Putative role of cytokinin in differential ethylene response of twolines of antisense ACC oxidase cantaloupe melons

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OULOUSE

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OATAO

)

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Eprints ID : 11478

To link to this article : doi:10.1016/j.postharvbio.2013.07.040

URL :

http://dx.doi.org/10.1016/j.postharvbio.2013.07.040

To cite this version : Goncalves, Ciane Xavier and Tiecher, Aline and

Chaves, Fábio Clasen and Nora, Leonardo and Li, Zhengguo and

Latché, Alain and Pech, Jean-Claude and Rombaldi, César Valmor

Putative role of cytokinin in differential ethylene response of twolines

of antisense ACC oxidase cantaloupe melons. (2013) Postharvest

Biology and Technology, vol. 86 . pp. 511-519. ISSN 0925-5214

Any correspondance concerning this service should be sent to the repository

administrator:

staff-oatao@listes-diff.inp-toulouse.fr

Putative

role

of

cytokinin

in

differential

ethylene

response

of

two

lines

of

antisense

ACC

oxidase

cantaloupe

melons

Ciane

Xavier

Gonc¸

alves

_,

_Aline

_Tiecher

_,

_Fábio

_Clasen

_Chaves

_,

_Leonardo

_Nora

_,

Li

Zhengguo

_,

_Alain

_Latché

_,

_Jean-Claude

_Pech

_,

_Cesar

_Valmor

_Rombaldi

a_{UniversidadeFederaldePelotas(UFPel)–CDTec(CentrodeDesenvolvimentoTecnológico),CaixaPostal354,CEP90010-900Pelotas,RS,Brazil} b_{UFPel,FaculdadedeAgronomiaEliseuMaciel,DepartamentodeCiênciaeTecnologiaAgroindustrial,CaixaPostal354,CEP90010-900Pelotas,RS,Brazil} c_{CollegeofLifeSciences,ChongqingUniversity,Chongqing400030,China}

d_{UMR990INRA/INP-ENSAT,LaboratoiredeGenomiqueetBiotechnologiedesFruits,ChemindeBorderouge,BP107,31326CastanetTolosanCedex,France}

Keywords: Cucumismelo Hormones Fruitquality Aroma ACCsynthase ACCoxidase

a

b

s

t

r

a

c

t

Twotransgeniclinesof‘Cantaloupe’melonderivedfromthesamewildtypegenotypewerepreviously generatedusingACCoxidaseantisenseconstructsfrommelon(pMEL1AS)andapple(pAP4AS).Bothlines yieldedfruitwithreducedethyleneproductionandlowACCoxidase(ACCO)expression.ACCOantisense fruitalsoexhibitedlowerexpressionofACCsynthasegenes,ACCS1andACCS3,indicatingthatthesegenes arepositivelyregulatedbyethyleneandparticipateintheautocatalyticethyleneproductionprocess.In contrast,ahigherexpressionofACCS5wasobservedinantisenselineswhencomparedtothewildtype indicatinganegativefeedbackregulationofACCS5byethylene.Fruitofbothtransformedlinesexhibited delayedripeningandreductioninestervolatileproductionbutdifferedintheirresponsetoexogenous ethylenesupply.WhilepostharvestethyleneapplicationfullyrestoredtheripeningprocessinpMEL1AS melon,itonlyrestoredfleshsofteningofpAP4ASmelonbutnotrindcolorchangeoraromavolatile pro-duction.Up-regulationoflipoxygenasepathwayassociatedgenes(hydroxyperoxidelyase,lipoxygenase, andalcoholacyltransferases1,3and4)occurredinethylene-treatedpMEL1ASfruitbutnotinpAP4AS melons.Polygalacturonase1genetranscriptaccumulationincreasedinpMEL1ASandpAP4ASfruitupon ethylenesupply.Zeatinandzeatinribosidecontentofrootsandfruit(rindandflesh)ofpAP4ASplants were5-foldhigherthanthewildtypeandpMEL1AScounterparts.Higherrelativetranscript accumula-tionofageneinvolvedinthecytokininsynthesisandageneinvolvedincytokininresponsewerealso foundintherootsandfruitofpAP4AS.Inaddition,polyamines,whichareknowntoreduce sensitiv-itytoethylene,remainedunchangedinallfruit.Collectivelytheresultssuggestaputativeroleforthe increasedendogenouscytokinincontentincounteractingethyleneactioninsomeaspectsofthefruit ripeningprocess.

1. Introduction

Melon(CucumismeloL.)var.cantalupensisNaudcv.

Vedran-tais is a typical climacteric fruit with a negative relationship

betweenethyleneproductionandshelflife,andapositive

relation-shipbetweenethyleneandaromaproduction.Suchphysiological

behaviorhasbeenconfirmedbytransformation of melonusing

1-aminocyclopropane-1-carboxylicacidoxidase(ACCO)antisense

genes(Ayubetal.,1996;Silvaetal.,2004);Ayubetal.(1996)

uti-lizedanACCOantisensegenefrommelon(pMEL1AS),previously

isolatedandcharacterizedbyBalaguéetal.(1993),andSilvaetal.

∗ Correspondingauthorat:UniversidadeFederaldePelotas(UFPel)–CDTec (Cen-trodeDesenvolvimentoTecnológico),CaixaPostal354,CEP90010-900,Pelotas,RS, Brazil.Tel.:+555332757258.

E-mailaddress:cesarvrf@ufpel.edu.br(C.V.Rombaldi).

(2004)utilized anACCOantisensegene from‘RoyalGala’ apple

(pAPAS).Inbothcases,thewildtypegenotypewasthesameand

thetransformationresultedinACCOtranscriptionsuppressionand

lowethyleneproduction.

Among pAPAStransgenic events(Silva et al.,2004), pAP4AS

exhibitedphenotypicchangescharacterizedbyhighaxillarybud

growth,greenishcolor,andreducedleafsenescence.Thesefeatures

arecommonlyobservedinplantsexpressinghighcytokinin

con-tent;forexample,HOCArabidopsismutant(Catterouetal.,2002)

andtransgenicplantsoverexpressingIPTgene(Merewitzetal.,

2011;Zhangetal.,2010),orplantssubmittedtomoderatedrought

stress(Cogoetal.,2011).Becauseofthesecharacteristics,itwas

hypothesizedthatcytokinincontentcouldhavebeenaffectedin

pAP4ASmelon.

