genetic dissection of grape berry ripening and composition · fruit ripening Ø irreversible...

Genetic dissection of grape berryripening and composition

Sara Zenoni

Fruit ripening

Ø irreversiblephenomenon

Ø tightlycoordinatedwithseeddevelopment

Ø geneticallyandepigeneticallyprogrammedsystem

_phytohormone signallingpathways

_transcriptionfactornetworks

Fruitistheorganspecializedforseeddispersaland

thetransitionfromunripetoripefruitrepresentsacrucialsurvivalstrategy

TOMATO

modelforflesh fruit ripening

climateric fruit

Grape berry ripening

Ø nonclimateric fruit

Ø verylongripening,almost3months

Ø stronglyaffectedbyenvironment

Ø ripeninginthegrapeberryoriginatesinpulpnearthestylar end

Castellarin etal.,2011

Ø theonsetofripeningischaracterizedbyanaccumulationofspecificreactiveoxygenspecies(ROS)

Pilatietal.,2014

Rogiers etal.,2017

Chemical and physiological changes during berry development

Large-scale transcriptional changes during berry development

LUNDetal.,

ZAMBONIetal.,

ZENONIetal.,

PILATIetal.,

GRIMPLETetal.,

DELUCetal., FORTESetal.,

GUILLAUMIEetal.,RINALDOetal.,

SWEETMANetal.,

LIJAVETZKY etal.,

FASOLIetal.,

CRAMERetal.,

PALUMBOetal.,

AGUDELO-ROMEROetal.,

DALSANTOetal.,

ZENONIetal.,

DALSANTOetal.,

FASOLIetal.,

MASSONNETetal.,

GHANetal.,

DAVIESANDROBINSON

TERRIERetal.,

WATERSetal.,

BURGERetal.,

GRAPEVINEGENOMESEQUENCING

JAILLONetal.,

cDNA-AFLPmacroarrays microarrays

CONSTANTIMPROVEMENTOFTRANSCRIPTOMICPLATFORMS

RNA-seq

Genome-widetranscriptome profiling

Bioinformatictools

Systembiologyapproaches

Identification anddefinition of“GRIP”grape ripening-induced protein

DAVIESANDROBINSON

PILATIetal.,

ZAMBONIetal.,

DALSANTOetal.,

SWEETMANetal.,CRAMERetal.,

LUNDetal.,

FORTESetal.,RINALDOetal.,

ZENONIetal.,LIJAVETZKY etal.,

FASOLIetal.,ZENONIetal.,

GRIMPLETetal.,

DELUCetal.,

GUILLAUMIEetal.,


PALUMBOetal.,

DALSANTOetal.,

FASOLIetal.,

MASSONNETetal.,

GHANetal.,TERRIERetal.,

WATERSetal.,

BURGERetal.,


hydroxyproline-rich proteinGRIP3

early nodulin GRIP13

early nodulin GRIP15

unknown GRIP22

cell wall related GRIP28

DAVIESANDROBINSON

PILATIetal.,

ZAMBONIetal.,

DALSANTOetal.,


LUNDetal.,




GRIMPLETetal.,

DELUCetal.,

GUILLAUMIEetal.,


PALUMBOetal.,

DALSANTOetal.,

FASOLIetal.,

MASSONNETetal.,


WATERSetal.,

BURGERetal.,

Expression profile oftheprincipal molecular eventsduring berry development


Transcriptomic rearrangement during the ripening transition

○ Secondarymetabolism

○ Sugarmetabolism

○ Starchdegradation

○ Regulationofgene

expression

○ Bioticstressresponse

○ Cellwallmetabolism

○ Photosynthesis

○ Cell cycle

○ Cellularcomponent

organization

○ Hormone(auxin)signalling

responsivetranscripts

THESHIFTFROMTHEGROWTHTORIPENINGPHASEINBERRYINVOLVESAPROFOUNDTRANSCRIPTOMICREARRANGEMENT


DAVIESANDROBINSON

PILATIetal.,

ZAMBONIetal.,

DALSANTOetal.,


LUNDetal.,




GRIMPLETetal.,

DELUCetal.,

GUILLAUMIEetal.,


PALUMBOetal.,

DALSANTOetal.,

FASOLIetal.,

MASSONNETetal.,


WATERSetal.,

BURGERetal.,

Transcriptomicchangesduringberrydevelopmentinpulp andskinseparately

Ripening program is anticipated in pulp

Oxidative stressresponse

Oil bodyorganization

Waxbiosynthesis

Flavonoids/anthocyaninsbiosynthesis

Stilbenoidbiosynthesis

Ethylenesignallingandflavorpathways

PULP

SKIN

Lijavetzky etal.,2012

TRANSCRIPTOMICPROGRAMISANTICIPATEDINPULPINCOMPARISONTOTHESKIN

DAVIESANDROBINSON

PILATIetal.,

ZAMBONIetal.,

