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Geneexpressiondynamicsandinter-tissuerecognitionmechanismsduring1
tissuefusionattheArabidopsisgraftjunction2
3
CharlesWMelnyk1,2,AlexanderGabel3,ThomasJHardcastle4,SarahRobinson5,4
ShunsukeMiyashima6,IvoGrosse3,ElliotMMeyerowitz1,75
6
1-SainsburyLaboratory,UniversityofCambridge,BatemanStreet,Cambridge7
CB21LR,UK8
2–DepartmentofPlantBiology,SwedishUniversityofAgriculturalSciences,9
Almasallé5,75651Uppsala,Sweden10
3-InstituteofComputerScience,MartinLutherUniversityHalle–Wittenberg,11
06120Halle(Saale),Germany12
4–DepartmentofPlantSciences,UniversityofCambridge,DowningStreet,13
CambridgeCB23EA,UnitedKingdom14
5-InstituteofPlantScience,UniversityofBern,Altenbergrain21,3013Bern,15
Switzerland16
6–GraduateSchoolofBiologicalSciences,NaraInstituteofScienceand17
Technology,8916-5Takayama,Ikoma,Nara630-0192,Japan18
7–HowardHughesMedicalInstituteandDivisionofBiologyandBiological19
Engineering,CaliforniaInstituteofTechnology,1201EastCaliforniaBoulevard,20
Pasadena,CA91125,USA21
22
Contact:[email protected]
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ABSTRACT34
35Theabilityforcuttissuestojointogetherandformachimericorganismisa36
remarkableandagriculturallyimportantpropertyofmanyplants,however,itis37
poorlycharacterizedatthemolecularlevel.Tobetterunderstandgraftingwe38
monitoredtemporalandspatialchangesingeneexpressioningraftedtissues.39
Tissuesaboveandbelowthegraftrapidlydevelopedasymmetrysuchthatmany40
genesweremorehighlyexpressedononesidethantheother.Thisasymmetry41
correlatedwithsugarresponsivegenesandweobservedanaccumulationof42
starchabovethegraftthat,afterthreedays,decreasedalongwithasymmetry43
oncevascularconnectionswerere-established.Despiteaninitialstarvation44
responsebelowthegraft,manygenesassociatedwithcelldivisionandvascular45
formationwererapidlyactivatedingraftedtissuesbutnotincutandseparated46
tissuesindicatingarecognitionmechanismthatwasindependentoffunctional47
vascularconnections.Auxinresponseingraftedplantsdifferedfromthatincut48
andseparatedplantsasresponsewasenhancedandsymmetric,suggestingthat49
auxinwasperceivedbytherootwithinhoursofreattachmenttoactivated50
vascularregeneration.Sucharecognitionprocesscouldhavebroaderrelevance51
forwoundhealing,inter-tissuecommunicationandtissuefusionevents.52
53
INTRODUCTION54
Formillenniapeoplehavecutandrejoinedplantsthroughgrafting.Generating55
suchchimericorganismscombinesdesirablecharacteristicsfromtwoplants,56
suchasdiseaseresistance,dwarfingandhighyields,orcanpropagateplantsand57
avoidthedelaysentailedbyajuvenilestate(Goldschmidt,2014).Agriculturally,58
graftingisbecomingmorerelevantasagreaternumberofplantsandspeciesare59
graftedtoincreaseproductivityandyield(Leeetal.,2010).However,our60
mechanisticunderstandingofgraftingandthebiologicalprocessesinvolved,61
includingwoundhealing,tissuefusionandvascularformation,remainlimited.62
63
Plantshaveefficientmechanismstohealwoundsandcuts,inpartthroughthe64
productionofwound-inducedpluripotentcellstermedcallus.Thecallusfillsthe65
gaporsealsthewound,andlater,differentiatestoformepidermal,mesophyll66
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andvasculartissues(Melnyk,2017c).IngraftedArabidopsishypocotyls,tissues67
adhere1-2daysaftergraftingandthephloem,thetissuethattransportssugars68
andnutrients,connectsafterthreedays(Yinetal.,2012;Melnyketal.,2015).69
Thexylem,tissuethattransportswaterandminerals,connectsaftersevendays70
(Melnyketal.,2015).Planthormonesareimportantregulatorsofvascular71
formation,andatthegraftjunction,bothauxinandcytokininresponsesincrease72
inthevasculartissue(Yinetal.,2012;Melnyketal.,2015;Matsuokaetal.,2016).73
Auxinisimportantfordifferentiationofvasculartissueswhereascytokinin74
promotesvascularstemcells,termedcambium,todivideandproliferateina75
processknownassecondarygrowth(Matsumoto-Kitanoetal.,2008;Leyser,76
2011).Auxinisproducedintheupperpartsofaplantandmovestowardsthe77
rootsviacell-to-cellmovement.Auxinexporters,includingthePINproteins,78
transportauxinintotheapoplast,whereasauxinimporters,suchastheAUXand79
LAXproteins,assistwithauxinuptakeintoadjacentcells(Leyser,2011).80
Disruptingthistransport,suchasbymutatingPIN1,inhibitshealingofa81
woundedstem(Asahinaetal.,2011).Blockingauxintransportwiththeauxin82
transportinhibitorTIBA(2,3,5-triiodobenzoicacid)intheshootinhibitsvascular83
formationandcellproliferationattheArabidopsisgraftjunction(Matsuokaetal.,84
2016).Inadditiontoauxin,othercompounds,includingsugars,contributeto85
vascularformation.Thelocalisedadditionofauxintocallusinducesphloemand86
xylembutrequiresthepresenceofsugar(Wetmore&Rier,1963;Aloni,1980).87
Inplants,sugarsareproducedintheleavesandtransportedthroughthephloem88
totheroots(Lough&Lucas,2006).Theroleofsugarsinvascularformationand89
woundhealingisnotwellestablished,however,sugarspromotecelldivisionand90
cellexpansion(Wang&Ruan,2013),processesimportantfordevelopment91
includingvascularformation.92
93
Themolecularandcellularmechanismsforwoundhealing,tissuereunionand94
graftformationremainlargelyunknown.Oneemergingthemeisthatthetopand95
bottomofthecutdonotbehavesimilarly.Suchtissueasymmetryoccursinother96
planttissues,mostnotablyleaves.Developingleafprimordiahaveaninherent97
asymmetrythatisthoughttoderivefrompositionalsignalsfromtheshootapical98
meristemthatspecifiesdifferencesbetweenthetopandthebottomoftheleaf.99
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Themolecularmechanismsthatestablishasymmetryarenotwelldescribed,100
thoughonehypothesisisthatauxincontributestowardsoractsontheputative101
signal(Chitwoodetal.,2007).AsymmetryalsoappearsincutArabidopsis102
inflorescencestemswherethetranscriptionfactorRAP2.6Lexpresses103
exclusivelybelowthecutwhereasthetranscriptionfactorANAC071expresses104
exclusivelyabovethecut(Asahinaetal.,2011).Bothwereimportantforstem105
healingandANAC071andaclosehomologue,ANAC096,wereimportantforgraft106
formation(Matsuokaetal.,2016).Asymmetryalsoexistsingenetic107
requirements,sinceALF4andAXR1,twogenesinvolvedinauxinperception,are108
importantbelowbutnotabovethegraftjunctionforphloemconnection(Melnyk109
etal.,2015).However,ANAC071isexpressedsymmetricallyaroundthe110
hypocotylgraftjunctionthreedaysaftergrafting(Matsuokaetal.,2016)sothe111
extentofasymmetryandthemechanisticbasisforitduringwoundhealing112
remainslargelyuncharacterised.113
114
Previouseffortshavecharacterisedwoundhealingandtissuereunionusing115
transcriptomicanalyses.Mechanicalwoundingalteredapproximately8%ofthe116
Arabidopsistranscriptomeandshowedahighdegreeofoverlapwith117
transcriptomicchangeselicitedbypathogenattackandabioticstress(Cheonget118
al.,2002).Stemwoundingandwound-inducedcallusformationalteredthe119
expressionofhundredsorthousandsofgenes(Asahinaetal.,2011;Iwaseetal.,120
2011;Ikeuchietal.,2017),whereasgraftinggrapevines,lycheetreesand121
hickorytreesinducedhundredsorthousandsofdifferentiallyexpressedgenes122
involvedinhormoneresponse,woundresponse,metabolism,cellwallsynthesis123
andsignaltransduction(Zhengetal.,2010;Yinetal.,2012;Cooksonetal.,2013;124
Cooksonetal.,2014;Chenetal.,2017).Thesegraftingstudiesprovidelimited125
information,astissuesfromaboveandbelowthegraftjunctionwerenotisolated126
totestwhetherthesetissuesbehaveddifferently,andcontrolswerenot127
performedtodistinguishhowgraftingandtissuefusionmightdifferfroma128
responseassociatedwithcuttissuesthatremainedseparated.Here,weperform129
anin-depthanalysistodescribethespatialandtemporaltranscriptional130
dynamicsthatoccurduringhealingofcutArabidopsistissuesthatarejoined131
(grafted)orleftunjoined(separated).Wefindthatthemajorityofgenes132
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differentiallyexpressedareinitiallyasymmetricallyexpressedatthegraft133
junctionandthatmanyofthesegenesaresugarresponsive,whichcorrelates134
withseveringofthephloemtissueandtheaccumulationofstarchabovethe135
junction.However,genesassociatedwithcelldivisionandvascularformation136
activateonbothsidesofthegraftand,similarly,auxinresponsivenessactivates137
equallyonbothsides.Weproposethatthecontinuoustransportofsubstances,138
includingauxin,independentoffunctionalvascularconnections,promoted139
divisionanddifferentiation,whiletheenhancedauxinresponseandblockedof140
sugartransportprovidedauniquephysiologicalconditiontoactivategenes141
specifictograftformationthatpromotewoundhealing.142
143
RESULTS144
Genesareasymmetricallyexpressedaroundthegraft145
Previousanalysesrevealedanasymmetryinthegeneticrequirementsfortissue146
