new tools for evalua/on of cell-speciﬁc environmental plas...

NewToolsforEvalua/onofCell-SpecificEnvironmentalPlas/cityinRice,TomatoandMedicagoGermainPauluzzia,MauricioReynosoa,KaisaKajalab,DongxueWangc,DonnellyWestb,MarkoBajicc,MichaelCovingtonb,KrisAnaZumsteinb,

RogerDealc,SiobhanBradyb,NeelimaSinhab,andJuliaBailey-SerresaaCenterforPlantCellBiology,UniversityofCalifornia,Riverside,CA92521

bDepartmentofPlantBiology,UniversityofCalifornia,Davis,CA95616cDepartmentofBiology,EmoryUniversity,Atlanta,GA30322

Contacts:[email protected];[email protected];[email protected];[email protected]

FromseedgerminaAontoovuleferAlizaAon,plantdevelopmentis exquisitely orchestrated by geneAc processes that are fine-tuned by environmental cues. This plasAcity entails the preciseregulaAon of networks of genes in individual cells. Of all thestressesexperiencedbycrops,extremesinwaterareparAcularlydamagingtocropyieldstability.Weareasking:Howdoesgeneac/vity in stem cells (meristems) of roots and shoots differacrossspecies?Howdofloodinganddroughtstressesinfluencethedevelopmentofspecializedcelltypesintheroot?

To address these quesAons, we have refined the INTACT(IsolaAonofnucleitaggedinspecificcelltypes)andTRAP(Taggedribosome affinity purificaAon) technologies that enableexaminaAonof the epigenome, transcriptome, and translatomeofspecificcelltypes.Ourchallengesandsuccesseshavebeen:•  TotranslatethesesystemsfromArabidopsistootherdicots•  ToestablishAgrobacteriumrhizhogenes-promotedhairyrootsintomatoandMedicago

•  ToidenAfymeristemandrootcell-specificpromoters•  ToopAmizeINTACTinamonocot•  TointegrateINTACTwith“tagmentaAon”(ATAC-seq)•  ToadvancenuclearRNA,mRNAandribosomefootprintlibraryconstrucAonwithlimitedrRNAcontaminaAon

•  Toestablishpipelinesfordataanalysis

Abstract

1.ProjectWorkflow

Well-watered

Drought-stressed

Water-logged

Res

ourc

es

Transgenic plants with tagged nuclei or ribosomes in

specific cell-types

Pip

elin

es/D

ata

Met

hodo

logy

2.EstablishmentofINTACTandTRAPlinesinfourspecies

Identification and validation of cell type-specific promoters in each species

Generation of TRAP and INTACT vectors for each species

3.Purifica/onofnucleibyINTACTandpolysomesbyTRAPasillustratedinrice

✓ ✓

Root tip containing affinity tagged nuclei in one cell type

Nuclei preparation Add streptavidin-coated magnetic beads

Collect labeled nuclei on a magnet

Mixture containing affinity labeled nuclei (green)

Streptavidin beads (black ovals) bind to

biotinylated nuclei

!"#$%&'%())*+',*-./0&11-'$#'234536789'1$#*'






biotinylated nuclei

!"#$%&'%())*+',*-./0&11-'$#'234536789'1$#*'






biotinylated nuclei

!"#$%&'%())*+',*-./0&11-'$#'234536789'1$#*'

NucleipreparaAon

AddStreptavidin-

coatedmagneAcbeads

TaggedRootCells

AffinityPurifywith

anAbodyHomogenizeandlyseER

Collectlabelednuclei

onamagnet






biotinylated nuclei

!"#$%&'%())*+',*-./0&11-'$#'234536789'1$#*'

TRAP%expressing%cells%

Affinity%Purify%with%an89GFP%%






biotinylated nuclei

!"#$%&'%())*+',*-./0&11-'$#'234536789'1$#*'

