ncomms-tomato


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ARTICLE
Received6Mar2015
Accepted14Sep2015
Published26Oct2015
Multi-levelengineeringfacilitatestheproduction
ofphenylpropanoidcompoundsintomato
YangZhang
*,EugenioButelli
*,SalehAlseekh
,TakayukiTohge
,GhanasyamRallapalli
,JieLuo
PrashantG.Kawar
,LionelHill
,AngeloSantino
,AlisdairR.Fernie
&CathieMartin
DOI:10.1038/ncomms9635
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heuseoftranscriptionfactors(TFs)toactivateorsuppress
Shikimate
pathway
Pentose
phosphate
pathway
Figure1|Schematicrepresentationofthephenylpropanoidpathwayin
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Fig.1b).SinceSlMYB12isnotexpressedintomatoesh
,esh
offeredarobustcontrolonwhichtoestablishenrichmentinChIP
experiments(Fig.3b).ChIP-qPCRthatscannedbindingof
SlMYB12acrossthepromotersof
inpeel
ofWTtomatoes,showedthatSlMYB12bindstothesameDNA
motifsasAtMYB12(Fig.3c).Consistentwiththisresult,the
01234
5.005.0
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expressionof
PeelFlesh
PeelFleshPeelFlesh
PeelFlesh
PeelFlesh
SIENOSIENO
SICHS1SIF3HSIFLSproSICHS1
abc
Figure3|AtMYB12andSlMYB12binddirectlytothepromoterregionsof
SlDAHPS
)BindingofAtMYB12totheregionsof
SlDAHPS
wasanalysedbyChIP-qPCR.Pericarp(peelplusesh)samplesofWTand
AtMYB12
fruitwereharvestedat3daysafterbreaker.The
numbersonthehorizontalaxisbelowthebarscorrespondtotheleftandrightborderoftheampliedregionrelativetothetranscriptionstartsitean
dbars
indicatemeansands.e.m.s(
3);asterisksindicatesignicantdifferencescomparedwiththenegativecontrol(
value
0.05,Students
lMYB12
expressionispredominantlyinthepeelofWTtomatofruitandisassociatedwithelevatedexpressionofgenesinvolvedinbothprimaryand
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aminoacids(exceptphenylalanine)weresignicantlyhigherthan
inWTfruit(SupplementaryFigs2and3).Greatercarbonux
throughglycolysis,thepentosephosphatepathwayandtheTCA
cyclein
AtMYB12
tomatoes,wouldprovideadditionalATP,
123456
00.511.5
00.0040.008
00.020.04
00.0050.010.0150.02
00.00050.0010.0015
0123
00.0020.0040.006
Figure4|AtMYB12changestheuxofcarbonintomatofruit.
NATURECOMMUNICATIONS|DOI:10.1038/ncomms9635
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Arabidopsis
2.00.08.0
abc
Figure5|Co-expressionof
AtMYB12
withothertranscriptionfactorsintomatofruitenhancesphenylpropanoidproduction.
)PhenotypesofWT,
AtMYB12
Del/Ros1
andIndigo(
Del/Ros1
AtMYB12
)tomatofruit.Picturesweretakenatsevendaysafterbreaker.(
)RT-qPCRdataindicatedthat
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inavanone3-hydroxylaseactivity(F3H)(Fig.6a)
tomake
AtMYB12are
.Comparedwith
tomatofruithadonly30%avonolsintheirfruit
(SupplementaryFig.6).Wechosethe
linewhichhadthe
highestgenistincontentinfruit(SupplementaryFig.5)and
crossedittoan
AtMYB12are
tomatolineandselected
differentgenotypesintheF2generation(Fig.6aand
SupplementaryFig.7).Infruitexpressing
only,the
StSyMYB12StSy
StSyMYB12StSy
IFSIFS
IFSIFS
Phenylalanine
4-Coumarate CoA
Naringenin chalcone
Naringenin
Anthocyanins
Flavonols
Dihydroflavonol
Resveratrol
PAL
Total isoflavones
Total flavones
Total flavonol
Total resveratrol
Res-Glc isoform
DWmg g
DW
DW
DW
abc
Figure6|Co-expressionof
AtMYB12
withotherstructuralgenesintomatofruitenhancesnovelphenylpropanoidproduction.
)Schematic
representationofresveratrolandgenisteinbiosynthesis.Bothpathwaysarisefromavonoidbiosynthesis(inblack)bytheadditionof
VvStSy
respectively.Aftersynthesis,bothresveratrolandgenisteinareglycosylatedtoformpiceidandgenistin.Thenaturallyoccurring
mutantisdecientin
F3Hactivity.(
)ThecontentsofCGA,totalavonolsandtotalresveratrolcompoundsindifferentgenotypes.Fruitwereharvested10daysafterbreaker.
Errorbarsshows.e.m.(
3).(
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possibilitythatthelargeeffectsofAtMYB12areduesolelytothe
increasedsinkstrengthofthephenylpropanoidpathwaygiven
thatinhibitorstudiesorRNAiofPALindevelopingfruitwould
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aftercentrifugationat2,000
at4
Cfor10minandresuspendedagainwith1ml
Hondabuffer,repeatingthecentrifugationandresuspensionatleastthreetimes,
tovisualizethepeaksinthetomatogenome.
Feedingexperimentswith
C-and
C-labelledglucose
Pericarpdiscsof
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Aminoacidmeasurements
Fruitwereharvestedsevendaysafterbreakerand
at4
Cfor10min,and
centrifugationat11,000
at4
Cfor10min,wasevaporatedwithoutheating.
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36.Zhang,Y.,Butelli,E.&Martin,C.Engineeringanthocyaninbiosynthesisin
Curr.Opin.Plant.Biol.
8190(2014).
37.Bilal,I.,Chowdhury,A.,Davidson,J.&Whitehead,S.Phytoestrogensand
preventionofbreastcancer:thecontentiousdebate.
WorldJ.Clin.Oncol.
705712(2014).
38.Rallapalli,G.
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