UV-C radiation modifies the ripening and accumulation of ethylene response factor (ERF) transcripts in tomato fruit

O

pen

A

rchive

T

OULOUSE

A

rchive

O

uverte (

OATAO

)

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Eprints ID : 14569

To link to this article : DOI:10.1016/j.postharvbio.2015.02.001

URL :

http://dx.doi.org/10.1016/j.postharvbio.2015.02.001

To cite this version : Severo, Joseana and Tiecher, Aline and Pirrello,

Jullien and Regad, Farid and Latché, Alain and Pech, Jean-Claude and

Bouzayen, Mondher and Rombaldi, César Valmor UV-C radiation

modifies the ripening and accumulation of ethylene response factor

(ERF) transcripts in tomato fruit. (2015) Postharvest Biology and

Technology, vol.102. pp.9-16. ISSN 0925-5214

Any correspondance concerning this service should be sent to the repository

administrator:

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UV-C

radiation

_modifies

the

ripening

and

accumulation

of

ethylene

response

factor

(ERF)

transcripts

in

tomato

fruit

Joseana

Severo

a,

*

,

Aline

Tiecher

b

,

Jullien

Pirrello

c

,

Farid

Regad

c

,

Alain

Latché

c

,

Jean-Claude

Pech

c

,

Mondher

Bouzayen

c

,

Cesar

Valmor

Rombaldi

d

a_{IFF,InstitutoFederalFarroupilha,EixodeProduçãoAlimenticia,RuaFábioJoãoAndolhe,1100,BairroFloresta,CEP98590-000,CampusSantoAugusto,}

SantoAugusto,RS,Brazil

b_{UNIPAMPA,UniversidadeFederaldoPampa,CampusItaqui,RuaLuizdeJoaquimdeSáBritto,s/n,BairroPromorar,CEP97650-000,Itaqui,RS,Brazil} c_{UMR990INRA/INP-ENSAT,PoledeBiotechnologieVégétale,24ChemindeBordeRouge,P.O.Box107,31326CastanetTolosanCedex,France} d_{UFPel/FAEM,DepartamentodeCiênciaeTecnologia,CampusUniversitário,CaixaPostal354,CEP90010-900,Pelotas,RS,Brazil}

Keywords: Abioticstress Ripening Senescence Solanumlycopersicum 1-Methylcyclopropene UltravioletC ABSTRACT

Ultraviolet-C(UV-C) radiationisused asapostharvest treatmenttoprolongthe shelflifeoffruit. However,thisstressfulprocessmayalsoaffectethyleneproductionand,consequently,theexpressionof genesencodingethyleneresponsefactors(ERFs).Totestthishypothesis,MicroTomtomatoesharvested atthebreakerstageweresubjectedto:1–applicationof3.7kJm2_{UV-Cradiation,2}

–applicationof 2mLL1_{1-methylcyclopropene(1-MCP)followedbyUV-Cradiation;and3}

–without1-MCPorUV-C (controltreatment). Aftertreatmentall fruitwerestored for12dat 21 2!_{Cand 80 5% relative}

humidity(RH).AlthoughUV-CradiationincreasedACCoxidasetranscripts andstimulatedethylene production,theripeningevolutionwasdelayed.FruittreatedwithUV-Cshowedloweraccumulationof lycopene,b-carotene,lutein+zeaxanthinandd-tocopherol;butretainedhigherlevelsofchlorogenic acid,r-coumaricacidandquercetinafter6d.Additionally,UV-Ctreatedfruithadhighercontentsof polyamines(putrescineandspermidine).Amongthe14ERFsstudied,11(Sl-ERFA.1,Sl-ERFA.3,Sl-ERFB.1, Sl-ERFB.2,Sl-ERFB.3,Sl-ERFC.6,Sl-ERFD.1,Sl-ERFD.3,Sl-ERFE.1,Sl-ERFF.5,Sl-ERFG.2)exhibitedincreased transcriptaccumulation,2ERFs(Sl-ERFE.2andSl-ERFE.4)showeddecreasedtranscriptaccumulationand only1ERF(Sl-ERFE.3)wasnotsignificantlyaffectedbyUV-Ctreatment.Asexpected,thetranscript profilesof1-MCPand/orUV-C-treatedtomatoesdemonstratethatethyleneplaysanimportantroleinthe expressionofERFs.ThedelayinfruitripeningmaybecausedbytheactivationofERFsthatcouldactas regulatorsofmetabolicpathwaysduringripening.However,thishypothesisneedstobebettertested.In conclusion,arelationshiphasbeenestablishedbetweenUV-Ctreatmentandripeningdelay,correlated tochangesin13ERFtranscriptsevaluatedduringpostharvesttreatment.