reunionorgraftformationandidentifiedseveralgenesexpressedaboveacut147
thatwerenotexpressedbelow(Asahinaetal.,2011;Melnyketal.,2015).To148
investigatewhetherasymmetrywasacommonfeatureofgraftingandtissue149
reunion,wegeneratedRNAdeepsequencinglibrariesfromArabidopsisthaliana150
hypocotyltissuesimmediatelyaboveandimmediatelybelowthegraftjunction0,151
6,12,24,48,72,120,168and240hoursaftergrafting(HAG)(Figure1A).Prior152
toRNAextraction,weseparatedtopandbottomtissuesatthegraftjunction.We153
foundthatthestrengthrequiredtobreakapartthegraftjunctionincreased154
linearly(FigureS1)similarlytopreviouslyreportedbreakingstrengthdynamics155
ofgraftedSolanumpennelliiandSolanumlycopersicum(Lindsayetal.,1974;156
Moore,1984).Whenpullingapartgraftstoseparatetopandbottomforsample157
preparation,graftsbrokecleanlywithminimaltissuefromonehalfpresentin158
theotherhalf(FigureS1,MovieS1,S2).Wemeasuredtheamountoftissuefrom159
topsadherenttobottomsandviceversa(FigureS1)andfoundlessthan4%160
cross-contamination.Inadditiontografting,wealsopreparedlibrariesfrom161
ungraftedhypocotyls(“intact”treatment)andcutplantsthathadnotbeen162
reattached(“separated”treatment)(Figure1A).Wehereinrefertotissues163
harvestedabovethegraftjunctionorfromtheshootsideofseparatedtissueas164
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“top”andthatfrombelowthegraftorfromtherootsideofseparatedtissueas165
“bottom”(Figure1A).166
167
RNAsthatweredifferentiallyexpressedasaconsequenceofgrafting(compared168
tointacthypocotyls)equallyintopsandbottomsofgrafts(symmetrically169
expressed)orweremorehighlyexpressedinonetissuethantheother170
(asymmetricallyexpressed),wereidentifiedbyperformingapairwiseanalysisof171
theprotein-codingtranscriptomedatasetsthatweredifferentiallyexpressed172
relativetotheintactgroup.SeveralthousandRNAswereidentifiedthatfiteither173
patternofexpressionincludingthetranscriptofthecambialmarkersHCA2that174
wasinducedsymmetrically,andANTthatwasinducedasymmetrically(Figure175
1B-G,FigureS2).6to48hoursaftergrafting,thenumberofgraft-differentially176
expressedgenesthatwereasymmetricallyexpressedwasroughly3-foldgreater177
thanthosesymmetricallyexpressedindicatingthattissuesabovethecut178
changedtheirexpressiondynamicsrelativetobelowthecut.However,at72179
hoursthenumberswerenearlyequal,andby120hours,thenumberof180
symmetricallydifferentiallyexpressedgeneswas3-foldgreaterthanthose181
asymmetricallyexpressed(Figure1B).Asasecondapproach,weperformeda182
hierarchicalclusteringanalysisthatindicatedthatthegraftedtopandgrafted183
bottombecamemostsimilarafter72hours(FigureS3),consistentwiththe184
symmetryanalysis(Figure1B).Thus,grafthealingandtissuereunionpromoted185
ashiftfromasymmetrytosymmetry(Figure1B).186
187
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188Figure1.Graftingrapidlyactivatesgenesinbothasymmetricand189symmetricpatterns.(A)Intact,cutandseparated,orcutandgrafted190Arabidopsistissueswereharvestedapproximately0.5mmabove(top),0.5mm191below(bottom)thecutsiteorforintactplants1mmsegmentsspanningthe192regionwherecutsweremadeingraftedandseparatedplants.(B)Pairwise193analysisbetweenthegraftedtopandgraftedbottomidentifiedsetsofprotein-194codinggenessymmetricallyorasymmetricallyexpressedatthegraftjunction,195withanFDR<0.05andalikelihoodofsymmetric/asymmetricexpressionof196greaterthan50%.Asymmetricallyexpressedgeneswerefurtherdividedinto197thosewhoseRNAswerehigherinthetop(orangedottedline)orhigherinthe198bottom(purpledottedline),againwithanFDR<0.05andalikelihoodof199asymmetricexpressiongreaterthan50%.(C,D)Expressionprofilesfor200transcriptsofcambium-associatedgenesthatinitiallyactivatesymmetrically201(HCA)orasymmetrically(ANT)plottedforintact,separatedandgraftedsamples.202
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(E-G)HCA2transcriptionupregulatesabovethegraftjunction(E)andbelowthe203graftjunction.(F)pHCA2::RFPwasgraftedtoCol-0roots(E)orCol-0shoots(F)204toavoidambiguityofsignaloriginatthejunction.HCA2wasalsoupregulatedin205separatedshootsbutnotinintactsamplesorinhighlevelsinseparatedroots206(G).HAG,hoursaftergrafting.HAS,hoursafterseparation.Whitetriangle207denotesinitialfluorescentsignal,dashedlinesdenotethegraftjunction.208209Sugarresponsecorrelateswithasymmetricgeneexpression210
Theshiftfromasymmetrytosymmetrycouldbeduetophloemreconnectionat211
72hours((Melnyketal.,2015),Figure2A)andtheresumptionofhormone,212
proteinandsugartransport.Wetestedaroleforsugarbygraftinginthe213
presenceofexogenoussucrosewhichhaspreviouslybeenreportedtoaffect214
graftingsuccess(Marsch-Martinezetal.,2013).Lowlevelsofexogenoussucrose215
loweredgraftingefficiency(Figure2A),suggestingthatdifferentialsugar216
responsesatthegraftjunctionmightbeimportantforvascularreconnection.217
ExpressionofApL3,agenewhoseexpressionisinducedbysugar(Villadsen&218
Smith,2004),wasrapidlyupregulatedinseparatedtopsandgraftedtops,219
whereasexpressionofDIN6,GDH1andSTP1,geneswhoseexpressionis220
repressedbysugar(Thumetal.,2004;Villadsen&Smith,2004;Cordobaetal.,221
2015),wasrapidlyupregulatedinseparatedbottomsandgraftedbottoms222
(Figure2B,FigureS4).Theseobservationswereconsistentwithsugar223
accumulationinthegraftedtopandsugardepletioninthegraftedbottom.The224
expressionofthesegenesreturnedtolevelssimilartointactsamplesby120225
hoursand,withtheexceptionofApL3,thegraftedsamplesnormalised226
expressionmorerapidlythantheseparatedtissues.Genesassociatedwith227
photosynthesisincreaseexpressioninseparatedbottoms24hoursaftercutting,228
acommonresponsetostarvation(Wang&Ruan,2013),butlikelytoolateto229
affectsugarlevelsbefore24hours(FigureS4).Atranscriptionaloverlapanalysis230
withRNAsfromknownglucose-responsivegenes(TableS1)revealedastrong231
overlapwithgenesdifferentiallyexpressedbygrafting.RNAsfromknown232
glucose-inducedgeneswereupregulatedinseparatedtopsandgraftedtops,233
whereastranscriptsfromknownglucose-repressedgeneswereupregulatedin234
separatedbottomsandgraftedbottoms(Figure2C,FigureS4).Thistrendwas235
notobservedwithgenesdifferentiallyexpressedbymannitoltreatment(Figure236
S4),suggestingtheeffectwasspecifictometabolicallyactivesugars.Tofurther237
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investigatethiseffect,westainedgrafted,separatedandintactplantswithLugol238
solutiontoassayforthepresenceofstarch.Stainingabovethegraftjunction239
increased48-72hoursaftergrafting(Figure2D).By120hours,stainingwas240
equalonbothsidesofthegraftwhereasinseparatedtopsstainingbecame241
strongerafter72hours(Figure2D,FigureS4).Weconcludedthatstarch242
accumulatedabovethegraftjunction,butafter72hours,thisasymmetry243
disappeared.Totestwhethertheaccumulationofstarchandincreasedsugar244
responsivenesscouldexplaintheobservedtransitionfromasymmetryto245
symmetry,wecomparedourdatasetstopreviouslypublishedgenesthatare246
inducedbystarvationorareinducedbysucrosere-addition(TableS1).Inearly247
timepoints,20-31%ofasymmetricallyexpressedgenesaregenesknownto248
respondtosugarscomparedto2-5%ofsymmetricallyexpressedgenes(Table249
1).However,at72hours,theoverlapbetweenasymmetricallyexpressedgenes250
andsugarresponsivegenesreducedsubstantially(Table1).251
252Figure2.Asymmetricchangesinaccumulationofsugar-responsiveRNAs253andofstarchoccuratthegraftjunction;equalizingsucrosebetweenthe254graftedsegmentsreducesanddelaysphloemreconnection.(A)pSUC2::GFP-255expressingArabidopsisshootsweregraftedtoCol-0wildtyperootsandGFP256
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movementtotherootswasmonitoredover7daysforphloemconnectioninthe257presenceorabsenceofvariousconcentrationsofsucrose.(B)Expressionprofiles258fortranscriptsofasugar-repressedgene(GDH1),andasugar-inducedgene259(ApL3)wereplottedforintact,separatedandgraftedsamples.(C)260Transcriptionaloverlapbetweenpreviouslypublishedglucose-inducedor261glucoserepressedgenesandourdataset.Thenumberinbracketsrepresentsthe262numberofglucose-responsivegenesidentifiedinthepreviousdataset,and263overlapispresentedasaratiooutof1.0fordifferentiationexpressedgenes264(DEG)up-ordown-regulatedinourdatasetrelativetointactsamples.Asterisks265representasignificantdifference(p<0.05)betweentheratioofup-anddown-266regulatedgenesinapreviouslypublishedtranscriptomedatasetcomparedto267theratioofallup-anddown-regulatedgenesinourgraftingdatasetatacertain268timepoint.(D)Lugolstainingofgraftedplantsatvarioustimepointsrevealed269darkbrownstainingassociatedwithstarchaccumulation.HAG,hoursafter270grafting.271272