Yellowingoffruitrind,fleshfirmnessreduction,formationofa

peduncularabscissionzone,andreestablishmentofester

produc-tion,resultingfrompostharvestethyleneapplicationtopMEL1AS

melon, indicate that these are ethylene-dependent or partially

ethylene-dependentevents(Floresetal.,2002;Nishiyamaetal.,

2007).Ontheotherhand,fleshcolorandsugarcontentarenot

affectedby ethylene (Bauchotet al., 1998; Bower et al., 2002;

Guisetal.,1997;Pechetal.,2008).InthecaseofpAP4ASmelon,

ripeningwasnotcompletelyrecoveredwithethyleneapplication

(0.1–400mLL−1_{).Althoughthecauseforsuchbehaviorisnotclear,}

itisknownthathighcytokinin(Akhtaretal.,1999;Chenetal.,2001;

Cogoetal.,2011;Martineauetal.,1995)orpolyamine-containing

organs(Neilyetal.,2011)arelesssensitivetoethylene.

Transgenicmelon (pMEL1AS) (Ayubet al., 1996)and

trans-genicapples(pAE12AS)expressinganACCOantisense(Dandekar

etal.,2004),withsuppressedethyleneandestervolatile

produc-tion(Bauchotetal.,1998;Silvaetal.,2004;Yahyaouietal.,2002),

wereabletorestorevolatileproductionwhenethylenewas

exoge-nouslysupplied.Estervolatilesynthesisisdependentonfattyacid

metabolisminvolvinglipoxygenase(LOX),a-andb-oxidations,

fol-lowedbyreductionintoaldehydesandalcoholsassistedbyalcohol

dehydrogenase(ADH),andalcoholacyltransferase(AAT)that

cat-alyzesthelastesterificationstep(Beekwilderetal.,2004).Flores

etal.(2002)andYahyaouietal.(2002)noticedthatestervolatile

synthesisispromotedbyAATactivation.Inmelon,fourAAT(AAT1,

AAT2,AAT3andAAT4)cloneswereisolatedandcharacterized;with

AAT1andAAT4beingup-regulatedduringripeningunderethylene

control(El-Sharkawyetal.,2005;Yahyaouietal.,2002).

Thus, the aforementioned transgenic lines pMEL1AS and

pAP4ASserveasmodelforstudyingtheinteractionbetween

eth-yleneandcytokinininclimactericmelonripening. Itispossible

thathormoneaccumulationsuchascytokinin(Cogoetal.,2011;

Merewitzetal.,2011;Zhangetal.,2010)and/orpolyamines(Neily

etal.,2011)impactpostharvestmetabolism.Inordertotestthis

hypothesis,melonswithnormalripeningonthevineandafter

har-vest(NT),werecomparedtoothersthateitherdevelop(pMEL1AS)

orlack (pAP4AS)theclassical responsestoethylenetreatment.

Ripeningassociatedchemicalandphysiologicalvariablesaswell

astranscriptaccumulationofethylenebiosynthesis,cellwall

dis-assembling,chlorophyllbreakdownandesterbiosynthesisgenes

weremonitored.

2. Materialsandmethods

2.1. Plantmaterialandexperiments

Non-transformedCantaloupemelon(C.melovar.

Cantalupen-sis,Naudcv.Vedrantais)(NT)andmelontransformedwithACCO

antisensepMEL1AS(Ayubetal.,1996)and pAP4AS(Silvaetal.,

2004)cloneswerecultivatedaccordingtostandardpractices,

leav-ingnomorethanfourfruitperplant,followingCTNBio(Brazilian

regulatorycouncil)biosafetyregulationforgreenhousecultivation.

2.1.1. Ripeningonthevine

Inordertofollowripeningevolution,ethylene,fleshfirmness,

rindcolor,rindchlorophyllcontent,rindandfleshcarotene

con-tent,andsolublesolidscontent,wereevaluatedduringripeningon

thevineateverytwodaysstarting34dafteranthesis(DAA)until

44DAAforNTmelon,and52DAAforpMEL1ASandpAP4AS.Six

fruitwereevaluatedineachanalysis,totaling48NTfruitand60

pMEL1ASandpAP4ASfruiteachpertreatment.Transcript

accu-mulation of ethylene biosynthesis genes (ACCO and ACCS)was

quantifiedstartingat30DAAuntil48DAAforNT,pMEL1AS,and

pAP4ASfruit.

2.1.2. Ripeningafterharvest

Postharvest physiological and molecular changes were also

evaluated.NTfruit wereharvestedat 36 DAA,when abscission

zonestartedtoformandwerekeptat23±2◦_Cand80±5%

rel-ativehumidity.pMEL1ASandpAP4ASwereharvestedat44DAA

since they had a longer maturation cycle. Thirty-six fruit per

treatmentwerekeptat23±2◦_{C and36 morewereexposedto}

ethylene(100mLL−1_{)for120h, in7.2L flaskscontainingaKOH}

solution(150mL,1N).At every12htheflaskswere openedto

replacetheKOHsolutionandethyleneconcentrationwasadjusted

to100mLL−1_{. Postharvestethylene production, rindcolor,rind}

chlorophyllandcarotenecontent,andsolublesolidsanalyseswere

performedafter1,24,48,72,96and120hofharvestinfruitkept

atroomtemperature(NT,pMEL1ASand pAP4AS)andfruitkept

underethylene(pMEL1ASandpAP4AS).Volatilecompoundswere

evaluatedimmediatelyafterharvestandfortransformedfruitthe

measurementwasrepeated120hafterethylenetreatment.

Indi-vidualfruitwereconsideredbiologicalreplicatesandeachanalysis

wasperformedinduplicate.

2.1.3. Cytokinin(rootandfruit)andpolyamine(fruit)

accumulation

Cytokinincontentandrelativeaccumulationofgenesassociated

tocytokininsynthesisandresponsewerequantifiedinroottips

(sampledaftertheharvestofthesecondfruit),inadditiontofruit

rindandfleshofNT,pMEL1ASandpAP4ASplants.Uponcollection

sampleswerewashedwithwatercontainingdiethylpyrocarbonate

(DEPC),frozeninliquidnitrogenandstoredat−80◦_C.Sample

col-lectionforpolyamineanalysisfollowedthesameprotocolexcept

atthistimeonlyrindandfleshwerecollected.