1.Introduction

UV-Cradiation(100–280nm)isa treatmentwithgermicidal

capabilitiesthathasbeenusedtopreventpostharvestrotinfruits

andvegetables(Stevensetal.,1998;Liuetal.,2011;Syamaladevi

et al., 2014). Because it is a stressor, UV-C can also accelerate

ethylene production and therefore activate the expression of

ethyleneresponsefactor(ERFs)genes.Alteringtheexpressionof

ERF, either through hormonal induction or abiotic stress, can

induce secondary metabolic pathways; these pathways may

activatepathogenesis-related(PR)genesrelatedtothesynthesis

of phytoalexins, phenols and terpenoids (Maharaj et al.,1999;

Charlesetal.,2008a,b;Liuetal.,2011;Pomboetal.,2011).Pombo etal.(2011)reportedthatUV-Ctreatmentofstrawberrieshelps

prevent rotnot onlyby direct inoculum reduction,but also by

activatinggenesencodingenzymesinvolvedinplantdefense.The

beneficial effects of the application of UV-Ccan vary between

species, cultivars and time of application. Bu et al. (2013)

previouslyreportedthatUV-CmaintainedthefirmnessofCherry

tomatoes(SolanumlycopersicumL.cv.Zhenzhu1.),withdecreased

expressionofcellwalldegradingenzymes.Incomparison,Tiecher

et al. (2013) observed delay in fruit maturation without a

commensurateprolongationof tomatofirmness(S.lycopersicum

cv. Flavortop). Obande et al. (2011) reported maintained the

*Correspondingauthor.Tel.:+555332757258;fax:+555332757258. E-mailaddress:joseana.severo@iffarroupilha.edu.br(J.Severo).

firmnessofpreharvestUV-Ctreatmentoftomatoes(S.

lycopersi-cumL. cv.Mill.) withvaryingresults dependingontheapplied

dose.

It iswidelyknownthatthephytohormoneethylenecontrols

manyeventsrelatedtogrowthanddevelopmentinplants,andis

expressed in response toabiotic and biotic stressors(Cara and

Giovannoni,2008;Bapat etal.,2010).1-Methylcyclopropene

(1-MCP)isapotentinhibitorofethyleneperception,whichhasbeen

usedsuccessfullyinstudiestounderstandtheactionofethylenein

ripeningprocessandconsequentlytheexpressionofrelatedgenes

(Hoeberichtsetal.,2002;OpiyoandYing,2005).

Ethylene is formed from the amino acid methionine by

S-adenosyl-L-methionine(AdoMet)and1-carboxylic

acid-1-amino-cyclopropane. The enzymes that catalyze the conversion of

AdoMettoACCandACCtoethyleneareACCsynthase(ACS)and

ACC oxidase (ACO), respectively. During ripening of climacteric

fruit,this biosynthesispathwayis autocatalyticallyregulatedby

ethylene (Barry et al., 1996; Cara and Giovannoni, 2008). In

responsetoethylene,theexpressionprofileofseveraltranscription

factorsmaybealtered,whichresultsintheactivationofpathways

thatinduceordelaysenescence(Ohme-TakagiandShinshi,1995;

Chenetal.,2008;Erkanetal.,2008;Liuetal.,2009,2011).

After synthesis, ethylene is recognized by receptors (ETRs)

locatedinthemembraneoftheendoplasmicreticulum.Asignaling

cascadewhich includespositiveandnegativeregulators,

modu-latestheexpressionofERF,whicharesubsequentlyresponsiblefor

changesinthemetabolicpathwaysinvolvedinripeningandplant

defense (Barry et al., 1996; Bapat et al., 2010). This process

culminates in biochemical and physiological responses suchas

chlorophylldegradation,carotenoidaccumulation,softening,and

changesintomatoaromaand_flavor.Inaddition,therearechanges

in the levels of L-ascorbic acid, tocopherols and phenolic

compounds (Stevens et al.,1998; Cara and Giovannoni, 2008).