Table1.Sugarresponseoverlapswithasymmetry.Symmetricallyand273asymmetricallydifferentiallyexpressedgeneswerecomparedtopreviously274publishedsugar-responsivegenes(Osunaetal.,2007)1andthepercentoverlap275calculated(*p<.05).HAG,hoursaftergrafting.276277
278279
Vascularformationandcelldivisionactivatesonbothsidesofthegraft280
Sincethegraftedbottomsamplesexhibitedastarvationresponseupto48hours281
aftergrafting,wereasonedthatpathwaysassociatedcelldivisionandcell282
differentiationwouldbedelayedorinhibited.Welookedattheexpressionof283
markersassociatedwithvascularformationandcelldivisioninthe284
transcriptomedatasets.Cambium,phloemandprovascularmarkersactivated285
within6hoursingraftedtopsamples,butactivationwasonlydelayed6-24286
hoursingraftedbottomsamplesdependingonthegene(Figure1B-C,Figure3,287
FigureS5,FigureS6).Expressionofphloemmarkerspeakedinbothgraftedtops288
HAG Sugar Induced1 Graft Bottom=Top Overlap % Graft Top>Bottom Overlap % Graft Bottom>Top Overlap %6 2243 4988 263 5 6679 1403 21* 4971 99 212 2243 3473 165 5 7111 1444 20* 5657 231 424 2243 4135 171 4 6873 1414 21* 4942 195 448 2243 3689 197 5 6601 1241 19* 4915 218 472 2243 10421 1005 10* 2459 228 9* 2019 138 7120 2243 15012 1063 7 1510 220 15* 941 115 12*
HAG Sugar Repressed1 Graft Bottom=Top Overlap % Graft Top>Bottom Overlap % Graft Bottom>Top Overlap %6 1998 4988 107 2 6679 53 1 4971 1563 31*12 1998 3473 68 2 7111 112 2 5657 1526 27*24 1998 4135 88 2 6873 72 1 4942 1525 31*48 1998 3689 113 3 6601 93 1 4915 1427 29*72 1998 10421 530 5 2459 111 5 2019 538 27*120 1998 15012 979 7 1510 84 6 941 210 22*
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andgraftedbottomsat72hours(Figure3,FigureS5,FigureS6),thetimewhen289
phloemreconnectionsformingraftedArabidopsis(Figure1,(Yinetal.,2012;290
Melnyketal.,2015)).Notably,theearlyphloemmarkerNAC020activatedbefore291
themidphloemmarkerNAC086whichactivatedbeforethelatephloemmarker292
NEN4,consistentwiththedynamicsofphloemtranscriptionalactivationduring293
primaryrootdevelopmentandleafvascularinduction(FigureS5)(Furutaetal.,294
2014;Kondoetal.,2016).Certainmarkersassociatedwithxylemformation,295
suchasVND7andBFN1,activatedearlyinthegraftedtop.Otherxylemmarkers,296
suchasIRX3andCESA4,activatedlateingraftedsamples.By120hoursafter297
grafting,xylemmarkerswereactivatedintopandbottom,consistentwithwhen298
thefirstxylemstrandsdifferentiateatthegraftjunction(Melnyketal.,2015).299
Genesassociatedwithcelldivisionwereactivatedby12hoursinthegraftedtop300
andby24hoursinthegraftedbottom(Figure3,FigureS5).Ontheotherhand,301
controlgeneswhoseexpressiondoesnottypicallyvarybetweentissuesand302
treatments(Czechowskietal.,2005)werenotdifferentiallyexpressedingrafted303
topsorbottoms(FigureS6).TheRNAseqexpressiondataappearedtocorrelate304
wellwithtranscriptionalfluorescentreportersforbothactivationdynamicsand305
thelocalisationofexpression(Figure1D-F,FigureS2).306
307
Thesimilaractivationdynamicsofvasculardifferentiationgenesingraftedtops308
andgraftedbottomspromptedustotestwhetherthisphenomenonoccurred309
withotherknowndevelopmentalprocesses.Weobtainedlistsofgeneswhose310
expressionisassociatedwithvariousbiologicalprocessesfromprevious311
publications(TableS1)andtestedhowmanyofthegenesdifferentially312
expressedinourtranscriptomesoverlappedwiththepreviouslypublishedlists.313
Differentiallyexpressedgenesingraftedsamplesandseparatedtopspartially314
overlappedwiththosewhoseexpressionisassociatedwithphloem,xylemand315
procambiumformation(Figure4,FigureS7).Therewasahighoverlapbetween316
Arabidopsisinflorescencestemhealingandgrafting,aswellasbetweenvascular317
inductioninleafdiskculturesandgrafting(Figure4).Variousgenesexpressedin318
acelltype-specificmanneralsoshowedahightranscriptionaloverlapwith319
grafting,includingphloem,endodermisandprotoxylem(Figure4,FigureS7).In320
nearlyallcases,theseparatedtop,graftedtopandgraftedbottomsamples321
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showedsimilaractivationdynamics.Theseparatedbottomsampleswere322
exceptionalthoughsincegeneexpressionassociatedwithvasculardevelopment323
andcell-specificprocesseswasdownregulated(Figure4,FigureS7).Wealso324
comparedourdatasetstoRNAsexpressedinlongitudinalcrosssectionsofthe325
Arabidopsisroot(Bradyetal.,2007).Therewaslittleoverlapbetweengrafted326
bottomsandsectionsfromtherootmeristemiczone,whereasoverlapexisted327
betweengraftedtopsandtherootmeristemiczoneinearlytimepoints,and328
betweengraftingandtherootmaturationzone(FigureS8).Ouranalysisalso329
revealedthattwogenesexpressedincambium,WOX4andPXY,wereinducedby330
graftingbuttheprimaryrootmarkerWOX5andlateralrootmarkerLBD18were331
not(Figure3,FigureS5,FigureS6).332
333Figure3.Transcriptionaldynamicsofgenesassociatedwith334provasculature,phloem,xylemandcelldivision.Expressionlevelswere335plottedovertimeforintact,separatedandgraftedsamples.336337
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338Figure4.Transcriptionaloverlapbetweenpreviouslypublishedvascular339datasetsandthegraftingdatasets.Geneswhosetranscriptsareassociated340withvariouscelltypesorbiologicalprocessesweretakenfrompreviously341publisheddatasets(seeTableS1)andcomparedtothetranscriptomicdatasets342generatedhere.Thenumberinbracketsrepresentsthenumberofcelltype-343specificorprocess-specificgenesidentifiedinthepreviousdataset,andoverlap344ispresentedasaratiooutof1.0fordifferentiationexpressedgenes(DEG)up-or345down-regulatedinourdatasetrelativetointactsamples.Asterisksrepresenta346significantdifference(p<0.05)betweentheratioofup-anddown-regulated347genesinapreviouslypublishedtranscriptomedatasetcomparedtotheratioof348allup-anddown-regulatedgenesinourgraftingdatasetatacertaintimepoint.349350Auxinresponseissymmetricatthegraft351
Therapidactivationofvascularmarkersinthegraftedbottomsdespitethe352
starvationresponsepromotedustoinvestigatedwhetherothermobile353
substancessuchasphytohormonescouldplayaroleingeneactivation.We354
comparedlistsofgenesknowntorespondtocytokinin,ethyleneormethyl355
jasmonate(Nemhauseretal.,2006)andfoundnosubstantialoverlapbetween356
theselistsandgenesdifferentiallyexpressedbygrafting(FigureS9)(TableS1).357
Abscisicacid-responsiveandbrassinosteroid-responsivegenesshowedoverlap358
withgenesdifferentiallyexpressedinourdatasets,butthisoverlapwasofa359
similarmagnitudeinbothseparatedandgrafteddatasetssuggestingtheeffect360
wasnotspecifictografting(FigureS9).Auxinresponsivetranscriptswere361
exceptionalthough,astheyshowedasubstantialoverlapwithRNAs362
differentiallyexpressedinourdatasetsthatvarieddependingonthetreatment363
(Figure5A-B,FigureS9).Auxin-inducedgeneswereupregulatedinseparated364
tops,graftedbottomsandgraftedtopswhereastheywererepressedin365
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separatedbottoms(Figure8).AuxinresponsivegenessuchasIAA1andIAA2366
(Abeletal.,1995)wereinducedtosimilarlevelsingraftedtopsandgrafted367
bottomsat24hours.Tofurtherinvestigatewhetherauxinresponsewasuniform368
betweengraftedtopsandgraftedbottoms,wegraftedtheauxin-responsive369
fluorescentreporterp35S:DII-Venuswhosefluorescentproteinisdegradedinthe370
presenceofauxin(Brunoudetal.,2012).DII-Venusfluorescedintheseparated371
bottomsbutdidnotfluoresceingraftedbottoms14hoursaftercutting(Figure372
5C)indicatingseparatedbottomshadalowlevelofauxinresponsebutgrafted373
tops,graftedbottomsandseparatedtopshadahighlevelofauxinresponse.374
375Figure5.Auxinresponseissymmetricatthegraftjunction.(A,D)Expression376profilesforvariousauxin-responsivegenes(IAA1,IAA2)orauxintransporter377genes(PIN1andABCB1genes)wereplottedforintact,separatedandgrafted378samples.(B)Overlapbetweenpreviouslypublishedauxin-inducedorauxin-379
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repressedRNAsandourdataset.Thenumberinbracketsrepresentsthenumber380ofauxin-responsivegenesidentifiedinthepreviousdataset,andoverlapis381presentedasaratiooutof1.0fordifferentiationexpressedgenes(DEG)up-or382down-regulatedinourdatasetrelativetointactsamples.Asterisksrepresenta383significantdifference(p<0.05)betweentheratioofup-anddown-regulated384genesinapreviouslypublishedtranscriptomedatasetcomparedtotheratioof385allup-anddown-regulatedgenesinourgraftingdatasetatacertaintimepoint.386(C)Graftedandseparatedplantsexpressingtheauxinresponsivep35S::DII-387Venustransgenethatisdegradedinthepresenceofauxinrevealareductionof388auxinresponseincutbottoms,butnotingraftedbottoms.HAG,hoursafter389grafting.390391
Tissuefusionimpartsauniquephysiologicalresponsethatdiffersfrom392
tissueseparation393
Wehypothesizedthatthesymmetricauxinresponseandasymmetricsugar394
responseatthegraftjunctioncouldallowauniquetranscriptionalresponse395
