Avi Prsntation

8/19/2019 Avi Prsntation

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The Tomato FRUITFULL HomologsTDR4/FUL1 and MBP7/FUL2 RegulateEthyleneInde!endent

"s!e#ts o$ F%u&t R&!en&ng

Marian Bemer, Rumyana Karlova, Ana Rosa Ballester, YuryM. Tikunov, Arnaud G. Bovy, Mieke Wolters-Arts, Priscilla de

Barros Rossetto, Gerco C. Anenent, and Ruud A. de Maad!The Plant 'ell( )ol* 24+ 44,7-

44.1

Pa!e% em&na% By

")I R"I0"D"


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"ntroduction and literature

• t#e ene reulatory net$orksinvolved in t#e develo%ment o& !ot#dry and 'es#y &ruits may !e similar,

alt#ou# t#e outcomes aremor%#oloically very di(erent.

• Ara!ido%sis t#aliana - )ruit&ull*&ul+

siliue mutants s#ort and !um%y %oor develo%ment o& one o&de#iscence- de&ect in seed dis%ersal.


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Researc# on 'es#y &ruit &ormation #as %rimarily&ocused on t#e model system tomato, $#ic#%roduces a true !erry derived &rom t#e ovary

• Autoin#i!itory et#ylene %roduction system /

*AC0/A+, AC01, AC02,

3*AC4/+, AC45, AC42

Autocatalytic %roduction system 1

3

Ri%enin


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T#e 6AM, ATA)/, C7C1 *6AC+-domain &amily %rotein646R"P86"6G *64R+, t#e 097AM40A %romoter!indin %rotein *0BP+ C4:4R:800 646-R"P86"6G

*C6R+, and t#e MA;0 domain %rotein R"P86"6G"6


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Pleiotro%ic %#enoty%e- rin mutant-distur!ances o& di(erent MA;0 com%le=es

• TAG:/, TAG://, )7:/, )7:1 interact $it#R"6

• Bindin o& rin to %romoters de%end oncnr activity

• TAG:/ knockdo$n yello$ orane&riuts- decreased e=%ression o& R"6taret AC01.


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Tomato contains > MA;0 Bo= enest#at !elon to 097AM40A?AP/ clade

• Ara!ido%sis tomato 8=%ression

• AG:@ MBP/,MBP1veetative tissues

• AP/ MA;0-MCin'orescences

• )7: T;R2, MBP@ &ruit

•Because o& seuence similarity !et$een-

• At )7: and 0" T;R2 *@>+ - )7:/

• At )7: and 0" MBP@ *@@+ - )7:1


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T#e Tomato )7: Genes ArePredominantly 8=%ressed durin )ruit

;evelo%mentRelat&e e!%ess&on !%o3les o$ FUL1 and FUL2 &n d&e%ent MTt&ssues o5ta&ned 5y 6RTP'R* FL( o8e%9 I( .mm $%u&ts9 II( 1#m$%u&ts9 L( lea$9 FL( o8e%9 I( .mm $%u&ts9 II( 1#m $%u&ts9 M:(matu%e g%een9 B%( 5%ea;e%9 B%*

*


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T#e Tomato )7: Genes Are Predominantly8=%ressed durin )ruit ;evelo%ment

=B> Relat&e e!%ess&on !%o3les o$ lFUL1 and lFUL2 &nse!a%ated $%u&t t&ssues o$ %ed %&!e $%u&ts* . seeds9 Pu .!ul!9 ' . #olumella9 Pl . Pla#enta9 ! . se!ta9 M .meso#a%!9 E


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4!Dectives

• To understand t#e ene reulatorynet$ork involved in tomato ri%enin

•

To kno$ t#e &unction o& )7:/ and)7:1 #omolos

• To com%are dry and 'es#y &ruitdevelo%ment net$ork conservationand diverence


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0euence o& study

• )unction o& )7:/ and )7:1 #omolos &ul mutants

•

Relation o& &ul #omolos $it# C6R,R"6 and TAG:/

• Position o& &ul #omolos in reulatorynet$ork

• ;iscussion

• conclusion


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)unction o& &ul #omolos

• ;ou!le knockdo$ns- &ul/?1 R6Ai

%lants

•

0inle knockdo$ns &ul/ and &ul1R6Ai %lants


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;ou!le knockdo$ns

• sta!le transenic MT lines

• 5>0E)7:/ R6A inter&erence *R6Ai+construct

• codin seuences- @F identical,

• t#e R6Ai &rament$as am%lied &romt#e 5H end o& )7:/, includin a %art o&

t#e 5H untranslated reion *7TR+, $#eret#e identical stretc#es did not e=ceed1 nucleotides


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Phenoty!es

*A+)ruits o& )7:/R6Ai lines R/-/and R/-1> and$ild-ty%e *WT+ MTat stae Br I @.