2.2. Analyses

2.2.1. Ethylene

Ethyleneconcentrationofindividualfruitwasmonitoredonthe

vineasdescribedbyAyubetal.(1996),andresultsexpressedas

mLL−1_{.Afterharvest,melonswereenclosedin7.2Lflasksatroom}

temperature(23±2◦_{C).After30min,1mLoftheheadspacewas}

sampledandinjectedintoaGC(Varian3300),asdescribedbySilva

etal.(2004).Ethyleneproductionafterharvestwasexpressedin

nmolkg−1_s−1_.

2.2.2. Firmness

Fleshfirmnesswasdetermined usinga texturometer(TA.XT

plus)witha2mmprobe,with50%penetrationat1mms−1_incut

openedfruit.ResultswereexpressedinNewtons(N)(Silvaetal.,

2004).

2.2.3. Solublesolids

SolublesolidscontentwasdeterminedusinganAbbe

refrac-tometer(ATAGO-N1)anddatawasexpressedaspercentage(Silva

etal.,2004).

2.2.4. Color

Colorwasdeterminedusingacolorimeter(Minolta

Chromome-terCR300,D65,Osaka,Japan),with8mmapertureandstandard

CIE-L*a*b*.a*andb*valueswereutilized.Measurementswere

per-formedonoppositesidesofthefruitattheequatorialregion(Silva

etal.,2004).

2.2.5. Titratableacidity

Determined by titration using NaOH 0.1N. Results were

expressedin%ofcitricacid(Silvaetal.,2004).

2.2.6. Chlorophyllcontent

1g of fruit rind wasground in 5mL acetone (80%v/v) and

leftstirringfor15min.Themixturewascentrifuged(10,000×g;

10min;4◦_{C)andthesupernatanttransferredtoa25mLvolumetric}

adjustedto25mLwithacetone(80%v/v).Absorbancewas

mea-suredat645nm(chlorophyllb–Chla)and662nm(chlorophylla

–Chlb)andconcentrationswerecalculatedaccordingtothe

formu-lasdescribedbyLichtenthaler(1987)(Chla=12.25A662−2.79A645;

Chlb=21.50A645−5.10A662),andresultswereexpressedas

chloro-phyllperfreshweightmass,mgkg−1_.

2.2.7. Carotenoidcontent

Totalcarotenoid contentwas determined using10gof rind

or flesh ground up in liquid nitrogen, following the same

procedure described for the chlorophyll content evaluation.

Absorbance was measured at 470nm. Results were

calcu-lated using the equation described by Lichtenthaler (1987)

(C=1000A470−1.82Ca−85.02Cb/198) and expressed on a fresh

weightbasisasmgkg−1_.

2.2.8. Estervolatiles

Ester volatile analysis followed protocol described by

Bauchot et al. (1998) except that here SPME carboxen-PDMS

(0.75mm×1cm,Supelco,USA)wasusedastheadsorbentmatrix.

All analyses were performed on a Varian 3800 gas

chromato-graphinterfacedwithaShimadzuQP-50000massspectrometer.

Volatileswereidentifiedbycomparisontospectraofstandardsand

toreferencecollections(NIST98/EPA/NIHMassSpectraldatabase).

2.2.9. Cytokinincontent

Zeatin(Z)andzeatinriboside(ZR)wereseparatedusingHPLC

anddetectedusinganimmunoenzymaticassayaccordingtoZieslin

andAlgom(2004)withsomemodifications.Rind,flesh,androot

tissuesfromNT,pMEL1AS,andpAP4ASmelonweregroundin

liq-uidnitrogenandcytokininswereextractedwithethanolduring

30minincubation.Ninevolumesofammoniumacetatesolution

(40mM,pH6.5)wereaddedtotheextract,whichwasthen

fil-tered througha 0.22mmfiltermembrane and purified through

apolyvynilpolypyrrolidonecolumn.Elutedcytokininswere

sepa-ratedbyHPLC(ShimadzuHPLCsystem)usinganEC250/4Nucleosil

100-5C18columnandmonitoredbyaUVdetectorsetat254nm.

Cytokinincontainingfractionsweresubmittedto

immunochem-icaldetectionusingmonoclonalantibodiesforzeatinandzeatin

riboside.Cytokinincontentwasexpressedasmassofzeatinand

zeatingribosideperfreshweightmassoffruit,mgkg−1_.

Commer-cialstandardswereusedforcalibrationandtherecoveryobtained

was89.87%.

2.2.10. Polyaminecontent

Extractionoffreepolyamineswasperformedasdescribedby

Haoet al. (2005)withminor changes:1g ofrind or fleshwas

homogenizedinperchloricacid(5%, 5mL)and extractedonice

for30min.Aftercentrifugationat12,000×g,4◦_{Cfor15min,the}

supernatant was transferred to another tube and kept on ice.

The pelletwas extracted again with perchloric acid(5%, 1mL)

onicefor30minandthencentrifugedat12,000×gand4◦_Cfor

15min.ThesupernatantswerecombinedandadjustedtopH7.0

withsaturatedNa2CO3.SeparationwasperformedinaBondapak

C18column,300mm×3.9mmi.d.,10mm(Waters,Milford,

Mas-sachusetts,EUA),andputrescineandspermidinewerederivatized

post-columnwitho-phthalaldehydeandmonitoredusinga

fluo-rescencedetector(Ex:340nm;Em:445nm).RefertoVieiraetal.

(2007)fora moredetaileddescriptionoftheanalytical method.

Resultswereexpressedonafreshweightbasisasmgkg−1_.

2.2.11. Transcriptaccumulation

Transcript accumulation was evaluated by quantitative PCR

(q-PCR).RNAwasextractedfrom0.1gofmelonfleshaccording

tomanufactureinstructionsusingPureLinKTM_{reagent(PlantRNA}

Reagent – InvitrogenTM_{). Total RNA was treated with DNAse I}

(InvitrogenTM_{), and RNA quality was confirmed in agarose gel}

(2%,w/v),byPCR,andspectroscopicallyquantified.cDNAswere

obtainedfrom2mgofRNAusingSuperScriptFirst-StrandSystem

(InvitrogenTM_{). Specific primers were designed for GenBank}

depositedsequencesusingVectorNTIAdvance10(InvitrogenTM_).

