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$#*'
Root tip containing affinity tagged nuclei in one cell type
Nuclei preparation Add streptavidin-coated magnetic beads
Collect labeled nuclei on a magnet
Mixture containing affinity labeled nuclei (green)
Streptavidin beads (black ovals) bind to
biotinylated nuclei
!"#$%&'%())*+',*-./0&11-'$#'234536789'1$#*'
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$#*'
NucleipreparaAon
AddStreptavidin-
coatedmagneAcbeads
TaggedRootCells
AffinityPurifywith
anAbodyHomogenizeandlyseER
Collectlabelednuclei
onamagnet
Root tip containing affinity tagged nuclei in one cell type
Nuclei preparation Add streptavidin-coated magnetic beads
Collect labeled nuclei on a magnet
Mixture containing affinity labeled nuclei (green)
Streptavidin beads (black ovals) bind to
biotinylated nuclei
!"#$%&'%())*+',*-./0&11-'$#'234536789'1$#*'
TRAP%expressing%cells%
Affinity%Purify%with%an89GFP%%
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$#*'
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