TRAP%expressing%cells%

Affinity%Purify%with%an89GFP%%

INTACTIsolaAonofNucleiTAggedinspecificCellTypes

TRAPTranslaAngRibosomeAffinityPurificaAon

AnA-HisorAnA-FlagMagneAcBead

Promoter' HIS' GFP' RPL18'FLAG'

Tags'for'Pull'Down'

Fluorescence'Visualiza@on'

Ribosomal'Subunit'

AtAc#n#BirA(

Bio#n(Ligase(

Promoter' WPP' GFP' BLRP'

Nuclear'Envelope'Targe9ng'

Fluorescence'Visualiza9on'

Bio9n'Ligase'Acceptor'Pep9de'

Nuclear'Targe9ng'Factor' Epidermis)

Cortex)

Endodermis)

Pericycle)

Quiescent)Center)

Columella)

Lateral)Root)Cap)

Stele)

Epidermis)

Cortex)

Endodermis)

Pericycle)

Quiescent)Center)

Columella)

Lateral)Root)Cap)

Stele)

Epidermis)

Cortex)

Endodermis)

Pericycle)

Quiescent)Center)

Columella)

Lateral)Root)Cap)

Stele)

Epidermis)

Cortex)

Endodermis)

Pericycle)

Quiescent)Center)

Columella)

Lateral)Root)Cap)

Stele)

Epidermis)

Cortex)

Endodermis)

Pericycle)

Quiescent)Center)

Columella)

Lateral)Root)Cap)

Stele)CortexMeristemaAccortex

Epidermis)

Cortex)

Endodermis)

Pericycle)

Quiescent)Center)

Columella)

Lateral)Root)Cap)

Stele)

Endodermis&QuiescentCenter

QuiescentCenter Vasculature

AtPEP:TRAP

AtPEP:NTF35S:TRAP

SlRPL11C:NTF SlWOX5:NTF

SlRPL11C:NTF

SlCO:NTF35S:NTF SlSHR:NTF

SlWOX5:NTF

SlSCR:TRAP

MeristemaAcZone

ConsAtuAve

Solanu

m

lycope

rsicum

T1ro

ots

Solanu

m

penn

ellii

T1ro

ots

Med

icag

otrun

catula

T0ro

ots

35S:TRAP AtWOX5:NTF AtSCR:NTFAtPEP:TRAP AtSHR:TRAPAtCO:TRAPAtRPL11C:NTF

6.FutureWork

4. Valida/on of common submergence condi/ons inseedlingsforthreecropspecies

5.Plas/cityofdevelopmentinresponsetodroughtandwaterloggingasillustratedintomatoandrice

SAMconsAtuAve

T1sh

oots

RSS1:TRAP CMZ:TRAP QHB:NTF EMZ:TRAP35S:NTF OsSHR1:NTF

Oryza

sa6v

aT0orT

1roots

S.lycopersicumrootsshowdevelopmentalplas6cityonrootsystemandcelltypelevelinresponsetowaterstressesC.Totalrootbiomassisnotaffectedby12-daystress,butbiomassshiistothesurfaceduetogrowthofhypocotyl-derivedrootsinresponsetowaterlogging(orangearrow).D.StainingrootsecAonsforsuberinwithfluorolyellowshowsthatindrought,suberinaccumulatesincelltype-specificfashioninexodermis(redarrow),waterproofingtheroot.

CONTROL

DROUGHT

WATERLOGGING

C D

T-DNA mapping

technology to identify single insertion lines

TRAP and INTACT of whole shoot, whole root & root cortex

TRAP and INTACT of meristems

(+ all cell types)

Epigenome (ATAC-seq), nuclear transcriptome, and translatome

sequencing and analysis

Cross-species comparison of stress-responses and meristem gene

expression

Generation of orthologous gene sets

BrAD-seq: development of

a high-throughput RNA

and DNAseq protocol

✓

Optimization of constructs for each species and validation of

TRAP and INTACT

✓

✓

Well-watered

Drought-stressed

Water-logged

TRAP and INTACT of whole shoot / root, meristems & root

cortex

TRAP and INTACT of meristems

(+ all cell types)