DALSANTOetal.,


LUNDetal.,




GRIMPLETetal.,

DELUCetal.,

GUILLAUMIEetal.,


PALUMBOetal.,

DALSANTOetal.,

FASOLIetal.,

MASSONNETetal.,


WATERSetal.,

BURGERetal.,


RNA-seq approach todissect thetranscriptionalcomplexity during berry development

Massonnet etal.,2017

Ripening transcriptomic program in red and white grapevine varieties

Number of expressed genes and biomarkers

THENUMBEROFEXPRESSEDGENESDECREASESDURINGBERRY

DEVELOPMENT

BIOMARKERSOFBERRYDEVELOPMENTSTAGESANDPHASESWEREDEFINED

Pea size

Pre-veraison Endofveraison

Harvest

Core transcriptomic traits during berry development


CORETRANSCRIPTOMICTRAITSWEREPROFILED


Relation with anthocyanin accumulation and ripening progress at transcriptional level

TRANSCRIPTOMICPROGRAMOFFRUITRIPENINGSEEMSMOREDIRECTLYRELATEDTOANTHOCYANINACCUMULATIONRATHERTHANSUGARCONTENT

Relation with anthocyanin accumulation and ripening progress at transcriptional level

DEGs

Secondary metabolic processTransport

Carbohydrate metabolismTranscription factor activity

About6000 genesareresponsible fortranscriptionaldifferencesamongred

varietiesatharvest


Rinaldoetal.,2015

Syrah

ANTHOCYANINLEVELSMAYINFLUENCEMANYOTHERPROCESSES• INCREASEOFSKINOPACITYTOSUNLIGHT

• INVOLVEMENTINSIGNALLINGPATHWAYSNOTYETDESCRIBED

Correlation of gene expression between genotypes increasesas haplotype distance decreases

Magrisetal.,unpublished

ü Sequencingoftengenomes

ü Pairwisecomparisontodefinethehaplotypedistance

ü IdentificationoflocalIBD(segmentidenticalbydescent)

Sharedchromosomesegmentsbetween

‘Garganega’and‘Passerina’

Sharedchromosomesegmentsbetween

‘Passerina’and‘Vermentino’

Two shared haplotypes IBD2

One shared haplotype IBD1

Noshared haplotypes IBD0

Correlation of gene expression between genotypes increasesas haplotype distance decreases

• GenesforwhichthepairofindividualsareIBD0(Sharing0)• Genesforwhichthepairofindividualsshareonehaplotype(IDB1-Sharing1)• Genesforwhichthepairofindividualssharetwohaplotypes(IDB2-Sharing2)

Correlationoftranscriptexpressionlevel

Fractionofnondifferentiallyexpressedgenes

Developmentalstages

HAPLOTYPESHARINGACCOUNTSFORCORRELATIONOFGENEEXPRESSION

Magrisetal.,unpublished

DAVIESANDROBINSON

PILATIetal.,

ZAMBONIetal.,

DALSANTOetal.,


LUNDetal.,




GRIMPLETetal.,

DELUCetal.,

GUILLAUMIEetal.,


PALUMBOetal.,

DALSANTOetal.,

FASOLIetal.,

MASSONNETetal.,


WATERSetal.,

BURGERetal.,


Molecular dissection of the grapevine GXE interaction

DalSantoetal.,2018

Changesinperformanceofgenotypes indifferentenvironmentsaredefinedasgenotypeXenvironment(GXE)interaction

Cabernet S.

Sangiovese

Cabernet S.

Sangiovese

Sangiovese

Cabernet S.

Pre-Veraison Mid-RipeningPeaSize Ripe

2GenotypesSangiovese andCabernetsauvignon

4Developmentalstages

2Years

3AreasAdjacentVineyardspereachArea

CabernetSauvignon

Sangiovese

Molecular dissection of the grapevine GXE interaction

Screening

Profiles definition

Profiles characterization

WholeTranscriptome↓

18122 genes

VariableImportanceMeasure (VIM)measureofhow eachvariableaffectsthe

expressionSTAGE

GENOTYPEYEARAREA

K-meansclustering→300clusters

DataMining Pipeline

DalSantoetal.,2018

SANGIOVESERESULTEDMORERESPONSIVETHANCABERNETSAUVIGNON

Variable-specific clusters

DalSantoetal.,2018

MedianVIMofeachVariable

Photosynthesis

MedianVIMofeachVariable

R-proteins

GxE clusters are enriched in secondary metabolism, signal transduction and abiotic stress response