Criteria for primer selection were: size of amplified fragment

between100and 230bp; %CG basesbetween40and 60%;not

morethan twoC orGbases amongthelast fivenucleotidesat

the3′_{end;annealingtemperature60–65}◦_{C;accordingtoApplied}

Biosystemsguidelines.Ampliconswereevaluatedinagarosegel

(2%, w/v) and sequenced prior toRT-qPCR. Dissociation curves

were evaluatedand onlyprimers yielding one peak, indicating

specificamplificationoftherespectivetargetgene,wereutilized.

A standard curve was prepared for each gene using six cDNA

dilutions and only genes with amplification efficiency close to

100% were used. For each cDNA, ˇ-actin and 18S transcripts

were usedas normalizer for each cDNA,given their consistent

transcription level in fruit samples in which theCt varied less

than1.4.q-PCRwasperformedina7500Real-TimePCRSystem

(Applied Biosystems) using fluorescent SYBR Green.

Amplifica-tion reaction wasperformed in 25mL total volume containing

2mMofeachprimer,12.5mLofthePCRMasterMixSYBRGreen,

4ng of cDNA (1mL), and water to make up the total volume.

PCR conditions were: denaturing at 50◦_{C for 2min and 95}◦_C

for 10min,followed by40 cyclesof three steps(95◦_{C for 30s,}

57◦_{Cfor1minand72}◦_{Cfor1min),andfinalextensionat72}◦_C

for 5min. Since reaction efficiency was high (close to 100%),

relativetranscriptaccumulationwascalculatedusingtheformula

2−11Ct_{(LivakandSchmittgen,2001).Forrelativequantification}

ofACCOandACCStranscripts,theexpressionlevelofNTfruitat

34 DAA (on the vine) wasselected asthe baseline. For all the

other genesstudied afterharvest, NTfruit expression

immedi-ately afterharvestservedasbaseline expression.Thefollowing

geneswereselectedbasedonspecificityandefficiency:ethylene

synthesis (ACCO – F: 5′_{-AATCCGCACAAACCAAATCTTGTAC-3}′_/R:

5′_{AAGGATCCTAAGCTGAAAGTGAATA-3}′_{;ACCS1– F:5}′_-GAAAGCG

TAC GATAACGATCCG-3′_{/R: 5}′

-CGGTATAAATAGAGGCTTTCGGAA-3′_;ACCS2–F:5′_{-GATGTCTCTCTAAATATTAAACAG-3}′_/R:5′_-CATTAT

CGTTGCTAGGAAACAAGTC-3′_{; ACCS3 – F: 5}′_-GGTCTGGCAGA

GAATCAGCTATCA-3′_{/R: 5}′_{-GTAGCGCCAGCTGTAAGGACTAT-3}′_;

ACCS4–F:5′_{-TATGACATAATTAAGGTCACTAAT-3}′_/R:5′_-TGATTAGT

GGAATATATAGGTTTTAT-3′_{; ACCS5 – F: 5}′_{-GACGCCTTTCTT}

CTGCCCACCCCCTAC3′_{/R: 5}′

CAATGTGAACTTGTTTACGGATTACGA-3′_{);cytokininsynthesisandresponse(CYP735A2–F:5}′_-CTTCAACGT

CTTTGTGTCCAAG-3′_{/R: 5}′_{-CTACTCCGACCGATCTCTACAC-3}′_{; ARR1}

–F:5′_{-TTCATATGCCTGACATGGACGG-3}′_/R:5′_{-AACCGCACCGTGCG}

TTACTCCC-3); flesh firmness (PG1 – F: 5′_{-CACGCCTTGACT}

GCTGCTGCTG-3′_{/R: 5}′_{-CGGCTTGGCTCCAAGATTGACG-3}′_{); ester}

synthesis (LOX – F: 5′_{-AGAAGG CACTCCTGAGTATGAG3}′_{/R: 5}′

-CTTCCAGCTTCTTTCTAAAATCCT-3′_;HPL–F:5′_{-GCATGGCGCCGCCG}

CGAGCCAACT-3′_{/R: 5}′_{-CAGCGCGCGCCGCCGCTTGACACT-3}′_{; AAT1}

–F:5′_{-CCACAGGGGCCAGAATTACA-3}′_/R:5′_{-TGGAGGAGGCAAGCA}

TAGACTT-3′_{; AAT2 – F: 5}′

-CTATAATTGGAGGGTGTGGAATTATC-3′_{/R: 5}′_{-AACATTTGCCCTAAATCTTTCCAT-3}′_{; AAT3 – F: 5}′_-CG

CTTGATGACATGGCACAT-3′_{/R: 5}′

-GGCCTTACGGATAGCAGAGATC-3′_{; AAT4 – F: 5}′_{-CAGTTGTACCCCCGTCGAGTA-3}′_{/R: 5}′_-AATAT

CGCTTCTGATCGGAACAC-3′_{); and constitutive expression}

(ˇ-actin – F: 5′_{-GTGATGGTGTGAGTCACACTGTTC-3}′_{/R: 5}′_-ACGACC

AGCAAGGTCCAAAC-3′_{; 18S – F: 5}′_{-AAAACGACTCTCGGCAACGG}

ATA-3′_/R:5′_{-ATGGTTCACGGGATTCTGCAATT-3}′_).

2.2.12. Experimentaldesignandstatisticalanalysis

Alltreatments had sixbiological replicatesand two

analyti-calduplicates,andweredistributedinacompletelyrandomized

Table1

Cytokinin(zeatinandzeatinriboside)content(mgkg−1_{)inroot,rindandfleshof}

non-transformed(NT)andtransformedCantaloupemelon(Cucumismelovar. Can-talupensis,Naudcv.Vedrantais),usingACCOpMEL1ASandpAP4ASantisensegenes.