ATAC-seq, nuclear transcriptome, translatome

2 h submerged and control Seedlings 35S:INTACT 35S:TRAP

TRAP and INTACT of root tips

ATAC-seq, nuclear transcriptome, translatome and Ribo-Seq

Cross-species comparison

0 2 4 6 8 100.1

1

10

100

0 2 4 6 8 100.1

1

10

100

1000

0 2 4 6 80.1

1

10

100

0 2 4 6 8 100.01

0.1

1

10

100

ADH

RelaAvetranscrip

tlevel

Hourstreatment

S.lycopersicumcv.M82

S.lycopersicumcv.M82hairyroots

S.pennellii O.sa6vacv.Nipponbare

M.truncatulacv.JemalongA17hairyroots

0 2 4 6 80.1

1

10

100

1000

Alcoholdehydrogenase(ADH)mRNAlevels,encodinganenzymerequiredforanaerobicmetabolism,werequanAfiedbyqRT-PCR.ValueswerenormalizedtoACT2mRNAlevelsforS.lycopersicumandS.penelli,RPL2forM.truncatula,andUBCIIforO.saPva.The2hour/mepointwasselectedforinter-speciescomparisonofnucleartranscriptome,translatomeandepigeneAcregulaAonaffectedbythestress.

Seedlings

RootAps(1cm)collectedfrom0to10h

FloodingorControl

DROUGHTCONTROL WATERLOGGING

LYR:TRAPSAM

35S:NTF

Leafprimordia

Transpira

AonRa

te

(mmolH

2Operm

2 perse

c.)

Normal

Insufficientwater

Waterlogging

PennLycoPennLyco

20-

15-

10-

5-

0-

PhotosyntheA

crate

(µmolCO2p

erm

2 perse

c)

ShootresponsesunderstressesA.Tomatoshootsexperienceobviousturgorlossandchlorosisaswellasprematurelossofleaves.B.LICORmeasurementstaken12daysaieronsetofstressrevealdifferenttranspiraAonandphotosyntheAclevelsbetweenspeciesandstresses.

A

LICORDataB

IdenAtybyexpression:ToApotentcellsfromthemeristem(M)expressSHOOT-MERISTEMLESS(LET6).CellsbegintodifferenAateintoleaves(P0)byexpressingantagonisAcLYRATE(LYR)toformleafprimordia.LYRexpressionalsomarksleaflets(*)inolder,compoundleafprimordia(P2).FurtherleafAssueidenAtyisdefinedatthisstagebytheexpressionofBLADE-ON-PETIOLE(BOP)whichrepresseslaminarAssuedifferenAaAon,creaAngpeAolesandpeAolules.Evenatthisstage,cellsremainplasAcintheirfate.CellularidenAtycommitmentfinallyoccurswiththeexpressionofAthB8whichiniAatesvasculaturedevelopment.

P0

**

P1

P2

M

✓

35S:TRAP

SucroseGradientSedimenta/on

Absorban

ce(2

54nm)

40S

60S

80S

polysomes

55

Westernα-FLAG

α-RPS6

kDa

HF-GFP-RPL18isincorporatedintopolysomes

RPS6isacomponentofthe40Ssubunit

RPL18isacomponentofthe60Ssubunit

RT-PCRACTIN1

Total

TRAP

25S18S

5.8S5S

SizeM

arker Wildtype35S:TRAP

TRAPyieldsrRNAsandmRNAs(noteabsenceoforganellarrRNA)

**

**35

Total

TRAP

UnboundInput

Wildtype

35:TRA

P

Wildtype

Wildtype

TRAP

HF-GFP-RPL18ispurifiedbyTRAP

35:TRA

P

35:TRA

P

kDa55

α-FLAG

PolysomescanbeimmunopurifiedbyTRAPfromO.sa6va

nucleoluscytoplasm

35S:OsNTFa

nucleus

I 35S

:OsW

PP 2.2

B 35S:O

sWPP 2.

