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MAPPINGTHETOMATOROOTGENEEXPRESSIONINDROUGHTANDWATERLOGGINGKAISAKAJALA1,2,DONNELLYWEST1, MAGGIEWOODHOUSE,JIAWENZENG1,2,NIBANIRMAL1,2,REINAMARIESANZ1,2,AYUMIGOTHBERG1,2 ,KRISTINAZUMSTEIN1,NEELIMASINHA1,SIOBHANBRADY1,21DEPARTMENTOFPLANTBIOLOGY,UCDAVIS,USA.2GENOMECENTER,UCDAVIS,USA.

Solanumpennellii“PENN”

DROUGHT-TOLERANT!

Solanumlycopersicumvar.M82

CULTIVATED

0"

50"

100"

150"

Control' Drought' Waterlogging'

'Dry'W

eight'(mg)'

A"B"C"D"

M82 M82 M82CONTROL NOWATER WATERLOG

M82CONTROLB M82NOWATERB

SUBERINSTAININGWITHFLUOROLYELLOWSHOWSSUBERINACCUMULATIONINEXODERMISAFTERNOWATERTREATMENTINM82,WHILEEXODERMALSUBERINISCONSTITUTIVEINS.PENNELLII.

@kaisakajala@bradylabs

Tomatohas

• asequencedgenome

• drought-tolerantinter-crossablespecieswithdevelopmentaldifferences

Weareinves]ga]ngtheresponsestodroughtandfloodingin

•  Specificcelltypes

•  Differentregulatorylevelsofgeneexpression

•  Rootsandshoots

•  Mul]pledifferentspeciesCross-sec]onof

M82root

Cross-sec]onofPENNroot

Wetestedtheeffectoflong-termdroughtandwaterloggingonroots.

Plantsgrownonclaymedia.21dayswellwatered+12daystreatment

•  control(wellwatered)•  nowater• waterlogging(rootballsubmerged)

Samplestakenin6cmsegments.

FloodingControl

1234

5

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Epidermis

CortexMiddleCortexEndodermisPericyclePhloemProcambiumXylem

Exodermis

HYPOCOTYL-DERIVEDADVENTITOUSROOTS(AR)INCREASEINWATERLOGGING.ARSHAVEMORECORTEXLAYERSWITHMOREEXTRACELLULARSPACEAFTERWATERLOGGING.

M82CONTROLAR M82WATERLOGGEDAR

*p=0.0013

INTACT:ISOLATIONOFNUCLEITAGGEDINCELLTYPES1)  Useapromotertoexpressanuclearbio]ntag.

2)  Pulldownthenucleiwithstreptavidinbeads.3)  Subsequentanalyses:ATAC-seq(AssayforTransposonAccessibleChroma]n)comparabletoDNase-seqbuteasier!(Buenrostroetal.,2013)

RNA-seq–weestablishedapipelinetosequencenuclearmRNA.NucleihavelowamountsofpolyAmRNA,sototalnuclearRNAissubjectedtorRNAdegrada]on.

TRAP:TRANSLATINGRIBOSOMEAFFINITYPURIFICATION1) Useapromotertoexpressaribosomeflagtag.

2) Pulldowntheribosomeswithα-flagbeads.

3)  Subsequentanalyses:RNA-seq–rapidrandom-primedlibraries(Townlseyetal.,2015)

aregeneratedfrompolysomalmRNAtoquerywhichtranscriptsareassociatedwithpolysomes

Ribo-seq–RNAimmunoprecipita]onallowsiden]fica]onofexactribosomeloca]onsonmRNA.