Melon Plantpart Zeatin Zeatinriboside

NT Root 4.87±2.01a _5.01±1.25 Rind 5.02±1.25 6.23±1.58 Flesh 2.52± 1.07 5.25± 2.01 pMEL1AS Root 5.68±1.89 6.69±0.87 Rind 9.14±2.31 8.75±1.11 Flesh 4.02±1.24 7.98±2.01 pAP4AS Root 7.87±2.34 9.78±2.12 Rind 21.01±3.25 36.84±3.69 Flesh 8.25±2.05 30.63±4.25

a_{Meanofsixbiologicalreplicates ± standarderror.}

(eachplateasablock),withsixbiologicalreplicatesandthree

ana-lyticalduplicates.Data was subjectedtoan ANOVA,performed

usingtheFtestatthe5%significancelevelandmeansof

treat-mentwerecomparedusingTukey’stestatthe5%significancelevel

(p≤0.05),usingSASversion9.2forWindows(SASInstitute,Cary,

NC).

3. Results

3.1. Physicochemicalandphysiologicalchangesinmelonfruitduringripeningon thevine

Inordertoestablishanassociationbetweenphysiologicalresponsestoethylene andhormoneslevels,cytokininszeatin(Z)andzeatinriboside(ZR)weremeasuredin roots,fruitfleshandrind(Table1),andpolyaminesspermidineandputrescinewere determinedintherindandfleshoffruit(Table2).pAP4ASfruitthatstayedgreen hadhigherZandZRcontentintheroots,fruitfleshandrind(Table1).Spermidine andputrescine,althoughhigherintherindwhencomparedtofruitflesh,showed nodifferenceamonggenotypes(Table2).Thesesresultsindicatethatgenetic trans-formationofpAP4ASthatsuppressedethyleneproductionalsoaffectedcytokinin synthesiswithnoeffectonpolyaminecontent.pAP4AS(lowethyleneproduction andhighcytokinincontent)wasthencomparedtopMEL1AS(lowethylene produc-tionandnormalcytokinincontent)andNTmelons(highethyleneproductionand normalcytokinincontent)inordertostudytheinteractionbetweenethyleneand cytokininsonthephysiologicalandmolecularchangesduringripeningandunder ethylenetreatment.

NTmelonsshowedatypicalclimactericbehavior,withariseinethylene pro-ductionafter34DAAandapeakat42DAAaccompaniedbysignificantfleshfirmness reduction(Fig.1B)Fig.1.Incontrast,transgenicmelondidnotpresentapeakof eth-yleneproduction(Fig.1A)andshowedsmallerchangesinfleshfirmness.NTflesh firmnessreduceddrasticallyduringripening,varyingfrom57Nat34DAAto5N at46DAA(Fig.1B).At48DAANTfruitwerecompletelysoft.AlthoughpMEL1AS andpAP4ASfruitalsoshowedareductioninfleshfirmness,itoccurredtoalesser extentgoingfrom65Nat34DAAto40Nat52DAA(Fig.1B).pMEL1ASandpAP4AS stayedgreenerandlessyellowthanNTduringripeningonthevine(Fig.1Cand D).NTfruitcolorvariationduring34DAAthrough42DAA(Fig.1D)wasassociated withchangesinthemajorcompoundsresponsibleforrindcolor:chlorophyll con-tentdeclined(Fig.1E)andtotalcarotenecontentincreased(Fig.1F).InpMEL1AS andpAP4ASfruit,thesecompoundsdidnotvaryasmuch.Fleshcarotenoidcontent wasnotaffectedbytreatments(datanotshown),reaching453.5mgkg−1_oftotal

carotenoidatharvest.

Table2

Polyaminecontent(spermidineandputrescinemgkg−1_{),atharvest,oftherind}

andfleshofnon-transformed(NT)andtransformedCantaloupemelon(Cucumis melovar.Cantalupensis,Naudcv.Vedrantais),usingACCOpMEL1ASandpAP4AS antisensegenes.

Melon Plantpart Spermidine Putrescine

UNT Rind 2.35± 0.31 a _{7.53± 0.72} Flesh 1.25± 0.29 4.56± 0.29 pMEL1AS Rind 2.06±0.45 8.01±0.94 Flesh 1.15± 0.45 4.98± 0.45 pAP4AS Rind 2.23±0.36 7.85±0.89 Flesh 0.96±0.37 5.01±0.67

a_{Meanofsixbiologicalreplicates±standarderror.}

3.2. Physicochemicalandphysiologicalchangesinmelonfruitduringripening afterharvest

NTmelonswereharvestedat36DAA(beginningofclimacteric)andpMEL1AS andpAP4ASat 44DAA(whenan abscissionzonewas observedandsoluble solidscontentwasapproximately16%).Afterharvest,ethyleneproductionofNT melonincreasedandreachedamaximum(7.65nmolkg−1_s−1_{)at72hafterharvest}

(Fig.2A).pMEL1ASandpAP4ASfruitdidnotshowapeakinethyleneproduction (Fig.2A),andlevelswere0.04nmolkg−1_s−1_{,99.5%lowerthanNTfruit.}

pMEL1ASandpAP4ASkeptatroomtemperaturewithoutethylenetreatment hadareductioninfleshfirmnessfrom50Nto32N,whilefruittreatedwithethylene showedavariationgoingfrom50Nto9N(Fig.2B).NTfruitwithoutethylene appli-cationshowedareductioninfleshfirmnessgoingfrom47Nto5N.Thus,confirming thatmelonfleshsofteningisaphysiologicalchangeacceleratedbyethylene.