DalSantoetal.,2018

Stilbene Sythases (VvSTSs)

Linalool Sythases (VvSPSs)

IDENTIFICATIONOFSEVERALCANDIDATEGENESTHATCOULDBEUSEDASMARKERSOFBERRYQUALITYTRAITSINGXEINTERACTIONS

DAVIESANDROBINSON

PILATIetal.,

ZAMBONIetal.,

DALSANTOetal.,


LUNDetal.,




GRIMPLETetal.,

DELUCetal.,

GUILLAUMIEetal.,


PALUMBOetal.,

DALSANTOetal.,

FASOLIetal.,

MASSONNETetal.,


WATERSetal.,

BURGERetal.,

Expression profile oftheprincipal molecular eventsduring postripening phase


Transcriptional changes during berry post-harvest

SyrahCabernetSauvignonOseleta

SangioveseCorvina

Merlot

~30%weightloss

Zenonietal.,2016

Transcriptional changes during berry post-harvest

DOWNREGULATION

UP REGULATION

LACsStress

LACsStress

LACsStress

TFsNucleic Acidmetabolism

STSsTPSs

StressLipid

Corvina Sangiovese Merlot

Syrah Oseleta Cabernet

Zenonietal.,2016

DURINGTHEPOST-HARVESTPHASETHEREISANACTIVEMETABOLICREARRANGEMENTANDNOTONLYAPASSIVECONCENTRATION

What triggers the ripening transition?

Berry softening is one of the earliest ripening events

Castellarin etal.,2016

(I)(II)

(III)

Fasoli etal.,2012

54samples representingdifferent plant organs duringdevelopment

Grapevine expression atlas as a starting point

Mature/woody Vegetative/green

Identification of putative regulators of ripening transition

THEPROFOUNDTRANSCRIPTOMICREARRANGEMENTINBERRYFROMIMMATURETO

MATUREPHASEWASOBSERVEDFORALLGRAPEVINEPLANTORGANS

Palumboetal.,2014

Identification of putative regulators of ripening transition

Differentially expressed genes between vegetative/green and mature/woody samples

Topological propertiesandroleswere analysed

DURINGTHETRANSITIONTOMATUREPHASEMANYPROCESSESAREINHIBITEDRATHERTHANACTIVATED

Identification of switch genes

Palumboetal.,2014

Heat cartography map

SWITCHGENES

• interacthighlywithgenesoutsidetheirownmodule

• interactpoorlywithgenesinsidetheirownmodule

• mainlyanti-correlatedwiththeirpartners

Switch genes model of action

ü Allswitchgenesaredownregulatedduringgrowthphaseandupduringmaturephase

ü Switchgenescouldactasanelectricswitchabletoswitch-offtheexpressionofvegetative-related

genesandtoswitch-ontheexpressionofmature-relatedgenes

Palumboetal.,2014

SWITCHGENES

Switch genes in red and white berries


5913181

190 212BERRYSWITCHGENES

GENE_ID GENE_description GENE_Name Atlas

VIT_17s0000g00430 basic helix-loop-helix (bHLH) family bHLH075

VIT_15s0046g00150 DOF affecting germination 1 DAG1

VIT_06s0004g07790 Lateral organ boundaries domain 15 *

VIT_03s0091g00670 Lateral organ boundaries protein 38

VIT_13s0158g00100 putative MADS-box Agamous-like 15a VviAGL15a

VIT_07s0031g01930 myb TKI1 (TSL-KINASE INTERACTING PROTEIN 1)

VIT_02s0033g00380 R2R3MYB transcription factor VvMybA2 (C-term)

VIT_02s0033g00390 R2R3MYB transcription factor VvMybA2


VIT_14s0108g01070 NAC domain-containing protein VvNAC11


VIT_19s0027g00230 NAC domain-containing protein VvNAC33 *

VIT_08s0007g07670 NAC domain-containing protein VvNAC60 *

VIT_07s0005g01710 WRKY Transcription Factor VvWRKY19

VIT_05s0020g04730 Zinc finger (C3HC4-type ring finger)

VIT_08s0040g01950 Zinc finger (C3HC4-type ring finger) *


VIT_03s0091g00260 Zinc finger protein 4

Transcription factors among switch genes


Cabernetsauvignon

Pinotnoir

Fasoli etal.,2018inpress

FIRSTTRANSITION SECONDTRANSITION

VERAISON

Two transcriptional transition mark the onset of ripening

WeeklysampledThreevintages

Fasoli etal.,inpress

Identification of markers of the onset of ripening

Positiveandnegativemarkers ofthetwo transitions define important transcriptional changes during thetwo weeksbefore verasion