20

100000

200000

300000

Nuc

lei y

ield

Input INTACT

35S:OsNTFa

5540

70

α-H3

Western

15

Wildtype

35S:OsN

TFa

INPUT

kDa

INTACT

Wildtype

35S:OsN

TFa

nucleus

INTACT

beads

dT Random0.000

0.002

0.004

0.006

0.008

UBCIIADH2QHB1

Tran

scrip

t lev

els

35S:OsNTFaINTACTcDNA

35S:OsNTFaINTACT-ChIPDNA

0

1

2

3

4

5

ACT1 H3K4me3ACT1 H3K27me3

% In

put

OsNTFaandH3arepurifiedbyINTACT

OsNTFaisassociatedwiththenuclearenvelope

INTACTyieldsmRNAs

α-Strept

INTACTyieldschroma/nthatcanbeChiP’ed

NucleicanbeisolatedbyINTACTfromO.sa6va

NucleipurifiedbyINTACT

INTACTandTRAPwasalsoestablishedbyourprojectteamfortomato(Ronetal.,PlantPhys2014)andMedicago(Reynosoetal.,2012andunpublisheddatabyBajicetal.,EmoryU)

oo

=Submergence=Control

Water

O. sa6va (cv.Nipponbare)root system response towaterextremeA:3week-oldricerootsystemB,C,D:PrimaryrootsExodermissuberizaAonshownbyFluorolYellowstainedtransecAon(2cmfromtheroot/shootjuncAon(redarrow))(falsecolorgreen).E,F,G:LateralrootstainedwithYellowFluorolshowenhancedsuberizaAonduringdroughtandwaterlogging.

ExEp

Sc

C

E

S

Ex

Ep

Sc

C

E

S

EpEx

Sc

C

E

S

A B C D

E F G

CONTROL DROUGHTWATERLOGGING

B, E: Control, plants grown 3 weeks in regular water regime. C, F:Waterlogging, plants grown 2 weeks as control then 1 week waterlogged(rootsystemcompletelysubmerged).D,G:Drought,plantsgrown2weeksascontrolthenwaterwasremovedfromtraysfor1week.Ep:Epidermis,Ex:Exodermis,Sc:Sclerenchyma,C:Cortex,E:Endodermis,S:Stele.

LEA3

Log10Re

la/v

emRN

Alevel

RAB16A

Log10Re

la/v

emRN

Alevel

AP37

Rela/v

emRN

Alevel

DAY0 DAY9

Shootdrought/mecourseexperimentPlantsweregrowninpotsfor2weeksandwateredeverydayunAlDay0whenwaterwasremovedfromtrays.Rolledupleaveswereobservedaier8dayswithoutwater.qRT-PCRanalysisofthreedroughtmolecularmarkersinriceshootsatdifferentAmepoints.LEA3andRAB16Aarehighlyinducedaier7dayswithoutwater.

INTACTisola/onofnuclei-op/mizedforrice

35S:OsNTFa 35S:OsNTFb 35S:OsNTFc

α-H3

Western

INTACT

Wildtype

35S:OsN

TFa

α-Strept

35S:OsN

TFc

35S:OsN

TFb

INPUT

Wildtype

35S:OsN

TFa

35S:OsN

TFc

35S:OsN

TFb

5540

70

15

kDa

INTACT comparison roots

Nipp

OsWPP

OsWIP

8

OsWIP

9Nipp

b

OsWPP b

OsWIP

8 b

OsWIP

9 b0

50000

100000

150000WildtypeOsNTFaOsNTFbOsNTFc

87%56%

13%2%

INTACT

Input

Nuc

lei y

ield

ExpressionofdifferentNuclearTaggingFusion(NTF)proteinstocapturenuclei

Nucleiquan/fica/on:InputandaherINTACT

HistoneH3ispresentincapturednuclei

Dayswithoutwater

lateralroot

-0.5

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0 3 7 9 11 14-0.5

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0 3 7 9 11 140

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0 3 7 9 11 14

SlCO:TRAP

new tools for evalua/on of cell-speciﬁc environmental plas...

Documents