*C+ Binocularmicrosco%y%ictures o&#andmade%ericar% sectionso& $ild-ty%e andR/-/ &ruits atstae Br I @.


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*B+ Com%arison o& t#e carotenoid levels in $ild-ty%e andR/-/ &ruits at stae MG, BI@ *Br I @ d+, and BI/ *Br

I / d+. A7, a!sor!ance unitsJ )W, &res#

$ei#t.


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Phenoty!es*;+ Water loss in $ild-

ty%e and R/-/ and R/-1 &ruits a&ter #arvestinat stae Br I @. T#e ya=is de%icts t#e%ercentae o& $ei#tloss since t#e day o&#arvestin.

*)+ Cuticle t#ickness in

$ild-ty%e and R/-/ Br I@ &ruits, measured &romt#e to% o& t#e e%idermalcell to t#e sur&ace


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Phenoty!es

*8+ Cuticlet#ickness o& $ild-ty%e and R/-/ BrI @ &ruits is visi!le

in li#t microsco%y%ictures o& t#in%ericar% sectionsstained $it# 0udan

". T#e side %anelss#o$ t#edesiccatedtomatoes 2 da&ter #arvestin.


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st%ongest!henoty!es sho8n5y these

*;+;o$nreulation o&)7:/ and )7:1 int#e )7:/ R6Ai

lines R/-/, R/-1>, R/-1, andR/-5 com%ared$it# t#e $ild ty%e

*WT+. T#e relativee=%ression levels$ere o!tained !yRT-PCR.


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0inle knockdo$ns

• - )7:1R6Ai construct 5H7TR and %art o& 5H4R)

• T#e %lants all e=#i!ited an orane-ri%e &ruit%#enoty%e similar to t#e &ruits o& t#e )7:/ R6Ailines

• - s%ecic )7:/R6Ai only 5H7TR

• - varia!le levels o& )7:/ and no do$nreulation o&)7:1

•

- s%ecic )7:1 R6Ai only 5H 7TR• - )7:/ is reduced to L o& WT.


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;o$n reulation

o& )7:/ and )7:1in t#e )7:1R6Aiand s%ecic )7:/and )7:1 lines.


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Phenoty!es

". elly o& redri%e &ruits&rom lines

Conclusion )7:/ and)7:1 actreduntantly


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)unctions o& )7: #omolos

• Caroteinoid !iosynt#esis- lyco%enereduced and its %recursor %#ytoenedecreases

• :utein remain same

• Beta carotene increases at !r I /stae o& R/-/

• 6ormal %ericar%

• ;ecreased lutamate content


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Relation $it# C6R, R"6 and TAG:/

• M"CR4ARRAY analysis o& R/-/ andR/-5 &rom !r stae

• Conrms uantitative RTPCR resultsand s#o$s several di(erentiallye=%ressed enes, $#ic# ives clues&or relation !et$een )7: #omolos

and u%stream ri%enin reulators.


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Microarray results

:ene/en?yme

FUL1/2 #n% %&n T":L1&len#ed

P0Y/ N - - 3

;O0/ 3 - - -

PG1A 3 3 - -

OY: 3 3 - -

PAT - -

GA;1, GA;5 - -


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• Tal/ rnai and rin %lants - ri%enindecient

• - AC01, AC02 and AC4/ do$nreulated

• Cnr mutants ri%enin decient

• - AC4 do$nreulated

• )7:/?1 R6Ai %lants no do$nreulationo& t#ese enes.*microarray analysis+

T 3


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To #on3%mth&s

Br or Br I 5 stae&ruits &romtransenics and$ild ty%e are

#arvested andet#ylene $asmeasured


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Position o& )7: %aralos inri%enin reulatory net$ork

=%ress on o a t ree


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=%ress on o a t ree ,)7:1 and TAG:/

do$nreulated


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Also et a(ected in rinmutants


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* *


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•

Rin and tal/ u%reulated 1-5 times neative &eed!ack

•

Cnr remains unc#aned


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;"0C700"46


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0trent# and $eakness o&study

• 7se o& microarray analysis to conrmRTPCR results

• ;oesnt clari&y t#at $#y t#ere is nodo$nreulation *even sli#t+ o& )7:1in s%ecic )7:/ R6Ai %lants.


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)uture %ros%ectives


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Re&erences


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"#;no8ledgement

" t#ank ;r. Marian Bemer and #is colleaues $#odid t#is $onder&ul $ork.

" am #i#ly t#ank&ul to ;r.


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Thank you

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