TheERFsbelongtotheAP2/ERFfamilyoftranscriptionfactorsthat

arecharacterizedbythepresenceofaDNAbindingdomaincalled

AP2/ERF, which is present exclusively in plants. This family of

transcription factorshasa 58-59amino acidconserveddomain

(ERFbindingdomain)thatcanbindtotwocis-elements:(i)

GCC-box, which is present in the promoter regionof PR-genes that

conferaresponsetoethylene,and(ii)C-repeat

(CRT)/dehydration-responsive element(DRE),whichisinvolvedintheexpressionof

genesrelatedtodehydrationand responsetolowtemperatures

(Singhetal.,2002;Xuetal.,2008,2011).Whereassomeofthese

transcription factorsbindtoonlyoneoftheseciselements(Gu

etal.,2002;Singhetal.,2002),othersmaymodulateresponsesto

stresstolerancethroughinteractionswithboth(GCC-boxandDRE)

ciselements(Huangetal.,2004;Zhangetal.,2004;Xuetal.,2007,

2011).

Since the first ERF binding domain was identified in four

tobaccoproteins(Ohme-TakagiandShinshi,1995),newERFgenes

have been identified in other plant tissues (Zhou et al., 1997;

Tournieretal.,2003;Wangetal.,2007;Xuetal.,2007;Zhangetal., 2010;Yinetal.,2012; Girardietal.,2013).Severalstudieshave

soughttorelatetheinfluenceofbioticandabioticstressorstothe

expression of these transcription factors (Singh et al., 2002;

GuttersonandReuber,2004;Xuetal.,2007,2011;Yinetal.,2012).

In general, studiesthat havemodified ERFexpressionin plants

havedemonstratedanincreasedtolerancetosalinity(Huangetal.,

2004; Wang et al.,2004; Zhang et al., 2004;Pan et al., 2010),

drought(Chenetal.,2008;Zhangetal.,2010),temperature(Chen

et al., 2008; Zhang and Huang et al., 2010) and/or pathogen

infection(Heetal.,2001;Panetal.,2010).Yinetal.(2012)showed

that 13 ERFs sequences are differentially expressed during

postharvestabioticstresses(lowtemperature,hightemperature,

highCO2andhighwaterloss)inKiwifuit.Liuetal.(2011),using

microarraytechniques,determinedthatUV-Cirradiationinduced

the expression of defense response genes (such as PR related

proteins,

b

-1,3-glucanase and chitinase), signal transduction

genes(such asethylene relatedgenes,IAA receptorproteinand

calmodulin) and protein metabolism genes. At the same time,

somegenesrelated tocell walldisassembly (such asexpansin,

pectinesterase and endo-

b

-1,4-D-glucanase), photosynthesis

(such as chlorophyll a/b binding protein precursor) and lipid

metabolism(suchaslipoxygenase)seemtobesuppressedinthe

tomatofruitafterUV-Cradiation.

The tomato is one model for the studyof the relationships

betweenstress,hormonalresponsesandfruitquality.Tomatoes

are a good model because their structural genomics are

well-known,theirtranscriptomeandproteomedatabasesarerelatively

rich,andbecausetheyareaspeciesofgreateconomicimportance

(CaraandGiovannoni,2008;Bapatetal.,2010;Barsanetal.,2010).

ThegoalofthisresearchwastounderstandhowUV-Caffects

thetranscriptionalprofilesofACO1andERFsaswellaslevelsofthe

majorsecondary metabolitesintomatoes.Theapplication of

1-MCPpriortoUV-Ctreatmentwasusedtodistinguishiftheeffectof

UV-C treatmenton gene expressionwas mainlydependent on

ethylene.

2.Materialandmethods

2.1.Plantmaterial

Tomato plants (S. lycopersicum Mill., “MicroTom”) were

cultivatedinpotswithpeatsubstrate(Klasmann-Deilmann,R.H.