sinceneitherseparatedplantsnorintactplantshadsimilarresponsedynamics396
tosugarsandauxinasmeasuredbygenome-widegeneresponse,starch397
accumulationandp35S::DII-Venusexpression(Figure2,Figure5).Touncover398
protein-codinggenesdifferentiallyexpressedspecificallybygrafting,we399
performedanempiricalBayesiananalysis(Hardcastle&Kelly,2010)whereat400
eachtimepointwecomputedlikelihoodsforeachgeneforeachpossiblepattern401
ofdifferentialexpressionintheintact,graftedtop,graftedbottom,separatedtop402
andseparatedbottomtissues(Figure6A).Theselikelihoodswereusedtodefine403
asimilarityscorebetweenpairsofgenes,whichwasusedtoclustergeneswith404
similarpatternsofgeneexpressionacrossthefivetissues(Hardcastle&405
Papatheodorou,2017).Clusterswerethenidentifiedbythepredominantpattern406
ofgeneexpressionobservedwithinacluster.Thisanalysisproduced113407
clusterscontainingatleast10RNAsatasingletimepoint(TableS2;DatasetS1).408
Approximately6000genesweredifferentiallyexpressedinatleastonetissue409
whereasbetween1000and4000geneswerenotdifferentiallyexpressed410
(Figure6B).411
412
Tosimplifytheanalysis,weconsideredclusterswheregeneexpressionwas413
groupedintopatternsconsistingofonecomparison.Atearlytimepoints,the414
clustercontaininggenesdifferentiallyexpressedingraftedtops,graftedbottoms,415
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separatedtopsandseparatedbottomshadhighnumbersthatdecreasedwith416
timeandcouldrepresentaninitialgeneralwoundresponse(Figure6C).417
Similarly,clusterscontainingseparatedtopsandgraftedtopsorseparated418
bottomsandgraftedbottomsinitiallyhadhighnumbersthatdecreasedwith419
time.Thisobservationindicatedthegraftedtopwasinitiallytranscriptionally420
similartotheseparatedtop,whereasthegraftedbottomwasinitially421
transcriptionallysimilartotheseparatedbottom.Afterthe48-hourtimepoint,422
clusterscontaininggenesonlydifferentiallyexpressedinseparatedtopsoronly423
differentiallyexpressedinseparatedbottomsincreasedinnumbers,suggesting424
thesetissuesgainedauniquepatternofgeneexpression.Theclustercontaining425
genesdifferentiallyexpressedingraftedtopsandgraftedbottomsincreasein426
numbersthroughoutthehealingprocess(Figure6C).Wesearchedforcluster427
categoriesthatcontainedgenesonlydifferentiallyexpressedbygraftingand428
foundveryfewgenesdownregulatedbygraftingorupregulatedonlyinthe429
graftedtop(Figure7A).Instead,therewereclustersthatcontainedseveral430
hundreddifferentiallyexpressedgenespresenteitherinthegraftedbottomonly,431
orpresentinbothgraftedbottomandgraftedtop(Figure7A).Geneswhose432
expressionchangedonlyinthegraftedbottomsamplewereprevalentearly433
duringgraftingandweremostcommonat48hours,whereasgenesactivatedin434
bothtopandbottombecameprevalentat48hoursandweremostcommonat435
120hours(Figure7A).Weperformedageneontology(GO)analysisonthegenes436
whoseexpressionwasgrafting-specificandfoundthatgenescodingforRNAs437
differentiallyexpressedonlyinthegraftedbottomsamplewereenrichedinthe438
immuneresponseandchitinresponsebiologicalprocesscategories(TableS3).439
Previouslypublishedchitin-inducedRNAshadahighproportionofoverlapwith440
differentiallyexpressedgraftbottom-specificgenes(Figure7E,FigureS10).441
Grafting-specificRNAsexpressedsymmetrically,inboththegraftedtopand442
graftedbottom,wereenrichedinvascular-relatedbiologicalprocesses(Table443
S3).Previouslypublishedphloem-enriched,endodermal-enriched,vascular-444
inductionandstem-woundingassociatedRNAshadahighproportionofoverlap445
withdifferentiallyexpressedgraft-specificgenes(Figure7C-D,FigureS10).Since446
fewgenesweregrafting-specificandgraftedtissueswereinitially447
transcriptionallyquitesimilartoseparatedtissues(Figure6C,Figure7A),we448
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reasonedthattissuesseparatedforshortperiods(<48hours)couldbegrafted449
withsimilarreconnectiondynamicsastissuesthathadbeengrafted450
immediately.Totestthishypothesis,plantswerecutatday0,andgraftedat1-5451
daysafterseparation.Separatedtissuesdidnotspeedupvascularreconnection,452
andinstead,italwaystookthreedaysfromthepointoftissueattachmentfor453
vascularconnectionstoform(FigureS10).Furthermore,theshootlost454
competencetograft2-3daysafterseparationwhereastherootremained455
competenttograftupto5daysafterseparation(FigureS10).Together,it456
appearsthegraftedshootandroothaveauniquephysiologicalresponsethat457
differfromseparatedplantsandthattissueattachmentisrequiredtoactivate458
graftformationandtissuefusion.459
460Figure6.Clusteringthetranscriptomeateachtimepoint,basedon461likelihoodsofallpossiblepatternsofdifferentialexpression.(A)Cartoon462depictingtheBayesiansegmentation.(B)Analysisofdifferentialbehaviour463produced113categoriescontainingatleast10geneswhoseexpressionwas464expressedinaspecificdifferentialpatterninatleastonetimepoint(seeTable465
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S2).Onegroupiscomprisedofgeneswhosetranscriptlevelsarenot466substantiallychangedinthefivetissues(unchanged)whereastheothergroupis467comprisedofthesumoftheother112groups(geneswhosetranscriptlevels468changedaftertreatmentinaleastonetissue)overthetimepointstested.(C)469MajorcategoriesinthesegmentationrevealedRNAswhoselevelschangedinall470thetreatmentslistedrelativetointactsamples.Notethatagenecanbe471representedonlyinonecategoryforagiventimepoint,thatcategoryinwhich472thetranscriptlevelchangesbestfitthecategory.473474
475Figure7.Asubsetofgenesisdifferentiallyexpressedonlyduringgraft476formationwhencomparedto0hourgrafts,orintactorseparatedtissues.477(A)Certaingeneswereonlydifferentiallyexpressedingraftedtops,grafted478bottomsorbothingraftedtopsandgraftedbottoms.(B)Expressionprofilesfor479agraftbottom-specific(ERF6)oragrafttopandbottomdifferentiallyregulated480gene(RTM2)wereplottedforintact,separatedandgraftedsamples.(C)481Grafting-specificgenesarealsoexpressedinotherprocessesoftissuefusion,482suchasstemhealing.Here,246previouslypublishedRNAswhosedifferential483expressionisassociatedwithstemhealingwerecomparedtoourdatasetto484assesstranscriptionaloverlapwiththegrafting-specificgenes.Asterisks485representasignificanthighoverlap(p<0.05)ofpreviouslypublishedgenesets486thatarealsodifferentiallyexpressedinthegraftedsamplesatacertaintime487point.(D-E)Genesdifferentiallyexpressedingraftedtopsandgraftedbottoms488showhighoverlapto324previouslypublishedgeneswhosetranscriptsare489associatedwithphloem,whereasgenesexpressedingraftedbottomsshowhigh490overlapto286previouslypublishedgeneswhoseencodedRNAsareassociated491withchitintreatment.Asterisksrepresentasignificanthighoverlap(p<0.05)of492previouslypublishedgenesthatarealsodifferentiallyexpressedinthegrafted493samplesatacertaintimepoint.494495
496
497
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DISCUSSION498
Tobetterunderstandhowplantsgraft,weanalysedindepthanRNAdeep499
sequencingdatasetthatspatiallyandtemporallydistinguishedgenesactivated500
bycuttingandtissueattachmentversusgenesactivatedbycuttingand501
continuoustissueseparation.Weobservedthatduringgraftformation,tissues502
hadaveryhightranscriptionaloverlapwithgenesdifferentiallyexpressedby503
inflorescencestemhealingandbyvascularinductioninleaves(Figure4,Figure504
S7)(Asahinaetal.,2011;Kondoetal.,2016)suggestinggraftingiscloselyrelated505
totheseprocesses.Graftformationhadlittletranscriptionaloverlapwithlateral506
rootformation(FigureS6)(Bradyetal.,2007)andappearedtofollowapathway507
similartosecondaryrootgrowth(FigureS6,FigureS8)sincecambium-specific508
markersWOX4andPXYwereexpressedduringgraftingandsecondarygrowth509
butarenotexpressedintheroottip(DeRybeletal.,2016)(FigureS5).Grafted510
topsinitiallyshowedsometranscriptionaloverlapwithgenesexpressedduring511
primaryrootformationwhichmayberelatedtotheaccumulationofsubstances512
activatingadventitiousrootformation,acommonresponseingraftsthatdonot513
properlyform.However,thisresponsewasshortlastingingraftedtopswhere514
adjoiningtissuescouldformvascularconnectionstore-establishsubstance515
transport.Thus,webelievethatgraftingproceedsviaapathwayinvolving516
secondarygrowthwithradialmeristemsactivatinginthematurecambiumto517
healthewound.Vascularformationgenesincludingthosespecifyingcambium518
andphloemwereactivatedearly,followedbyanactivationofcelldivisiongenes,519
suggestingthatthestartofcellulardifferentiationprecededactivationofcell520
division.Xylemidentitygenesshowedanearlyandalateactivationpeak(Figure521
3,FigureS5).Thereisnovisiblexylemdifferentiationatthegraftjunctionduring522
thefirstpeakofexpression(Melnyketal.,2015)andthesegenesmightbe523