Followingsimilarripeningbehaviorobservedonthevine(Fig.1C),NTmelon showeddegreening(colorparameter“a”changingfrom−33to−19)after har-vest(Fig.2C).pMEL1ASandpAP4ASfruitshowedsignificantlylessrinddegreening (“a”from−38to−33).Responseswerevarieduponethyleneapplication;pMEL1AS showeddegreeningsimilartoNTfruit,whilepAP4ASfruithadonlyaslightchange ingreencolorwith“a”values(green/redscale)closeto−30(Fig.2C).Inaddition, fruitthathadahigherdecreaseingreencolorafterharvest(NTandpMEL1AS+C2H4)

alsoshowedhigheryellowing(Fig.2D).Rindcolorvariationwassimilarbetween pMEL1ASandpAP4ASduringripeningonthevine(Fig.1CandD),butdiffered fromfruitexposedtoethyleneafterharvest(Fig.2CandD).HarvestedpMEL1AS fruittreatedwithethyleneshoweddegreening(Fig.2C)andyellowing(Fig.2D), associatedwithhigherreductionofchlorophyllcontent(Fig.2E)andincreasein rindcarotenoidcontent(Fig.2F).InpAP4ASthesechangesoccurredonlypartially, indicatingalowersensitivitytoethylene.Fleshcarotenoidcontentdidnotchange significantlyafterharvest(datanotshown),stayingatapproximately449mgkg−1_,

despiteethylenesupply.InhibitionofethyleneproductioninpMEL1ASandpAP4AS fruitresultedin93%reductionofestervolatileproductionfrompMEL1ASand87% frompAP4AS(Fig.3).Reductionofvolatileproductionoccurredforallcompounds evaluated,includingcompoundswithlowodorvalues(forexample,methylpropyl acetate)andpotentodorants(methylpropanoate)(Fig.3).Fivedaysafter ethyl-enetreatmentpMEL1ASfruitrestorednormalesterproduction.However,pAP4AS fruitonlypartiallyrestoredestervolatileproductionreachingabout26%ofthe productionobservedfromNTfruit.

3.3. Genetranscriptaccumulationduringripeningonthevine

DuringNTfruitripeningonthevineACCOtranscriptsaccumulatedbeyond32 DAA,withmaximumaccumulationat38DAA(Fig.4A),followedbysubsequent increaseinethyleneproduction(Fig.1A).TransformedfruithadlittletonoACCO transcriptsdetected,inagreementwithareductionofmorethan99.5%inethylene productionfromthesefruit.

ACCStranscriptaccumulationvariedamongACCS1(Fig.4B),ACCS2(Fig.4C), ACCS3(Fig.4D)andACCS5(Fig.4E).ACCS4transcriptswerenotdetected.ACCS1 (Fig.4B)andACCS3(Fig.4D)hadhighertranscriptaccumulationinNTthanin pMEL1ASandpAP4ASfruit,andtheobservedvariationshadhighpositive correla-tionwithACCOtranscriptaccumulation(Fig.4A)andethyleneproduction(Fig.1A). Theseresultsindicatethatthesegeneshavestrongassociationwithripening evo-lution.Ontheotherhand,highACCS5transcriptaccumulationinpMEL1ASand pAP4ASfruit(Fig.4E),demonstratedthatthisgeneisnegativelyregulatedby eth-ylene.ACCS2mRNAaccumulationseemstobeindependentofethylenesincethere wasanup-regulationofACCS2inallthreegenotypes(Fig.4C).

3.4. Genetranscriptaccumulationduringripeningafterharvest

ACCOmRNAaccumulationdecreasedovertimewhenNTfruitharvested36DAA werekeptatroomtemperaturetocompleteripening(Fig.5).Thisisinagreement withthemaximumtranscriptaccumulationobservedat36and38DAA(Fig.4A). pMEL1ASandpAP4ASmelonevenafterethyleneapplicationdidnotshowincrease inACCOtranscriptlevels.Asexpected,PG1transcriptsofNTfruitaccumulatedduring ripeningandwereup-regulatedbyethyleneinpMEL1ASearlierandmoreintensely whencomparedtopAP4AS.

LOX,HPL,AAT1,AAT3andAAT4,allesterbiosynthesisassociatedgenes,had ele-vatedtranscriptaccumulationbetween24and72hinNTfruit(Fig.5).pMEL1AS andpAP4ASfruit,however,showedconsiderablylowtranscriptaccumulationof thesamegenes.OnlyAAT2hadanincreasedmRNAcontentintransformedfruit whencomparedtoNT.pMEL1AStreatedwithethyleneshowedageneralinduction oftranscriptaccumulation.Ingeneral,theseresultsindicatethatthosefruit produc-ingmoreestervolatiles(NTandpMEL1AS+C2H4)(Fig.3)alsohadincommonan

increasedmRNAcontentofHPL,LOX,AAT1,AAT3andAAT4.

CYP735A2,acytokininhydroxylasegene,showedhighertranscript accumula-tionintheroot,andfruitofpAP4ASwhencomparedtopMEL1ASandNT(Fig.6).The highestrelativeexpressionofthisgenewasobservedintherootsofpAP4AS. Sim-ilarly,ARR1knowntobeatranscriptionfactorinducedbycytokinins,alsoshowed highertranscriptaccumulationinthepAP4ASgenotype,speciallyinthefruit(Fig.6).

Fig.1.Physicochemicalandphysiologicalchangesinnon-transformed(NT)andtransformedCantaloupemelon(Cucumismelovar.Cantalupensis,Naudcv.Vedrantais)using ACCOpMEL1ASandpAP4ASantisense,duringripeningonthevine.(A)Ethyleneconcentration(mLL−1_{);(B)fleshfirmness(N);(C)rindcolor(-a);(D)rindcolor(b);(E)rind}

chlorophyllcontent(mgkg−1_{);(F)rindcarotenoidcontent(mgkg}−1_{).Verticalbarsrepresentstandarderrorofthemean(n=6).DAA(daysafteranthesis).}

Fig.2. Physicochemicalandphysiologicalchangesinnon-transformed(NT)andtransformedCantaloupemelon(Cucumismelovar.Cantalupensis,Naudcv.Vedrantais) usingACCOpMEL1ASandpAP4ASantisense,duringpostharvest.Ethyleneproduction(nmolkg−1_s−1_{)(A),Fleshfirmness(N)(B),Rindcolor(-a)(C);Rindcolor(b)(D);Rind}

4. Discussion

InhibitingethyleneproductionbythetransgenicACCOantisense

approachprolongsripeningandshelf-lifeinfruit(Ayubetal.,1996;

Defilippietal.,2005;Silvaetal.,2004).ACCOantisensepMEL1AS

andpAP4ASmelon studiedherealsohad anextended ripening

period(10d).pMEL1ASandpAP4ASdidnotdifferregarding

mat-uration,ethyleneandesterproduction,fleshfirmness,rindcolor,

rindchlorophyllandcarotenoidcontent,andpolyaminecontent

duringripeningonthevine.However,afterharvest,upon

ethyl-eneapplicationonlypMEL1ASrecoveredestervolatileproduction

andcolorchangesand developedapeduncularabscission zone,

confirmingpreviousfindings(Bauchotetal.,1998;Guisetal.,1997).