P.15).Growing conditionswere:a14:10hlight/darkcyclewith

temperatures of 25!_{C during the dayand 20}!_{C overnight, 70%}

relativehumidity(RH)andalightintensityof250

m

molm2_s1_.

Tomatofruitwereharvestedatthebreakerstageoftheripening

processandtransportedatroomtemperature(RT)fortreatment.

Theaveragetimebetweenharvestandtreatmentwas30min.

2.2.UV-Ctreatment

ForUV-Ctreatment, theharvestedtomatoes werepackedin

traysandplaced underUV-Clamps(TUVG30T8, 30W,Philips).

Fourlamps were placed at a distance of 30cm from thefruit,

providing a UV-C dose of 3.7kJm2 _{as measured by a digital}

radiometer(ModelMRUR-203,Instrutherm1_{).Toachievethetotal}

dose,4minofexposurewererequiredoneachofthefoursidesof

the fruit, totaling 16min of treatment. To isolate the effect of

ethylene,atreatmentof1-MCPwasappliedtothefruitinthe

1-MCP+UV-C group at a concentration of 2

m

LL1 _{before UV-C}

treatment.TheseconditionswerepreviouslyoptimizedbyTiecher

et al. (2013). Thus, the experimental design contained the

followingtreatments:1–UV-C:fruitwereharvestedandtreated

withUV-Cat3.7kJm2_{andstoredatRT(20 3}!_{Cand80 5%RH)}

for12d.2–1-MCP+UV-C:fruitwereharvestedandtreatedwith

1-MCPat2

m

LL1_{for12h,followedbytreatmentwithUV-Cas}

describedaboveandstoredatRTfor12d.3–Control(untreated

fruit):fruitwereharvestedandimmediatelyplacedatRTfor12d.

2.3.RNAextraction,cDNAsynthesisandrealtimePCR(qPCR)

Theexocarpsoftheharvestedtomatofruitwereusedtostudy

the transcriptional expression of ACO1 and ERF genes by

quantitative PCR (qPCR).The samples described in Section 2.2

werecollectedafter6hofstorage.TotalRNAwasextractedusing

PureLinkTM_{reagent(Invitrogen}1_{)accordingtothe}

manufacturer’s

instructions.ThequalityandconcentrationofRNAextractswere

evaluatedusinganAgilent2100Bioanalyzer1_(Agilent

Technolo-gies,CA),inwhichonlyRNAsamplesthathadRIN(RNAintegrity)

Fig.1.EffectsofUV-CtreatmentonrelativeaccumulationofACO1(A)andERF(B–O)genetranscriptsin“MicroTom”tomatofruitafter6hofstorage.Therelative quantificationoftranscripts(RQ)isrelativetocontrolfruitandnormalizedwithb-actintranscripts.Verticalbarsrepresentthestandarddeviation.

2

m

gofRNAextractwastreatedwithDNase(Qiagen,Valencia,CA,

USA). Reverse transcription of mRNAwas completedusing the

OmniscriptReverseTranscriptionkit(Qiagen,Valencia,CA,USA),

resultinginatotalvolumeof20

m

L.ForqPCR,2

m

LofcDNAwas

addedto25

m

Lofreactionagent-SYBRGREENPCRMasterMix

(PE-AppliedBiosystems,FosterCity,CA,USA),andanABI7900ht

sequence-detection system was used. The Sl-ACO1 gene (Barry

etal.,1996)and14ERFgenes(Sl-ERFA.1–Pirrelloetal.,2012;

Sl-ERFA.3–Zhouetal.,1997;Sl-ERFB.1–Pirrelloetal.,2012;Sl-ERF

B.2–Pirrelloetal.,2012;Sl-ERFB.3–Tournieretal.,2003;Sl-ERF

C.6–Zhouetal.,1997;Sl-ERFD.1–Pirrelloetal.,2012;Sl-ERFD.3–

Pirrelloetal.,2012;Sl-ERFE.1–Tournieretal.,2003Sl-ERFE.2– Zhang etal., 2004; Sl-ERFE.3 –Wang etal., 2004; Sl-ERFE.4 – Pirrelloetal.,2012;Sl-ERFF.5-Tournieretal.,2003;Sl-ERFG.2