Chromatin

TRAP Translating Ribosome

Affinity Purification

INTACT Isolation of Nuclei

TAgged in Cell Types

mRNA

Polysomal mRNA

AAAA…

nucleus

AtPEP:INTACT

SlCO2:INTACT SlCO2:TRAP

Cortex

Meristem

a]c

cortex

S.lycopersicumM82 S.pennellii

35S:INTACT

S.lycopersicumcv.M82

S.pennellii

S.lycopersicum S.pennellii

SlRPL11C:INTACT

Dividingcells(meristema]c)

SlRPL11C:INTACT

SlWOX5:INTACT

Quiescentcenter&ini]als

SlWOX5:INTACT

TRANSGENICMARKERLINESTOSTUDYUNDERLYINGGENEEXPRESSION

TRANSGENICMARKERLINESUSEDINPROFILINGMULTIPLELEVELSOFGENEEXPRESSION

S.lycopersicumHairyrootculture

35S:TRAP 35S:TRAP 35S:TRAP

35S:INTACT 35S:INTACT

S.lycopersicum S.lycopersicum S.lycopersicum S.lycopersicum S.lycopersicum

Endodermis&QC Vasculature Phloem Xylem Nearcons]tu]ve Lateralrootcap,epidermis&exodermis

S.lycopersicum S.lycopersicum

S32:TRAP S18:INTACTSlSHR:INTACTSlSCR:TRAP AtWER:TRAP AtWER:INTACTSlACT2:TRAP

S.lycopersicum

Cortex(noexodermis)

AtPEP:TRAP

SlPEP:TRAP

S.lycopersicumM82&S.pennellii:7daysonver]calplates

Horizontalsealedcontrolplates

Horizontalsubmergedplates

EXPERIMENTALSET-UP&TIMECOURSEOFRESPONSE HAIRYROOTCULTURES•  inducedbyAgrobacteriumrhizogenes

•  ausefultoolforgenera]ngrapidtransgenictomatorootstostudyrootdevelopment(Ronetal.,2014)Hairyrootcultures:

9daysofgrowthonhorizontalplates

ADH

Rela]vetranscrip

tlevel

Time(h)

S.lycopersicumcv.M82primaryroot

S.lycopersicumcv.M82hairyroots

S.pennelliiprimaryroot

SubmergenceControl

GENEEXPRESSIONPATTERNSCANDIFFERBETWEENLEVELSOFREGULATION

HAIRYROOTSUBMERGENCERESPONSEISDISTINCTFROMPRIMARYROOTRESPONSE

S.lycopersicum S.pennellii

MDSplotofuniquelymappedreadsfromTOTALmRNAlibraries

UNLIKEPRIMARYROOTS,HAIRYROOTSUBMERGENCE-RESPONSIVEGENESAREENRICHEDIN

•  transcrip]onfactors•  lateralrootini]a]onrelatedgenes

NEXTUP:DIFFERENTIALLYACCESSIBLECHROMATINREGIONS!COMPARISONOFDIFFERENTREGULATORYLEVELS!COMPARISONSACROSSTOMATOES,MEDICAGO&RICE!

Submergenceresponsivegenes–TOTALmRNA

Primaryroot Hairyroot

Submergenceresponsivegenes–NuclearmRNA(INTACT)

Primaryroot Hairyroot

Hairyrootsubmergenceresponsivegenes

PolysomalmRNA(TRAP)

NuclearRNA(INTACT)

TotalRNA CONTROL

HAIRYROOT

SUBMERGEDHAIRYROOT

CONTROLPRIMARYROOT

SUBMERGEDPRIMARYROOT

CHROMATINACCESSIBILITYOFTRANSPOSABLEELEMENTSISGLOBALLYHIGHINCONTROLHAIRYROOTS,BUTISREDUCEDTOPRIMARYROOTLEVELINSUBMERGENCE

TRANSPOSABLEELEMENTLOCATIONSMAPPINGREFERENCELOCATIONS

ATAC-SEQREADSREADCOUNTVS.BACKGROUNDRED(HIGH)àBLUE(LOW)ATAC-SEQREADSREADCOUNTVS.BACKGROUNDRED(HIGH)àBLUE(LOW)

TRANSPOSABLEELEMENTLOCATIONSMAPPINGREFERENCELOCATIONS

ATAC-SEQREADSREADCOUNTVS.BACKGROUNDRED(HIGH)àBLUE(LOW)ATAC-SEQREADSREADCOUNTVS.BACKGROUNDRED(HIGH)àBLUE(LOW)