ExposureofpAP4ASfruittoethylenefor120hdidnotresult

incompletegreencolorreductionorinrindyellowing,andester

volatileproductionwasonly26%ofNT.Thehighcytokinincontent

observedinthesefruitmayberesponsibleforreducingtheir

sen-sitivitytoethylene.Thisexplanationissupportedbystudiesusing

Arabidopsismutantswithhighcytokinincontentthathaveslowed

senescence (Catterou et al., 2002). In addition, plants

express-inglowlevelsofpheophorbideoxidasegeneorcultivatedunder

moderatewater stresshaveshown inducedcytokinin synthesis

resultinginlesssensitivitytoethyleneandbetterpreservationof

greencolor(Buchanan-Wollastonetal.,2005;Cogoetal.,2011;

Pruzinskaetal.,2003).

Although carotenoid synthesis in melon is thought to be

ethylene-independent (Guis et al., 1997), in pMEL1AS rind

carotenoidcontentwasaffectedbyethyleneleadingtoyellowing

ethyl acetate methylpropyl acetate

butyl acetate hexyl acetate

methyl propanoate ethyl propanoate meth yl butanoate

ethyl butanoate 2-methyle

thyl butanoate

Ester volatile production (

µ g kg -1) 0 20 40 60 80 100 120 140 160 180 NT pMEL1AS w/out C2H4 pMEL1AS with C2H4 pAP4AS w/out C2H4 pAP4AS with C2H4

Fig.3. Estervolatileproduction(mgkg−1_{)bynon-transformed(NT)and}

trans-formed(usingACCOpMEL1ASandpAP4ASantisense)Cantaloupemelon(Cucumis melovar.Cantalupensis,Naudcv.Vedrantais),treatedwithethylene(withC2H4)and

measured120haftertreatmentornottreatedwithethylene(w/outC2H4)measured

immediatelyafterharvest.

0 500 1000 1500 2000 2500 30 32 34 36 38 40 42 44 46 48 Cm-ACCO DAA NT pMEL1AS pAP4AS 0 10 20 30 40 50 30 32 34 36 38 40 42 44 46 48 Cm-ACCS1 DAA NT pMEL1AS pAP4AS 0 5 10 15 20 25 30 32 34 36 38 40 42 44 46 48 Cm-ACCS2 DAA NT pMEL1AS pAP4AS 0 2 4 6 8 10 12 30 32 34 36 38 40 42 44 46 48 Cm-ACCS3 DAA NT pMEL1AS pAP4AS 0 100 200 300 400 30 32 34 36 38 40 42 44 46 48 Cm-ACCS5 DAA NT pMEL1AS pAP4AS A B E C D

Fig.4. Relativetranscriptaccumulationofgenesassociatedwithethylenebiosynthesisduringripeningonthevineofnon-transformed(NT)andtransformedmelon(Cucumis melovar.Cantalupensis,Naudcv.Vedrantais),usingACCOpMEL1ASandpAP4ASantisensegene.Relativetranscriptaccumulationcalculatedaccordingtotheformula2−11ct_.

Fig.5.Relativetranscriptaccumulationof1-aminocyclopropane-1-carboxylicoxidase(ACCO),hydroperoxidelyase(HPL),lipoxygenase(LOX),alcoholacyltransferase(AAT1, AAT2,AAT3,AAT4),and(PG1)genesofCantaloupemelon(Cucumismelovar.Cantalupensis,Naudcv.Vedrantais),non-transformed(NT)andtransformedusingACCOpMEL1AS andpAP4ASantisensetreatedornotwithethylene.Sampleswerecollectedat0,24,48,72,96and120h.Transcriptlevelisdescribedina0to10scale.Greencoloronthe leftindicatesminimalaccumulation,blackcolorinthemiddlerepresents5timesthemRNAcontentcomparedtogreenandredcolorontherighthandcornerrepresentsa 10foldincreaseinmRNAcontentcomparedtothegreenendofthescale.

offruitrind.Thisfindingindicatesthatcarotenoidsynthesisand

accumulationmayberegulateddifferentlyintherindandfleshof

melonfruit.Inaddition,highcytokinincontainingfruit(pAP4AS)

didnotshowthesamebehavior,i.e.,ethylenedidnotstimulate

chlorophylldegradationorcarotenoidaccumulationintherind.

Defilippietal.(2005)showedthatareductioninethylene

pro-ductionnegativelyaffectedestervolatileproduction.Inaddition,

Pechetal.(2008)indicatedthatselectionofgenotypesforincreased

shelf-lifeeitherbyclassicalbreedingoratransgenicapproachled

tofruitwithloweraromaticpotential.Aspreviouslyobservedby

Flores etal. (2002)and Guiset al.(1997), ethyleneapplication

to pMEL1AS restored volatileproduction (Fig. 3). In apple, the

samearomare-establishmentoccurredinadditiontothe

restora-tionofrindcolor andtheformation ofa peduncularabscission

zone(Defilippietal.,2005).However,inpAP4ASfruitnotallthe

classicalmaturationeventswererestoredwithethylene

applica-tion.ThiswasnotexpectedsincebothpMEL1ASandpAP4ASwere

transformedwithACCOantisenseclones frommelonand apple,

respectively,andhad94.3%homology.