-Zhouetal.,1997)wereused.Primerswereusedataconcentration

of50nM,andtheqPCRconditionswereasfollows:50!_Cfor2min,

95!_{Cfor10min,40cyclesat95}!_Cfor15s,60!_{Cfor1min, 1cycleof}

95!_{Cfor15sand1cycleof60}!_{Cfor15s.Analyseswereperformed}

in triplicate onplates witha capacity of 384reactions. The Ct

(threshold cycle) values were calculated for each sample. The

relative quantification (RQ) was calculated with the method

proposedbyLivakandSchmittgen(2001),using

b

-actin(Pirrello

etal.,2006)asaninternalstandard(nonaffectedby1-MCP+UV-C,

UV-C, fruit growth and development) and control fruit for

calibration.

2.4.Ethyleneproductionandfruitcolor

Fruitethyleneproductionwasquantifiedbygas

chromatogra-phy 1h, 6h, and 12h, and daily (up to 12d) after the UV-C

application.Thefruitineachgroupwasplacedina100mL

screw-capglassvial.After30minofincubation,1mLofheadspacewas

collectedtodeterminetherate ofethyleneproduction, andthe

resultswereexpressedinngkg1_s1_.

Thecolorofalleachofthesixfruitineachgroupwasmeasured

daily (upto 12d) on 4 sides with a colorimeter (Minolta

CR-300 TM),and the results wereexpressedas thehueangle “H”

[H=tan1_{(b/a)whena>0andb>0orh=180+tan}1_(b/a)when

a<0andb>0].

2.5.Levelsoflycopene,

b

-carotene,luteinandzeaxanthin

Extraction techniques and chromatographic analysis were

performed following methods described by Rodriguez-Amaya

(2001) followedbysaponificationoftheetherextract.Levelsof

lycopene,

b

-carotene,luteinandzeaxanthinwerequantifiedusing

a highperformanceliquid chromatography(HPLC) systemfrom

Shimadzuequippedwithanautomaticinjector,UV–visdetectorat

450nm, a RP-18CLC-ODS (5mm, 4.6mm"150mm, Shimadzu)

reverse-phase column and CLC-GODS (5mm, 2mm"4mm,

Supelco)guardcolumn.Separationwasperformedusingagradient

elutionsystemwithmethanol(solventA),acetonitrile(solventB)

andethylacetate(solventC)asthemobilephaseataflowrateof

16.7

m

Ls1_(i.e.1mLmin1_{).Theinitialphaseconsistedof30%A}

and70%B;after10minthecompositionwaschangedto10%A,80%

Band10%C;after35minthecompositionwaschangedagainto5%

A,80%Band15%C;theinitialcompositionwasrepeatedat40min

and maintainedfor 2.5mintorebalance thesystem.The peaks

were identified by comparison with the retention times of

standardsandquantifiedbycomparisonwithexternalcalibration

curves forlycopene,

b

-carotene,lutein andzeaxanthin (Sigma–

Aldrich1_{)standards.TheHPLCresultsareexpressedasmgkg}1_of

freshmaterial.

2.6.

d

-tocopherollevels

Tocopherolextractionwasperformedas describedby

Rodri-guez-Amaya (2001), using a method similar to that used for

carotenoid extraction. The tocopherols were separated and

quantifiedusingHPLCinamanneridenticaltothatdescribedin

item 2.5. The separation was performed by a gradient elution

systemwithamobilephaseof:methanol(solventA),isopropanol

(solventB),andacetonitrile(solventC)ata_flowrateof16.7

m

Ls1

(i.e. 1mLmin1_{). The gradient began with a ratio (A/B/C) of}

40:50:10 (v/v/v), which was changed linearly to 65:30:5 over

10min,thendecreasedto40:50:10overanother2minandheld

constantfor15min.Thepeakwasidentifiedbycomparisonwith

theretentiontimeofthestandardandquantifiedbycomparison

with an external calibration curve for

d

-tocopherol (Sigma–

Aldrich1_{).Resultsonafreshweightbasisareexpressedasmgkg}1_.