POSITIVEMARKERSOFTHEFIRSTTRANSITIONSEEMTOPLAYAMAJORROLEASTRIGGERS

GENE_ID GENE_description GENE_Name Atlas Marker of the first

transition

Marker of the second

transition

VIT_17s0000g00430 basic helix-loop-helix (bHLH) family bHLH075 *


VIT_06s0004g07790 Lateral organ boundaries domain 15 * *



VIT_07s0031g01930 myb TKI1 (TSL-KINASE INTERACTING PROTEIN 1) *

VIT_02s0033g00380 R2R3MYB transcription factor VvMybA2 (C-term) *


VIT_02s0033g00390 R2R3MYB transcription factor VvMybA2 *




VIT_19s0027g00230 NAC domain-containing protein VvNAC33 * *


VIT_07s0005g01710 WRKY Transcription Factor VvWRKY19 * *


VIT_08s0040g01950 Zinc finger (C3HC4-type ring finger) * *


VIT_03s0091g00260 Zinc finger protein 4 *

Switch genes are positive markers of the two transitions

Functional analysis of VvNAC33 and VvNAC60

Transientexpressionafter

48h35S VvNAC33 TER Prom eGFP TER

35S VvNAC60 TER Prom eGFP TER

Stable GFPexpressionafter2-3months

InvitroplantletsGreenhouse andphenotypicanalysis after 1-2years

Embryogenic calli

Overexpression of VvNAC33 in transgenic Syrah

CTRLA4A5A8

2

4

6

8

10

CTRL A4 A5 A8

µgChl/cm

2

2

2,4

2,8

3,2

3,6

CTRL A4 A5 A8

Chl/Car

**

*

* * *

CTRL35S::NAC33

D’Incà,unpublished

Overexpression of VvNAC60 in transgenic Syrah

CTRL35S::VvNAC60

0

1000

2000

3000

4000

5000

6000

7000

CTRL VvNAC60

Anthocyanins(ug/m

l)/gFW *

Clearsenescencesymptoms

“REGULATORYNETWORKBEHINDTHEBERRYRIPENING:THEROLEOFVITISVINIFERANAC60TRANSCRIPTIONFACTOR”

Poster P119ofChiaraForesti

ChIP-Seq analysis ofVvNAC60

Prom VvNAC33 VvNAC33-EAR TER Prom eGFP TER

Prom VvNAC60 VvNAC60-EAR TER Prom eGFP TER

PosterP148ofEdoardoBertini

“IDENTIFICATIONANDFUNCTIONALCHARACTERIZATIONOFMASTERREGULATORSOFTHEONSETOFBERRYRIPENINGINGRAPEVINE”

Transcriptomic analysis of VvNAC33 and VvNAC60 overexpressed plants

35S:NAC33 35S:NAC60

35S:NAC33 35S:NAC60

233 32 374

365 243 334

2Galactinol synthases2Lateral organ boundaries proteins

(LOB1,LOB39)3Nitrate transporters

VvMYB154ERF/AP2genes

3Methyl jasmonate esterases

VvMYBF1VvMYBPA1

16Auxin responsiveproteins3Auxin induced proteins

17Ankyrins4Calmodulin binding proteins

VvMYBC2-L26Gibberellin-regulated proteins (GASA4)

Auxin responsive/induced proteinsVegetativestorage protein

5MADS-boxesShortVegetal Phase (SVPs)5Photosystem reaction centers

4Light-harvesting chlorophyll binding(LHCB) proteins

VvMYBA1VvMYBA2

Anthocyanin permease (VvAnthoMATE1)VvMYB142STSs

Trans-resveratrol di-O-methyltransferase - VvROMT6ERF/AP2genes (VvERF075)

Lateral organ boundaries proteins (LOB38)5XHTs

MADS-boxAgamous 1(VvAG1)MADS-boxdelta2b(VvMADSD2b)5NACs (VvNAC11andVvNAC26)

VvNCED1

UPREG

ULAT

EDDO

WNREG

ULAT

ED

**

VvMYBA1p:LUC

AlessandraAmato

Tomato as model to study flesh fruit ripening

Ø Accessiblegermoplasm resources

Ø Simplediploidgenetics

Ø Efficientgreenhousepropagation

Ø Short lifecycle

Ø Easeoftransformation

Ø Recombinantinbredlines

Ø High-qualitygenomesequence

Ø Naturalmutantsofripening

Induced firstinlocules

Induced later inpericarp

Ø developmental window in which fruit responds toethylene that corresponds to seed maturation;