suppressedbyphloemdifferentiationgenessuchasAPLandCLE41thatactivate524
earlyaftergraftingandareknowntosuppressprotoxylemformation(Bonkeet525
al.,2003;Itoetal.,2006)(FigureS6)(TableS4).Alternatively,thefirstpeakcould526
representprogrammed-celldeaththatdoesnotleadtoxylemdifferentiation.527
Thesecondexpressionpeakofxylem-expressedgenesat120hoursoccurred528
afterthedifferentiationoffunctionalphloemandcoincidedwiththe529
differentiationofxylemstrandsatthegraftjunction(Melnyketal.,2015).530
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Previousstudieshighlightedtheimportanceofcallusandpericyclecellsduring531
regeneration(Sugimotoetal.,2010;Iwaseetal.,2011),butweseelittleevidence532
thatgenesexpressedinthepericycleorduringcallusformationhavehigh533
transcriptionaloverlapwithgenesdifferentiallyexpressedbygrafting(Figure534
S4).Expressionprofilesforallprotein-codinggenescanbefoundintableS4.535
536
Weobserveonlyaslightdelayinphloem,cambiumandcelldivisionactivation537
belowthegraftjunctioncomparedtoaboveit(Figure3,FigureS5,FigureS6)538
whereassomegenes,suchasHCA2andTMO6,activatedequallyinbothgrafted539
topandgraftedbottomat6hoursaftergrafting(Figure1D-G,Figure3).These540
dataindicatethat,atleasttranscriptionally,thegraftedrootrapidlyresponded541
tothepresenceofthegraftedshoot.Thisresponsewasnotpresentinseparated542
roots,indicatingthatattachmentwaskeyforrecognition.Sugarsareknown543
activatorsofcelldivisionandcellelongation(Wang&Ruan,2013)andinour544
datasets,alargeproportionofasymmetricallyexpressedgenesaregenesthat545
aresugar-responsive.However,sugarsaretransportedinthephloem(Lough&546
Lucas,2006)thatisseveredupongraftingwhereasthegraftedrootexhibiteda547
sugarstarvationresponseandshowedsimilarsugar-responsedynamicsasthe548
separatedroot.Instead,webelievethatnotsugarbutauxin,orsomeother549
molecularthatistransportedintheabsenceofvascularconnections,couldbe550
thesignalinthegraftedbottomthatactivatesHCA2,TMO6aswellascell551
division,phloemandcambiumrelatedgenes.552
553
Givenauxin’sroleinvascularpatterningandformation((DeRybeletal.,2016),it554
isastrongcandidatefortheactivatingsignal.Auxinresponsewaslargely555
symmetric,particularlyfrom12hoursaftergrafting(Figure6)consistentwith556
previousfindingsthattheauxin-inducibleDR5andANAC071genesare557
symmetricallyexpressedaroundthegraftjunction(Yinetal.,2012;558
Pitaksaringkarnetal.,2014;Melnyketal.,2015;Matsuokaetal.,2016).Oneidea559
isthatauxintransportwasnotsubstantiallyinterruptedbythegraftingprocess,560
andinstead,whereopposingtissuesadhered,auxinmovedregardlessof561
vascularconnectionssinceauxinistransportedfromcelltocellthroughthe562
apoplast(Leyser,2011).ThegenesencodingauxineffluxproteinsPIN1and563
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Page 21
ABCB1weretranscriptionallyactivatedabovethegraftjunction(Figure5D),564
similartoputativePisumsativumPIN1proteinaccumulatingaboveacutstem565
priortovascularreconnection(Saueretal.,2006),andcouldreflectarolefor566
theseproteinsinexportingauxinacrossthecut.Consistentwiththese567
observations,addinganauxintransportinhibitortograftedArabidopsisshoots568
preventedtheexpressionofgrafting-inducedgenesbelowthegraftjunction569
(Matsuokaetal.,2016).Sinceauxinisfundamentalforpatterningvasculature570
(DeRybeletal.,2016),itisplausiblethatauxinmovementacrossthegraft571
occurspriortosugarmovementsincetheflowofauxincouldassistwith572
reconnectingthephloem.Althoughauxinresponsewassymmetric,ourprevious573
workdemonstratedthattheauxinresponsefactorsALF4andAXR1affected574
graftingonlybelowthegraftjunction(Melnyketal.,2015).MutatingALF4below575
thegraftjunctionmorestronglyreducedauxinresponsethanmutatingALF4576
abovethejunction(Melnyketal.,2015).Thus,proteinssuchasALF4orAXR1577
mightenhanceorpromotearootstock-specificauxinresponseandcouldbe578
particularlyimportantwhenthereisincompleteattachment,cellulardamageor579
inefficienttransport.Allhigherplantstransportauxinfromshoottoroot,yetnot580
allplantspeciescanbesuccessfullygrafted(Melnyk,2017c)sotheresponseto581
auxinratherthanthetransportitselfmaybeadeterminingfactorintheabilityto582
graft.Aroleforsugarsisnotcompletelyruledoutthough,sincethemagnitudeof583
differentialexpressionwasoftenlowerinthegraftedbottomandexogenous584
sugarreducedgraftingefficiency(Figure2A).Pericyclecellsrequireauxinto585
divideandtheadditionofsugarsenhancestherateofdivisions(Skylaretal.,586
2011),suggestingthatthepresenceofsugarsinthegraftedtopenhancedcell587
divisionanddifferentiation.Anotherhypothesisthatwarrantstestingiswhether588
grafthealingactivatesviaamechanicalsignalprovidedbythephysicalpresence589
oftheopposingtissues.590
591
Ouranalysesidentifiedtwogroupsofgeneswhoseexpressionchangeswere592
uniquetograftformationinourexperiments(Figure7).Onegroupactivated593
shortlyaftergraftingbelowthegraftjunctionandwasenrichedinimmune-594
responsiveandchitin-responsivegenes(TableS3,Figure7,FigureS10).The595
breakdownproductsofcellwallsarepotentelicitorsofdefenceresponses596
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Page 22
(Souzaetal.,2017),soitispossiblethatthegraftedbottomupregulates597
pathwaysspecifictowounddamageresponse.Thisgroupwasnotupregulated598
inseparatedbottomsthough,sotheuniquephysiologicalstateofthegrafted599
root,indicatedbythepresenceofauxinresponsebuttheabsenceofsugar600
response,couldhavepromotedtheirupregulation.Thesecondgroupactivated601
bothaboveandbelowthegraftjunctionandbecamehighlyexpressedlater602
duringgraftformation(Figure7).ThisgroupwasenrichedinRNAsassociated603
withvasculardevelopment(TableS3,Figure7)andwesuggestthattheproducts604
ofthesegenesareinvolvedinthevascularreconnectionprocessesbetweenthe605
twotissues.Despitemanytranscriptionalsimilaritiesbetweenseparatedtops606
andgraftedtissues,tissueshadtobeattachedforatleastthreedaysforphloem607
connectionstoform,regardlessofwhencuttingoccurred(FigureS10).Thus,it608
appearsthatRNAsexpressedintheseparatedtoporseparatedbottomare609
insufficienttodrivegraftformation.Insteadattachmentandthegenesactivated610
bythisrecognitionprocessincludinggrafting-specificRNAchanges(Figure7A)611
orRNAsexpressedinthegraftedbottom,graftedtopandseparatedtop(Figure612
6C)arethosethatcontributetoaresponsethatdistinguishesattachedfrom613
separatedplanttissues.FutureworkshouldfocusontheseRNAstoidentifythe614
pathwaysrequiredforgraftingthatcouldbemodifiedtoimprovegraft615
formation,woundhealingandvascularregeneration.Likewise,therapid616
transcriptionalchangesbelowthegraftindicatearecognitionsystemthat617
promotestissueregeneration.Identifyingtheexogenouscuethattriggers618
recognitionandunderstandinghowitisperceivedshouldbepriorities,asshould619
understandingwhetherthisphenomenonappliesmorebroadlytointer-tissue620
communication,tissueregenerationortissuefusionevents,suchasparasitic621
plantinfections(Musselman,1980),epidermalfusions(Becraftetal.,1996;Lolle622
etal.,1998)orpetalfusions(Zhong&Preston,2015).623
624
METHODS625
Plantmaterialandgrafting626
ArabidopsisthalianaaccessionColumbiawasusedthroughout.Thep35S::GFP-ER627
(Nelsonetal.,2007),pSUC2::GFP(Imlauetal.,1999),pUBQ10::PM-tdTomato628
(Segonzacetal.,2012),pARR5::GFP(Yanaietal.,2005),pANT::H2B-YFP(Randall629
.CC-BY-NC-ND 4.0 International licensenot certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which wasthis version posted October 5, 2017. . https://doi.org/10.1101/198598doi: bioRxiv preprint
Page 23
etal.,2015),pLOG4::n3GFP(DeRybeletal.,2014),pCASP1::NLS-GFP(Roppoloet630
al.,2011),p35S::DII-Venus(Brunoudetal.,2012)lineshavebeenpreviously631
published.FortheconstructionofpHCA2::RFP,a2.9kb5'upstreamregionofthe632
HCA2genewasclonedintopDONRp4-p1Rdonorvector,andrecombinedwith633
tagRFPerintoadestinationvectorbytheMultisiteGatewaysystem(Siligatoet634
al.,2016).ThefollowingprimerswereusedforpHCA2cloning:attB4_HCA2(-635
)2958GGGACAACTTTGTATAGAAAAGTTGtcgatacgcgggacagatatac636
attB1_HCA2ProEndggggACTGCTTTTTTGTACAAACTTGttttgtgttctgtatgtttg.637
Arabidopsisthalianamicrograftingwasperformedaccordingtoapreviously638
publishedprotocol(Melnyk,2017a).Briefly,sevendayoldArabidopsisseedlings639
weregrownverticallyonMurashigeandSkoog(MS)medium+1%bactoagar640
(pH5.7;nosucrose)inshortdayconditions(8hoursof80-100µmolm-2s-1light)641
at20°C.Seedlingswereplacedononelayerof2.5x4cmsterileHybondN642
membrane(GEHealthcare)ontopoftwo8.5cmcirclesofsterile3ChrWhatman643
paper(ScientificLaboratorySupplies)moistenwithsteriledistilledwaterina644
9cmpetridish.Inalaminarflowhoodusingadissectingmicroscope,one645