In order tocorrelate physiological and molecularresponses,

ethylene and ester biosynthesis and flesh firmness associated

genes were monitored. First, transformation was efficient in

CS Root CR Root CS Rind CR Rind CS Flesh CR Flesh

RQ 0 2 4 6 8 10 12 14 16 18 pMEL1AS pAP4AS

Fig.6.Transcriptaccumulationintheroot,rindandfleshofgenesCYP735A2(CS) andARR1(CR)ofCantaloupemelon(Cucumismelovar.Cantalupensis,Naudcv. Vedrantais),transformedusingACCOpMEL1ASandpAP4ASantisenseVerticalbars representstandarderrorofthemean.RQ–relativequantitation.

suppressingmRNAsfromACCO,whichalsointerferedwithACCS

transcriptaccumulation.mRNAaccumulationofesterbiosynthesis

genesLOX,AAT1,AAT2,AAT3andAAT4washigherinNTmelonthan

in pMEL1AS and pAP4AS.Ethylene application induced a rapid

increaseofLOX,AAT1,AAT3andAAT4transcriptaccumulationin

pMEL1AS melonsbut not in pAP4ASmelons. pMEL1ASand NT

showedsimilarphysiologicalresponsestoethylene.Itisageneral

consensusthatestervolatilebiosynthesisinclimactericfruitisan

ethylene-dependenteventandthatinductionofAATexpressionis

necessaryandsufficientforvolatileproduction(El-Sharkawyetal.,

2005).ForpAP4ASmelonhowever,ethylenesupplywasunableto

restoreesterproductionforbothstrongandlowodorantintensity

compounds.

It is knownthat cytokinins, mostlysynthesized in theroots

and translocated to other plant parts, are affected by

ethyl-eneandretardleafandflowersenescence(Buchanan-Wollaston

et al., 2005; Martineau et al., 1995). Broccoli (Chen et al.,

2001) and tomato (Martineau et al., 1995) overexpressing

cytokinins are less sensitive to ethylene. Exogenous

applica-tion of 6-benzylaminopurine tobroccoli can alsolead to these

same effects (Downs et al., 1997). Application of cytokinins

(6-benzylaminopurine, 6-BAP and N-(2-chloro-pyridin-4-yl)-N′

-phenylurea,CPPU)tovegetativeplantpartsorfruitofNT,pMEL1AS,

andpAP4ASdidnotshowdifferencesinethyleneproduction,

firm-ness, soluble solids,titrable acidity,carotenoids,ester volatiles,

ormRNAcontentexceptthatNTandpMEL1AS plantshadfruit

that were 18% largerthan pAP4AS.Although the physiological

mechanismwasnotdescribed,anassociationbetweencytokinin

synthesis and increasedshelf-life(Zaicovski etal., 2008)or the

stay-greensymptom(Akhtaretal.,1999)hasbeenobserved.Inthis

study,highercytokinincontentwasfoundinroots,fruitrindand

fleshofpAP4ASmelon.Theseresultsareinagreementwithfindings

byMartineauetal.(1995),suggestingthatareductioninethylene

productionbeyondextendingshelf-life,prolongedthevegetative

cycleoftomatopromotingrootemissionandconsequentlyhigher

cytokininsynthesisandaccumulation,reducingresponsivenessto

ethylene.Theexaminationoftranscriptaccumulationoftwogenes

involvedinthecytokininsynthesisandresponse(CYP735A2and

ARR1) supportthis hypothesis. CYP735A2 involved in cytokinin

synthesisshowedhightranscriptaccumulationinpAP4ASroots.

On the other hand,ARR1 mRNA content,a transcription factor

affectedbyendogenouscytokinins,washigherinpAP4ASfruit.The

up-regulationofCYP735A2inpAP4ASrootsmayhaveledtothe

observedincreasedhormonelevel,whichmayconsequentlyhave

Itwaspossible forpAP4AStransgenicmelon tohave higher

polyaminecontentduetoahigherACCavailability,butthis

hypoth-esis wasnot confirmed.Spermidine and putrescine content, as

expected,werehigherinfruitrindthaninfruitflesh.Melon

trans-formationwithhighlyhomologousclonesledtolineswithdistinct

physiology.pMEL1ASwasmadetoripenwithethyleneapplication,

allowingforthecharacterizationofethylene-dependent,

indepen-dentandpartiallydependentevents.Unresponsivenesstoethylene

inpAP4ASmelonislikelyduetothehighcytokinincontent,

sug-gestinganinvolvementofplanthormonesotherthanethylenein

ripeningcontrol.

Insummary,thehighcytokinincontentfoundinpAP4ASmelons

(twofoldhigherthanpMEL1AS)affectedthefruitresponsivenessto

ethylene.Thetypicalmolecularandphysiologicalchangesknown

asethylene-dependent,includingtranscriptaccumulationofgenes

involvedintheestersynthesis,especiallyAAT1andAAT4(Lucchetta

etal.,2007;Pechetal.,2008),degreening(Goldingetal.,1998),and

peduncularabscissionzone(Guisetal.,1997)appeartobeaffected

byhighercytokinincontent.Ontheotherhand,fleshfirmness,a

partiallyethylenedependentevent(Nishiyamaetal.,2007)was

notaffectedbyhighcytokinincontent.Basedupontheseresultsit

isproposedthatfruitresponsestoethylenearepartiallyaffected

bycytokinincontent.

5. Conclusions

Melonsexpressing an antisense ACCoxidase (pMEL1AS and

pAP4AS)genedidnotdevelop normalripening.In addition,the

silencing of ACCO revealed regulation of members of the ACC

synthasegene family byethylene.ACCS1and ACCS3were

posi-tivelyregulatedbyethyleneandparticipateintheautocatalytic

ethyleneproductionprocess,andtheACCS5genewasnegatively

regulatedby ethylene. ACCS2 mRNA accumulatedcontinuously

duringripeningof NTand ethylene-suppressedfruit suggesting

thatthisgeneisethylene-independent.ThepMEL1ASandpAP4AS

melonsresponddifferentiallytopost-harvestethylenetreatment.

Polygalacturonase1respondedtoethyleneinbothpMEL1AS and

pAP4ASfruit,whilegenesoftheLOXpathwaywerestimulatedby

ethyleneonlyinpMEL1ASfruit.Asaconsequence,thepMEL1AS

restoredestervolatileproduction.pAP4ASrootsandfruitshowed

highaccumulationofcytokininbutnotpolyamines.Theelevated

transcriptaccumulationofgenesinvolvedinthecytokinin

synthe-sis(CYP735A2)and response(ARR1)supportsthatthis hormone

maybe responsible for the differential physiological responses

betweenpMEL1ASandpAP4AS.

Acknowledgments

ToCapes-Cofecub(631/2009),Fapergs(PQGaúcho2011–2012)

andCNPqforscholarships(301206/2010-4)andfinancialsupport

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