenoid biosynthesis, blue: lipid, yellow: inositol
polyphosphates. A: analysis of polar metabolites of LA1777; B: analysis of
polar metabolites of LA 4024. For abbreviations, see Supplemental
Dataset 3. R2X[1]: cumulative sum of squares of the entire X explained by
principal component 1 (X = log-normalized peak heights relative to LA1777
leaf matter); Q2X(cum): cumulative fraction of the total variation of X that
can be predicted by principal component 1 for all of its x-variables
(response variables: 1 = GT; 2 = leaves). Mutual predictability for both data
sets was demonstrated in Supplemental Dataset 4.
Figure 4. MapMan overview of the transcriptomics of cellular metabolism
in LA1777. The color scale (bottom right hand corner) corresponds to log2fold changes for trichome versus trichome-free leaves with red being
significantly overexpressed in trichomes and blue overexpressed in leaves (p
< 0.05, Fisher exact t-test). White squares: log2-fold changes between -1 and 1
or t-test p > 0.05).
Figure 5 . Transcript profiles of genes involved in sucrose
degradation and transport in tomato leaves and trichomes.
A: cell wall invertases; B: cytosolic invertases; C: cytosolic
sucrose synthases; D : invertase inhibitors; E: plastidic
invertases; F sucrose symporter. Data presented are normalized
fluorescence counts from the microarray data (see details in
Supplemental Dataset 5). All leaf versus trichome differential
expression within a species are significantly different (Student’s ttest p < 0.05) except those annotated by an asterisk *.
Figure 6. Distribution of the 2000 most highly expressed genes in
ontology groups in leaves, trichomes and trichomes versus leaves. In A
(leaves) and B (trichomes), the 2000 most highly expressed genes were
grouped according to Mapman ontology. The categories that represent less
than 2% of the cumulative expression of these 2000 genes were grouped
together under “other”. C: as in B, except the 2000 top expressed genes are
those with a fold change in expression of over 2 in GTs versus leaves. For
detailed information, see Supplemental Dataset 5. All data are based on the
average of n=3 individual hybridizations per group.
Figure 7. Cumulative transcript expression of
photosynthesis genes. A) light reactions, B)
carbon fixation and carbonic anhydrase. For
details, see Supplemental Dataset 5, PS1 –
photosystem I, PS2 - photosystem II.
Figure 8. Time course after 13C-pulse labeling of LA1777 with 13C-CO2 and
U13C-glucose. Panel A: 13C-CO2, panel B: U13C-Glucose in the presence of ambient
CO2. Blue: total pool sizes of 3-phosphoglycerate (3-PGA), ribulose-1,5bisphosphate (RU-1,5-BP), and sucrose normalized to sample dry weight. Red:
fraction of carbon labeled in these metabolites. Error bars represent the average ±
s.d. (n=6). The fraction of labeled carbon represents the sum of all labeled C from all
measured isotopologs. All the data have been corrected for the natural isotopic
abundance of 13C-isotopes. Relative isotopolog abundances are presented in
Supplemental Figure 8. Numbers above the red bars represent the relative 13C
enrichment in the respective metabolites. Differences in the relative 13C-enrichment
between leaf and trichome for a given time point are all significant based on
heteroscedastic t-tests (p < 0.05) unless indicated by *.
Figure 9. Expression map of the citrate-malate-pyruvate shuttle in GTs compared to
leaves. Log2-fold changes between trichomes and trichome-free leaves of LA1777, relative
transcript expressions (left boxes) and relative protein abundances (right boxes). Red indicates
significant (p <0.05; t-test) overexpression in trichomes, blue significant overexpression (p <
0.05; t-test) in leaves, and white non-significant changes (p > 0.05 ; t-test). The subcellular
localization of the respective enzymes was manually checked using the software tools listed in
the Methods section. The horizontal blue bar represents the mitochondrial envelope and the
yellow discs with dark blue circle the pyruvate-proton symporter (left) and the malate-citrate
antiporter (right). cPK: cytosolic pyruvate kinase; PEPC: phosphoenolpyruvate carboxylase;
PEPCK: phosphoenolpyruvate carboxykinase; cME: cytosolic malic enzyme; cMDH: cytosolic
malate dehydrogenase; mMDH: mitochondrial malate dehydrogenase; mCS: mitochondrial
citrate synthase; mPDH: mitochondrial pyruvate dehydrogenase.
Figure 10. A putative model of central carbon and energy metabolism in tomato
GTs. The figure represents a type VI glandular cell with its plasma membrane as a black
line. The three main compartments involved (chloroplast, cytosol, mitochondria) are
indicated in blue. The yellow star represents the sun which emits light of
photosynthetically active wavelengths (l). These allow the photosystems in thylakoid
membranes (represented by stacks of green horizontal bars) to produce chemical
energy (ATP) and reducing power (NADPH). Photosynthesis and metabolic activity are
accompanied by the production of ROS which are detoxified by PUFAs and glutathione.
CO2 is in red in reactions where it is released and in green in reactions where it is fixed.
Black arrows between metabolites represent either metabolic pathways or reactions
which are discussed in detail in the main text. Abreviations: AcCoA: Acetyl-CoA; C6:
hexose; CBB Cycle: Calvin-Benson-Bassham cycle; CIT: citrate; DMAPP: dimethylallyl
diphosphate; GA3P: glyceraldehyde-3-phosphate; GSH: glutathione; IPP: isopentenyl
diphosphate; MAL: malate; MEP: methylerythritol 4-phosphate pathway; MEV:
mevalonate pathway; OA: oxaloacetate; OPP: oxidative pentose phosphate pathway;
PUFAs: polyunsaturated fatty acids; PYR: pyruvate; Rib5P: ribulose-5-phosphate; ROS:
reactive oxygen species.
Multiomics of tomato glandular trichomes reveals distinct features of central carbon metabolism
supporting high productivity of specialized metabolites
Gerd Balcke, Stefan Bennewitz, Nick Bergau, Benedikt Athmer, Anja Henning, Petra Majovsky, José
M. Jiménez-Gómez, Wolfgang Hoehenwarter and Alain F Tissier
Plant Cell; originally published online April 13, 2017;
DOI 10.1105/tpc.17.00060
This information is current as of June 17, 2017
Supplemental Data
/content/suppl/2017/04/14/tpc.17.00060.DC1.html
/content/suppl/2017/06/05/tpc.17.00060.DC2.html
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