2.7.Levelsofp-hydroxybenzoicacid,p-coumaricacidandquercetin

The extraction and identification of individual phenolic

compoundswasperformedfollowingthemethodsdescribedby

Häkkinenetal.(1998).Phenoliccompoundswereextractedwith

methanol acidified with6M HCl and separated and quantified

usinganHPLCprocessidenticaltothatdescribedinitem2.5.The

mobilephaseconsistedofanelutiongradientwithaceticacidin

water(99:1)(solventA)andmethanol(solventB)ataflowrateof

15

m

Ls1_{(i.e.0.9mLmin}1_{).Thestartingpercentageof100%Awas}

graduallychangedto60%Aand40%Boveraperiodof25min,held

constantatthisratioforafurther2min,graduallychangedto95%

Aand5%Bat37min,heldconstantforanadditional5minand

thenreturned tothestarting proportionfora totalrun timeof

45min.Thephenoliccompoundswereidentifiedbycomparison

with the retention time of standards and quantified based on

calibrationcurvesofexternalstandardsforp-hydroxybenzoicacid,

p-coumaricacidandquercetin(Sigma–Aldrich1_{).Theresultsare}

expressedonafreshweightbasisasmgkg1_.

2.8.Polyaminelevels

Polyamine extraction and quantification was carried out

following Vieira et al. (2007) with minor changes. Polyamines

wereextractedwithtrichloroaceticacid(5%inwater)andanalyzed

byHPLCseparatedinaC18column(30cm"3.9mmi.d."10

m

m,

Waters).Polyamineanalysesusedanelutiongradientprogramin

which mobilephase Awas acetate buffer(0.1M) containing

1-octanesulfonicsodiumsalt(10mM),adjustedtopH4.9withacetic

acidandeluentBwasacetonitrile,ataflowrateof11.7

m

Ls1(i.e.

0.7mLmin1_{.Afterseparation,theamineswerederivatizedwith}

o-phthalaldehyde(OPA)anddetectedfluorometricallyat340nm

excitationand445nmemission.Resultswereexpressedonafresh

weightbasisasmgkg1_.

2.9.Experimentaldesignandstatisticalanalysis

Theexperimentaldesignwascompletelyrandomized,

consist-ingof3UV-Ctreatmentgroups(control,1-MCP+UV-C,UV-C)with

3 analytical replicates. Data was verified for normality using

Shapiro–Wilks’testandforhomoscedasticityusingHartley’stest.

ResultswereanalyzedusingANOVA,withaP#0.05 considered

significant.Post-hoc analysis was performed using Tukey’stest

(p#0.05).SASsoftwarewas usedforallstatisticalanalysis(Sas

3.Results

3.1.TheeffectsofUV-Ctreatmentonthetranscriptionalaccumulation

ofACO1andERFgenes

As revealed by the relative accumulation of ACO1 and ERF

transcripts,UV-Ctreatmentaffectedtheexpressionofmostgenes

investigated(Fig.1).Therewasanincreaseintheaccumulationof

ACO1 gene transcripts when fruits were treated with UV-C

(Fig.1A),andtheapplicationof1-MCPprior toUV-Ctreatment

reducedlevelsofACO1transcriptscompared toUV-Ctreatment

alone; however, levels were still above those observed in the

controlfruit.

Amongthe14ERFgenesstudied(Fig.1B–O),11ERFs(Sl-ERFA.1,

Sl-ERFA.3,Sl-ERFB.1,Sl-ERFB.2,Sl-ERFB.3,Sl-ERFC.6,Sl-ERFD.1,

Sl-ERF D.3, Sl-ERF E.1, Sl-ERF F.5, Sl-ERF G.2) increased withUV-C

treatment, 2 ERFs (Sl-ERF E.2 and Sl-ERF E.4) had decreased

transcriptaccumulationand1ERF(Sl-ERFE.3)wasnotaffectedby

UV-Ctreatment.Whentheethyleneactioninhibitor(1-MCP)was

applied prior to UV-C treatment, there was less transcript

accumulationcompared toUV-C treatment alone for allof the

ERFsstudied,withtheexceptionofSl-ERFG.2.