cotyledonwasremovedandatransverscutthroughthehypocotylwasmade646
withavasculardissectingknife(UltraFineMicroKnife;FineScienceTools).647
Graftswereassembledbyaligningthetwocuthalvesandjoiningthemtogether,648
afterwhich,thepetridisheswassealedwithparafilmandplacedvertically649
undershortdayconditionsat20°C.Forgraftingonamicroscopecoverslipto650
imagethegraftjunction,a10cmsquarePetridishmodifiedbygluinga651
microscopecoverslipinplaceofasectionofplasticfromtheback.Ontopofthe652
microscopecoverslipwasplaceda2.5x4cmrectangleofHybondNmembrane.653
AttheedgesandbaseofthePetridishthree3x8cmstripsofWhatmanpaper654
wereplaced.SterilewatermoistenedbothWhatmanpaperandHybondN.After655
which,rootswereplacedontheHybondNmembraneandhypocotylsonthe656
coverslip.Graftingthenproceededasabove.Graftjunctionswereimaged657
throughthecoverslipwithaPlan-Apochromat20X/0.8objectiveonaZeissLSM-658
700orLSM-780confocalmicroscope.659
660
Fluorescentassaysandmicroscopy661
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Page 24
Totesttheeffectofsugarsongrafting,ArabidopsisthalianaCol-0plantswere662
grownon1/2MS+1%bactoagar(pH5.7;nosucrose)forsevendaysinshortday663
conditions.WildtyperootsweregraftedtoscionsexpressingpSUC2::GFPusing664
theprotocoldescribedabovebuteitherwaterorwatercontaining0.25%,0.5%665
1%or2%sucrosewasaddedtothegraftedplates.Rootswereobservedfor666
fluorescence2-7daysaftergraftingwithaZeissV12dissectingmicroscope667
equippedwithaGFPfilter.Rootswerescoreddailyandthesameplantswere668
observedduringthe7-dayassayaspreviouslydescribed(Melnyk,2017b).669
670
FluorescentimagesweretakenonaZeissLSM-700orLSM-780confocal671
microscopewithaZeissPlan-Apochromat20X/0.8dryobjective.A488nmargon672
laser(Zeiss780)or488nmsolid-statelaser(Zeiss700)wasusedforexcitation673
ofGFPandYFP.A561nmsolid-statelaserwasusedforexcitationofthe674
tdTomatofluorescentprotein.AT-PMTdetectorobtainedbright-field675
transmittedlight.Blackandwhitefluorescentimagesofgraftjunctionswere676
takenonaZeissV12dissectingmicroscopefittedwithaHamamatsuEM-CCD677
cameraandRFPandYFPfilters.FIJIsoftware(Fiji.sc)wasusedtoprocess678
images.Imagecontrastandbrightnesswereadjustedforcontrolsandsamples679
equally.Forlongitudinalimagesofthegraftjunction,z-stackprojectionsare680
shownandmadewiththeaverageintensityfunctioninFIJIfromstacks681
containingthehypocotylvasculartissues,mesophyllandepidermis.682
683
RNAseqsampleandlibrarypreparation684
WildtypeArabidopsisthalianaaccessionCol-0weregraftedasabovetakingcare685
toswitchshootandrootbetweendifferentplants.Allgraftingandcuttingwas686
performedinthemorningtominimizecircadianeffects.Forthe0hourtime687
points,plantsweretransferredfrom1/2MSplatestothegraftingplatesand688
immediatelyharvested.Onlyintactplantswereharvestedatthe0hourtime689
sinceatthispoint,therewouldbeinsufficienttimetoreasonablyexpectthe690
separatedorgraftedsamplestobetranscriptionallydifferent(thetimebetween691
cuttingandfreezingislessthantwominutes).Forallothertimepoints,after692
cuttingplantswereleftongraftingplatesfortherespectiveamountoftime.693
Tissueswereharvestedandcaretakentoseparategraftsbygentlypullingplants694
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Page 25
apart.Approximately0.5mmoftissuewastakenaboveorbeloweachcutsite695
andkeptseparate.Intactplantshad1mmoftissuetakeninasimilarlocationon696
thehypocotylasseparatedorgraftedplants.Grafted,separatedorintacttissues697
werepooledintogroupsofapproximately24tissues(1plateswith24plants)698
whichwereimmediatelyplacedin96%ethanolondryice.Afterharvesting,699
microcentrifugetubeswerebrieflycentrifugedandtheethanolremovedbefore700
storingat-80°C.Plantsweregraftedovertwomonthstogetsufficientmaterial.701
702
Tissuesweregroundinthemicrocentrifugetubeusingamicrocentrifugepestle703
frozeninliquidnitrogen.RNAwasextractedusinganRNeasyKit(Qiagen,UK)704
followingthemanufacturer’sinstructionsincludingoncolumnDNasedigestion.705
RNAwaselutedfromthecolumnwith50ulofsterilewater.Qualityandquantity706
ofRNAwascheckedusinganAgilent2200TapeStationandHighSensitivity(HS)707
RNAscreentapes(Agilent,UK).AfterRNAextraction,twotofourbiological708
replicateswerecombined(50-100plants)togetsufficientRNA.90-100ngof709
RNAwasusedtoprepareRNAseqlibrariesusingtheTruSeq®StrandedmRNA710
LTkit(Illumina,UK)accordingtothemanufacturer’sinstructions.ThefinalPCR711
wasfor15cyclesandsampleswereresuspendedin23ulofdistilledwater.712
QuantityandqualityofDNAlibrarieswascheckedontheAgilent2200713
TapeStationusingD1000screentapes(Agilent,UK).Eachsamplehadtwo714
librariespreparedfromgraftedtissuesorseparatedtissuesatdifferenttimesso715
thatindependentbiologicalreplicatesweremade.Samplesweredilutedto10nM716
and11-12barcodedsamplesrandomlymixedtomakeatotalof7mixesfor7717
flowlanes,onemixperlane.SamplesweresequencedontheHiSeq4000718
platform(Illumina,UK)withPairedEnd75bptranscriptomesequencing(BGI719
TechSolutions,Shenzhen,China).720
721
Iodinestaining722
Arabidopsisseedlingswereplacedinafixationsolution(3.7%formaldehyde,723
50%ethanol,5%aceticacid)for1houratroomtemperature,thentransferred724
to70%ethanolfor10minutes.Afterwards,plantsweretransferredto96%725
ethanolandstoredat-20°Cforuptoaweek.Sampleswererehydratedin50%726
ethanolfor1houratroomtemperature,thentransferredtodistilledwaterfor727
.CC-BY-NC-ND 4.0 International licensenot certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which wasthis version posted October 5, 2017. . https://doi.org/10.1101/198598doi: bioRxiv preprint
Page 26
30minutes.SampleswerethentransferredtoasolutionofLugolsolution728
(Sigma)andstainedfor10minutesatroomtemperature.Plantswererinsed729
withwater,thenmountedonmicroscopeslides.ImagesweretakenonaZeiss730
Axioimager.M2microscopewithaPlanApochromat20xobjectiveandSPOTFlex731
camera(Imsol,UK).732
733
PairwiseandBayseqanalyses734
Thereadsacquiredthroughhigh-throughputsequencingwerequalitytrimmed735
withsickle(Joshi&Fass,2011)toincreasethereadqualitybeforemapping.736
Readswerealignedtoprotein-codinggenesequencesacquiredfromTAIR10737
usingBowtie2.ReadassignmentwasperformedusingtheeXpresstool,which738
alsoprovidedeffectivegenelengthsforuseinnormalisation.Libraryscaling739
factorswereinferredfromthesumofthenumberofreadsassignedtothegenes740
inthelowestseventy-fivepercentilesofexpressedgenesforeachlibrary741
(Hardcastleetal.,2012).742
743
AnalysesofthedatawerecarriedoutusingtheRpackagebaySeq(Hardcastle&744
Kelly,2010)andclusteringbasedontheposteriorprobabilitiesacquiredfrom745
thispackage.Foreachtimepoint,allpossiblepatternsofdifferentialexpression746
betweenthegrafttypeswereconsidered,wherea‘pattern’definessimilarityand747
differencebetweendifferentexperimentalconditions.Forexample,748‘{Col_cut_bottomGenes=Col:Col_bottomGenes=ungraftedGenes},{Col_cut_topGenes=Col:Col_topGenes}’749definesapatterninwhichgeneexpressionisequivalentintheseparated750
bottoms,thegraftedbottomsandtheintactplant,butdifferenttothe751
equivalentlyexpressedseparatedtopandgraftedtop.Thetotalnumberof752
possiblepatternsforfiveexperimentalconditions(asinthisanalysis)isfifty-753
two.754
755
Foragiventimepoint,posteriorlikelihoodsonthelikelihoodofeachpatternof756
expressionarecalculatedforeverygenewithgreaterthantenreadsacrossall757
experimentalconditions.Thepatternswerethenmodifiedtoincludeorderings758
(denotedby<or>),forexample,thepatterndescribedwouldleadtothe759
orderedpattern760
.CC-BY-NC-ND 4.0 International licensenot certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which wasthis version posted October 5, 2017. . https://doi.org/10.1101/198598doi: bioRxiv preprint
Page 27
‘{Col_cut_bottomGenes=Col:Col_bottomGenes=ungraftedGenes}>{Col_cut_topGenes=Col:Col_topGenes}’761inwhichgeneexpressionisequivalentintheseparatedbottoms,thegrafted762
bottomsandtheintactplantandgreaterthantheequivalentlyexpressed763
separatedtopandgraftedtop.Intotal,541orderedpatternsexistinthisdata764
set.Posteriorlikelihoodsforanorderedpatternwereassignedtothatofthe765
unorderedpatternforgenesinwhichthe(normalised)meanexpressionswithin766
theequivalentlyexpressedgroupsconformedtotheordering,andtozero767
otherwise.768
769
Basedontheposteriorlikelihoodsfortheorderedpatterns,asimilarityscoresij770
wasestablishedbetweentwogenesiandjasthesumovertheproductsoftheir771
likelihoodsofeachorderedpattern.Asingle-linkagglomerativeclusteringof772
genes,inwhichagenewilljoinaclusterifithasagreaterthan50%similarityto773
anygenewithinthatclusterwasthenperformedbasedonthesesimilarity774
scores.Welabeleachclusteraccordingtothepredominantorderedpatternwith775
highlikelihoodamongstthegenesthatcompriseit.Thechangeinsizeofthese776