Ø fruit tissues do not mature uniformly;

Ø a core set of ripening regulators has been defined,including RIN-MADS, NOR-NAC, CNR-SPL and TAGL1;

Ø hypomethylation contributes towards the start ofripening regulatory cascade

Tomato NOR mutant

EthyleneproductionimpairedRipeningstronglyinhibited

VvNAC33 and VvNAC60 partially complement the NOR function

nor/nor

Wildtype

#1

#2

35S::VvNAC33

0

0,1

0,2

0,3

MNE

#1#2WT VvNAC33#1VvNAC33#2

#1

#2

35S::VvNAC60

0

0,2

0,4

0,6

MNE

#1#2WT VvNAC60#1VvNAC60#2

VvNAC60 induces ethylene biosynthesis

nor/nor

Wildtype

35S::VvNAC33#1

35S::VvNAC60#1 0

2

4

6

8

10

12

BK BK+3 BK+5 BK+7

Ethy

lene

(nL/gfw/h)

WT

nor/nor

VvNAC33

VvNAC60

“FUNCTIONALCOMPLEMENTATIONOFnon-ripening (nor)TOMATOMUTANTWITHFOURNACTRANSCRIPTIONFACTORS,PUTATIVEMASTERREGULATORSOFTHEVEGETATIVE-TO-MATUREORGANTRANSITIONINGRAPEVINE”

PosterP145ofEricaD’Incà

GENE_ID GENE_description GENE_Name Atlas Marker of the first

transition

Marker of the second

transition

VIT_17s0000g00430 basic helix-loop-helix (bHLH) family bHLH075 *


VIT_06s0004g07790 Lateral organ boundaries domain 15 * *



VIT_07s0031g01930 myb TKI1 (TSL-KINASE INTERACTING PROTEIN 1) *

VIT_02s0033g00380 R2R3MYB transcription factor VvMybA2 (C-term) *


VIT_02s0033g00390 R2R3MYB transcription factor VvMybA2 *






VIT_07s0005g01710 WRKY Transcription Factor VvWRKY19 * *


VIT_08s0040g01950 Zinc finger (C3HC4-type ring finger) * *


VIT_03s0091g00260 Zinc finger protein 4 *

VvbHLH075 and VvWRKY19

Functional analysis of VvbHLH075 and VvWRKY19

35S YFP Term 35S WRKY19 Term

35S YFP Term 35S BHLH75 Term

Firstleaf Secondleaf

d.p.i.7

TheYFPfluorescence is mainly localized inthefirstandsecond leavesThompsonseedless plants

grown invitro

Firstleaf

Secondleaf

Transcriptomicanalysis

EdoardoBertini


Putative targets of VvbHLH075 and VvWRKY19


Putative hierarchy of transcription factors in the onset of berry ripening

bHLH075NAC60

WRKY19

NAC33

NAC26

BERRYRIPENINGMarkerofthefirsttransition

NAC17

Markerofthesecond transition

NAC11

MYBA1

Anthocyaninmetabolism

MYBA2

MYB14

Stilbenoidsynthesis

LOB38

Berrysize andshape

LOB15

LOB39

Switchgene

MYBF1 MYBPA1

Flavonolsynthesis

Proanthocyaninsynthesis

Conclusions

v Newtechnologicaladvancementsingeneexpressionanalysishavegeneratedahugeamountoftranscriptomicdatathatneedstobedeeplyinterpreted

v Theintricatetranscriptionalnetworkoftheonsetofripeninghasbeenpartiallydisentangledthroughco-expressionandstatisticalpipelines

v Biomarkersandputativeregulatorshavebeenidentified

v Functionalstudiesareneededtounderstandtheroleofthesecandidatesintriggeringripeningtransition

Next steps

Ø Identifydirecttargets

Ø Investigateroleofmethylationinberryripeningcontrol

Ø CharacterizepotentialmicroRNAsthatcouldcontrolexpressionofidentifiedregulators

THANKSFORYOURATTENTION

Theberriesareripeandmytimeis finished….Thanks to

Mario PezzottiGiambattistaTornielliSilviaDalSantoMelanieMassonnetEricaD’IncàAlessandraAmatoEdoardoBertiniChiara Foresti

LucioBrancadoroGabriellaDeLorensis

MicheleMorganteGabrieleDiGasperoGabrieleMagrisEmanueleDePaoli

NickDokoozlianMarianna FasoliChandra RichterJim Giovannoni

JuliaVrebalov

MassimoGardiman

Paola ZuccolottoMarcoSandri

Paola PaciLorenzoFarina

genetic dissection of grape berry ripening and composition · fruit ripening Ø irreversible...

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