clustersovertimeisshownforthemajorclusteringsinFigure6.777
778
Wecanalsofindlikelihoodsoncomparisonsbetweenpairsofexperimental779
conditionsbysummingthelikelihoodsovercombinationsofpatterns.Figure1B780
showsthenumberofgenesidentifiedateachtimepointinapairwiseanalysis781
betweenthegraftedtopandgraftedbottomsamples.Thelikelihoodof782
symmetricexpression(i.e.,expressionwhichisequivalentacrossthegraft783
junction)iscalculatedasthesumofthelikelihoodsofallpatternsinwhichthe784
graftedtopandgraftedbottomsamplesareequivalent.Conversely,asymmetric785
expressioniscalculatedasthesumofthelikelihoodsofallpatternsinwhichthe786
graftedtopandgraftedbottomsamplesarenotequivalent.Additionalsetscan787
beformedbyconsideringtheorderingofthegraftedtopandgraftedbottoms788
samples.SetsofgenesareidentifiedateachtimepointwithanFDRoflessthan789
0.05andalikelihoodofsymmetric/asymmetricexpressiongreaterthan50%.790
Genesinthisanalysiswereonlyincludediftheyweredifferentiallyexpressed791
relativetointactsamples.792
793
.CC-BY-NC-ND 4.0 International licensenot certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which wasthis version posted October 5, 2017. . https://doi.org/10.1101/198598doi: bioRxiv preprint
Page 28
Geneoverlapanalysesofupanddownregulatedgenes794
Tomeasureiftheratioofup-anddown-regulatedgenesfromapreviously795
publisheddatasetissignificantlydifferenttotheratioofup-anddown-regulated796
genesinourgraftingdatasetweonlytookintoaccountgenesthatare797
differentiallyexpressedatacertaintimepoint.Agenewascalleddifferentially798
expressedatacertaintimepointifthemarginallikelihood,calculatedbyBaySeq,799
wasgreaterthan0.9andiftheabsolutelog2-foldchangewasgreaterthan1.800
Hence,weonlyconsidergenesthataresignificantlytwo-foldup-ordown-801
regulated.Thisdefinitionofdifferentiallyexpressedgeneswasalsousedtofilter802
thepublisheddatasetsaccordingtotheexpressionvaluesinourtranscriptome803
dataset.Hence,somegeneswerefilteredoutfromtheoriginalpublished804
datasetsbecausetheydidnotshowasignificantup-ordown-regulationduring805
acertaintimepointinourexpressiondatabasedonourcriteria.Thehistograms806
(Figure2,4,5,S4,S7,S8,S9)showtherelativenumberofup-anddown-807
regulatedgenesfromthepublisheddatasetsduringacertaintimepointanda808
certaincondition(separatedtop,separatedbottom,graftedtop,graftedbottom)809
basedonthenumberofgenesinthepublisheddatasetafterfiltering.To810
calculatethesignificanceofthedifferenceoftheratiosbetweenthepublished811
DEGsandallup-anddown-regulatedgenes,weusedatwo-sidedFisher’sexact812
test.TocorrectformultipletestingweusedtheBenjamini-Yekutieli(BY)813
correctionmethod.Hence,theasterisksinthebarplotshighlightthatthe814
correctedp-valueisbelow0.05. 815
816
Dealingwithprobeidsfrommicroarraydatasets817
Duetothefactthatsomepublisheddatasetsonlyusedprobeidsinsteadofgene818
idstorepresenttheirdifferentiallyexpressedgeneswefirsthadtomatchthese819
probeidstotheircorrespondinggeneids.ThisstepwasdonewiththeRpackage820
biomartr(Drost&Paszkowski,2017).Ifoneprobeidmatchedmorethanone821
geneidweusedallthecorrespondinggeneidsandtestedafterwardsifthese822
geneswereactuallydifferentiallyexpressedinourdataset.Insomecases,one823
probeidwasrepresentedbymorethanonegeneid.Hence,somegenesets824
containedslightlymoregeneidsthanpublishedprobeids.Incontrast,some825
.CC-BY-NC-ND 4.0 International licensenot certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which wasthis version posted October 5, 2017. . https://doi.org/10.1101/198598doi: bioRxiv preprint
Page 29
probeidsdidnotmatchtocurrentlyexistinggeneids.Hence,somegenesets826
containedslightlyfewergeneidsthanpublishedprobeids.827
828
Geneoverlapanalysesofgenesetsinvolvedingraftformation829
ForthisanalysisweusedthepreviouslycalculatedgenesetsfrombaySeqof830
differentiallyexpressedgenesclusteredforeachtimepointintograftedbottom,831
graftedtop,andgraftedbottomandtopandcalculatedtheiroverlapto832
previouslypublisheddatasets.Thesignificanceofthenumberofoverlapping833
genesbetweenthegraftedsamplesandthepublisheddatasetswasdetermined834
byaone-sidedFisher’sexacttest,toproveiftheoverlapisgreaterthan835
expected.Theresultingp-valueswerecorrectedformultipletestingbyusingthe836
Benjamini-Yekutielimethod.837
ThisprocedurewasalsoappliedtogenerateTable1tostudytheoverlapsof838
symmetricallyandasymmetricallyexpressedgenesinthegraftingdatasetwith839
previouslypublishedsugar-responsivegenes.840
841
GOenrichmentanalysis842
Thegeneontologyanalysis(GO)enrichmentanalysisongrafting-specificgenes843
wasdonewithacustomizedRscriptusingthepackageGOstats(Falcon&844
Gentleman,2007).GeneontologyannotationwasusedfromtheBioconductor845
packageorg.At.tair.db(Carlson,2017).Thep-valuescalculatedbya846
hypergeometrictestwerecorrectedformultipletestingwiththeBonferroni847
correction.AGOcategorywascalledenrichedifthecorrectedp-valuewasbelow848
0.05.849
850
Adetaileddescription,therequireddataandRscriptstoreproducethe851
clustering(hierarchicalclusteringandPCA),thestatisticalanalysesregarding852
theoverlapstudies,andtheGOenrichmentanalysisareavailableviatheGitHub853
repository(https://github.com/AlexGa/GraftingScripts).854
855
Breakingforcemeasurements856
Amicro-extensometerwasusedforallbreakingforcegraftmeasurements857
accordingtoapreviouslypublishedprotocol(Robinsonetal.,2017).Briefly,858
.CC-BY-NC-ND 4.0 International licensenot certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which wasthis version posted October 5, 2017. . https://doi.org/10.1101/198598doi: bioRxiv preprint
Page 30
graftedCol-0plantswereattachedtoamovingplateusingtoughtags(0.94X0.50859
inches,white,679catalogno.TTSW-1000,DiversifiedBiotech)anda860
cyanoacrylateglue.Theplatesweremovedapartandtheforcewasmeasured861
usingaforcesensor(FutekLSB20010gloadcell,FutekInc.).Uponbreakingthe862
forcedropped.Themaximumforcemeasuredbeforebreakingwasrecorded.863
ImageswerecapturedusingaLeicaSP5confocalmicroscope.Duringthe864
experimentsinglez-placeimageswerecapturedandmadeintoamoviein865
ImageJ.866
867
Tomeasurelevelsoftissuecontaminationbetweengraftedtopandbottom,868
graftedgreenfluorescentprotein(p35S::GFP)andredfluorescentprotein869
(pUBQ10::PM-tdTomato)expressingplantswerepulledapartmanuallyand870
imagedonaZeissLSM700confocalmicroscope.Z-stackscomprisingthe871
majorityofthehypocotylweremadeandregionsapproximately0.5mmabove872
and0.5mmbelowthegraftjunctionweremadeintoprojectionsusingthe873
averageintensityfunctiononFIJIsoftware,andthenthemeanintensity874
quantifiedforredandgreenchannelsonFIJI.Themeanintensityofonecolourin875
thetissuetestedforcontaminationwasdividedbythemeanintensityofthe876
samecolourinthetissueexpressingthattransgenestogetapercentageof877
contamination.Aregionawayfromthecutsitewasalsoquantifiedtogeta878
percentageofspectraloverlapbetweenredandgreenchannels.Thepercent879
spectraloverlapwasthensubtractedfromthepercentagecontaminationtoget880
anoverallpercentageforhowmuchcontaminationwaspresent.881
882
ACKNOWLEDGEMENTS883
WethankNikoGeldner,DolfWeijers,PaulTarr,YkaHelariutta,RuthStadlerand884
TheArabidopsisInformationResourceforprovidingseeds.Fundingforthis885
workwasprovidedbyGatsbyCharitableTrustgrantsGAT3272/CandGAT3273-886
PR1,byaKnutandAliceWallenbergAcademyFellowshipKAW2016.0274(to887
C.W.M),andbytheHowardHughesMedicalInstituteandGordonandBetty888
MooreFoundationgrantGBMF3406(toE.M.M.)889
890
891
.CC-BY-NC-ND 4.0 International licensenot certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which wasthis version posted October 5, 2017. . https://doi.org/10.1101/198598doi: bioRxiv preprint
Page 31
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SUPPLEMENTARYMATERIAL1125
MovieS1.Breakingweightmeasurementsatthegraftjunction,relatedto1126figure1.Amicro-extensometerpulledapartanArabidopsisplant9daysafter1127graftingwhileforcewasmeasuredusingaforcesensorandimagescaptured1128usingaZeissSp5confocalmicroscope.Here,thetransmittedlightfieldisshown.1129Shootisontopandroottowardsthebottom.11301131MovieS2.Breakingweightmeasurementsatthegraftjunction,relatedto1132figure1.Amicro-extensometerpulledapartanArabidopsisplant9daysafter1133graftingwhileforcewasmeasuredusingaforcesensorandimagescaptured1134usingaZeissSp5confocalmicroscope.ThesameexperimentisshowninMovie1135S1,buthere,theGFPandRFPchannelsareshown.Shootisontop(p35S::GFP1136expressing)androottowardsthebottom(pUBQ10::PM-tdTomatoexpressing).11371138TableS1.Detailsofpreviouslypublisheddatasetsusedtocomparetothe1139graftingdatasets.11401141TableS2.NumbersandcategoriesassociatedwiththeBayseqanalysis.1142Categoriesaredefinedasgrafted(Col:Col),separated(Col_cut)orintact1143(ungrafted).11441145TableS3.GOanalysisforbiologicalprocess(BP).Shownarethetop20BPGO1146termsforthegrafting-specificgenes.Timepointselectedarethosewhenthere1147arethemostgenesingraftedbottomsamples(48hrs)orgraftedtop+bottom1148samples(120hrs).11491150TableS4.Normalisedreadsforalltheprotein-codinggenesinthedatasets.By1151enteringtheATGnumberofinterest,aplotismadewhichshowsadifferential1152geneexpressionprofileforthegeneofinterest.11531154DatasetS1.DetailsoftheBayesiansegmentationanalysisprovidingfiles1155containingtheATGsforeveryclusterforeverytimepoint.11561157
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1158FigureS1.Experimentalapproach,relatedtofigure1.(A)Thebreaking1159weightrequiredtopullapartgraftedplantsmeasuredbyamicro-extensometer.1160(B-C)Contaminationoftopinbottomorbottomintopwaslessthan4%as1161measuredbygraftinggreenfluorescentprotein-expressingplants(p35S::GFP)to1162tomatofluorescentprotein-expressingplants(pUBQ10::PM-tdTomato)and1163measuringamountsoffluorescenceatthedifferentwavelengthsofemissionin1164thetopsegmentsrelativetothebottom.Imagesshowdifferentplantspriorto1165andafterpullingwiththepercentcontaminationindicated.11661167
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1168FigureS2.ComparisonbetweenRNAseqtranscriptomeexpressionprofiles1169andtranscriptionalfluorescentreporters,relatedtofigure1.RNAseq1170expressionprofilesforvariousgenesupregulatedduringgraftformationwere1171plottedforintact,separatedandgraftedsamples(leftpanels).Transcriptional1172reporter-expressingplantswerecutandseparated,cutandgrafted,orleftintact.1173Aftercutting,plantswereimagedandz-projectionsmadeatvarioustimepoints1174(rightpanels).ForpCASP1::GFP,wedidnotobserveasignalinintactplants1175graftedonglassslides(seeMaterialsandMethods),butobservedasignalwith11763/5plants7daysaftergraftingonWhatman-nylonmembrane,thesame1177conditionusedfortranscriptomelibrarypreparation.HAG,hoursaftergrafting.1178HAS,hoursafterseparation.Dashedlinesdenotethegraftjunction.11791180
1181
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1182FigureS3.Hierarchicalclusteranalysisandprinciplecomponentanalysis1183(PCA)forthetranscriptomelibraries,relatedtofigure1.(A)Hierarchical1184clusteringofsamplesbasedonlog10transformedTPMvalues.Similarity1185betweensampleswasmeasuredby1–spearmancorrelationcoefficient.(B)1186PCAofexpressiondatashowsclusteringofsimilarsamples.Especiallythe1187graftedtopandgraftedbottomsamplesareverysimilarfrom120hours1188onwards.1189
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1190FigureS4.Asymmetryisafeatureobservedwithsugarresponseandstarch1191accumulation,relatedtofigure2.(A)ExpressionprofilesforRNAscodedby1192sugar-repressedgenes(DIN6,STP1)orphotosyntheticgene(RBSC3B)were1193plottedforintact,separatedandgraftedsamples.(B)Transcriptionaloverlap1194betweenpreviouslypublishedmannitol-induced,mannitol-repressed,sugar-1195inducedorsugar-repressedgenesandourdatasets.Thenumbersinbrackets1196representsthenumberofglucoseormannitol-responsivegenesidentifiedinthe1197previousdataset,andoverlapispresentedasaratiooutof1.0fordifferentially1198expressedgenes(DEG)upordownregulatedinourdatasetrelativetointact1199samples.Asterisksrepresentasignificantdifference(p<0.05)betweentheratio1200ofup-anddown-regulatedgenesinapreviouslypublishedtranscriptome1201datasetcomparedtotheratioofallup-anddown-regulatedgenesinour1202graftingdatasetatacertaintimepoint.(C)Lugolstainingofgraftedplantsat1203varioustimepointsrevealsdarkbrownstainingassociatedwithstarch1204accumulation.UppergraftedpanelsarethesameasthosepresentinFigure21205andshownheretocomparewithcontrols.HAG,hoursaftergrafting.1206
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1207FigureS5.Transcriptionaldynamicsofgenesassociatedwithcambium,1208phloem,xylemandcelldivision,relatedtofigure3.Expressionprofilesin1209graftedtopsorgraftedbottomsforvariousgenesofinterestwereplotted,1210normalisedtointactsamplesandplottedonalog2scale.12111212
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1214FigureS6.AccumulationofRNAfromgenesassociatedwithprovasculature1215andphloem,andofRNAsfromWOXtranscriptionfactors,relatedtofigure12163.(A)RNAaccumulationprofilesforvariousgenesofinterestwereplottedover1217timeasmeasuredingraftedtopsorgraftedbottoms,normalisedtointact1218samplesandplottedonalog2scale.Thegraftingdatasetscouldalsobeusedto1219investigatethetranscriptionaldynamicsofrelatedgenes,suchasthesequential1220activationofWOXtranscriptionfactorsatthegraftjunction.(B)Expression1221levelsofaprimaryrootspecifictranscript(oftheWOX5gene)oralateralroot1222specifictranscript(fromtheLBD18gene)wereplottedovertimeforintact,1223separatedandgraftedsamples.1224
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1225
1226FigureS7.Transcriptionaloverlapbetweenpreviouslypublisheddatasets1227andthegraftingdatasets,relatedtoFigure4.Geneswhosetranscriptsare1228associatedwithvariouscelltypesorbiologicalprocessesweretakenfrom1229previouslypublisheddatasets(seeTableS1)andcomparedtothedatasets1230generatedhere.Thenumberinbracketsrepresentsthenumberofcelltype-1231specificorprocess-specificgenesidentifiedinthepreviousdataset,andoverlap1232ispresentedasaratiooutof1.0fordifferentiationexpressedgenes(DEG)up-or1233down-regulatedinourdatasetrelativetointactsamples.Asterisksrepresenta1234significantdifference(p<0.05)betweentheratioofup-anddown-regulated1235genesinapreviouslypublishedtranscriptomedatasetcomparedtotheratioof1236allup-anddown-regulatedgenesinourgraftingdatasetatacertaintimepoint.1237
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1238FigureS8.Transcriptionaloverlapbetweenpreviouslypublishedroot1239transcriptomicdatasetsandthegraftingRNA-seqdataset,relatedtoFigure12404.Geneswhosetranscriptsareassociatedwithvariousrootregionsweretaken1241frompreviouslypublisheddatasets(seeTableS1)andcomparedtothedatasets1242generatedhere.Rootlayerisindicatedinthebottomrightofeachhistogram1243withreferencetothecartoon.Thenumberinbracketsrepresentsthenumberof1244rootlayer-specificgenesidentifiedinthepreviousdataset,andoverlapis1245presentedasaratiooutof1.0fordifferentiationexpressedgenes(DEG)up-or1246down-regulatedinourdatasetrelativetointactsamples.Asterisksrepresenta1247significantdifference(p<0.05)betweentheratioofup-anddown-regulated1248genesinapreviouslypublishedtranscriptomedatasetcomparedtotheratioof1249allup-anddown-regulatedgenesinourgraftingdatasetatacertaintimepoint.1250
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1253FigureS9.Transcriptionaloverlapbetweenpreviouslypublishedhormone1254responsiveRNAdatasetsandthegraftingRNAdatasets,relatedtoFigure5.1255Geneswhosedifferentialexpressionisassociatedwithvarioushormone1256responsesweretakenfrompreviouslypublisheddatasets(seeTableS1)and1257comparedtothegenesrepresentedintheRNA-seqdatasetsgenerated.The1258numberinbracketsrepresentsthenumberofcelltype-specificorprocess-1259specificgenesidentifiedinthepreviousdataset,andoverlapispresentedasa1260ratiooutof1.0fordifferentiationexpressedgenes(DEG)up-ordown-regulated1261inourdatasetrelativetointactsamples.Asterisksrepresentasignificant1262difference(p<0.05)betweentheratioofup-anddown-regulatedgenesina1263previouslypublishedtranscriptomedatasetcomparedtotheratioofallup-and1264down-regulatedgenesinourgraftingdatasetatacertaintimepoint.12651266
1267
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1268FigureS10.Asubsetofgenesisdifferentiallyexpressedonlyduringgraft1269formation,relatedtoFigure7.(A)pSUC2::GFPexpressingArabidopsisshoots1270weregraftedtoCol-0wildtyperoots0-5daysaftertransferringtografting1271platesandkeptintactuntilgrafting(intacttreatment).GFPmovementtothe1272rootsforphloemconnectionassayswasmonitoredover7days.(B)Plantswere1273cutatday0andgraftedatdays0-5aftercutting.(C)Shootswerecutatday01274andgraftedatdays0-5aftercutting.Rootswerekeptintactuntilimmediately1275beforegrafting.(D)Rootswerecutatday0andgraftedatdays0-5aftercutting.1276Shootswerekeptintactuntilimmediatelybeforegrafting.(E)Genes1277differentiallyexpressedingraftedtopsandgraftedbottomsshowoverlapwith1278previouslypublishedgeneswhosetranscriptsareassociatedwiththe1279endodermis,vascularinductionandgrafting(seeTableS1fortreatment1280information).Asterisksrepresentsignificanthighoverlap(p<0.05)ofpreviously1281publishedgenesetsthatarealsodifferentiallyexpressedinthegraftedsamples1282atacertaintimepoint.12831284
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