3.2.TheeffectsofUV-Ctreatmentonethyleneproductionandcolor

ThefruitsubjectedtoUV-Ctreatmentshowedhighethylene

production in the first hour after treatment. The evolution of

ethyleneproductioninalltreatmentsfollowedaclassicclimacteric

pattern,withincreasedethyleneproductioncorrespondingtothe

climactericpeak. However,the maximumclimacteric peak was

delayedby1dwithUV-Ctreatment(Fig.2A),and3to4dwiththe

application of 1-MCP prior to UV-C,as compared withcontrol

tomatoes(Fig.2A).

TheapplicationofUV-Chelpedtomaintainthegreencolorof

the fruit, and 1-MCP+UV-C treatment further inhibited color

change,and retaineda higher !_{Hue value(Fig.2B),despitethe}

increasedethyleneproductionofthisfruit(Fig.2A).Additionally,

bettervisualappearanceinUV-Ctreatedfruitwasobservedafter

12dofstorage(Fig.2C).

3.3.EffectofUV-Consecondarymetabolitelevels

TheUV-Ctreatmentdelayedtheripeningevolution,withlower

levels of lycopene,

b

-carotene, lutein and zeaxanthin and

d

-tocopherolobserved,aftersixdaysofstorage.Slower

accumu-lation was observed when 1-MCP was applied before UV-C

treatment(Table1).Thetreatmentalsoresultedinhigherlevels

ofallmeasuredphenoliccompounds(Table1,chlorogenicacid,

p-coumaricacidandquercetin).

Putrescine and spermidine were predominant among the

polyaminesdetected intreated fruit(Table 1).It wasclearthat

the application of UV-C promoted a greater accumulation of

putrescineandspermidineafter6doftreatment.Infruitthatwas

previously subjected to treatment with 1-MCP before UV-C,

polyaminelevelswerelowerthaninfruittreatedonlywith

UV-Cbuthigherthancontrol.

4.Discussion

Thereisalargebodyofresearchdemonstratingthebeneficial

effectsofUV-Cradiationtreatmentonfruit(Maharajetal.,1999;

González-Aguilaretal.,2007;Charlesetal.,2008a,b;Erkanetal., 2008;Liuetal.,2009;Pomboetal.,2011;Stevensetal.,1998;Liu etal.,2011;Tiecheretal.,2013;Maharajetal.,2014;Syamaladevi

etal.,2014).Theresultsobservedinthisworkhaveconfirmedthat

UV-Ctreatmentstimulatesethyleneproduction,especiallyinthe

first few hours after treatment (Fig. 2A, Maharaj et al., 1999).

Additionally, UV-C treatmentcauses an increase in ACO1 gene

transcripts(Fig. 1A)thatcodefortheenzymeACCoxidase,whichis

activeduringthelaststepofethylene biosynthesis(Barryetal.,

1996;CaraandGiovannoni,2008).Thisphysiologicalresponseis

consistentwiththefactthat UV-Cisa stressorand thatplants

Fig.2.EffectsofUV-Ctreatmentonethyleneproduction(A),!_{Hue(B)andtomatofruits(C):(i)control,(ii)1-MCP+UV-C,(iii)UV-C,in“MicroTom”tomatofruitsstoredfor}

generally increase ethylene production under stress, likely by

actingonsystem2autocatalyticethylene(González-Aguilaretal.,

2007;Liuetal.,2011;VandePoeletal.,2012;Tiecheretal.,2013).

UV-Cdelayedripeningintomatofruit,indespiteoftheincrease

in ethylene production (Fig. 2A) and ACO1 transcriptional

expression(Fig.1A).Infact,infruittreatedwithUV-Cradiation

thedevelopmentofcolorationwasslowerthaninthecontrolfruit,

andthetreatedfruitshowedthefewestsenescencesignals(Fig.2

B,C).This_findingisconsistentwithStevensetal.(1998),Maharaj

etal.(1999),Liuetal.(2009)andTiecheretal.(2013)whoalso

foundthat UV-C treatmentled toa reduction in ripening and

delayedtheonsetofredcolorationintomatoes.TheeffectofUV-C

on the development of fruit coloration may be due to its

interferencewithcarotenoids(Table1),whicharethe

predomi-nantpigmentsintomatoes (Stevensetal.,1998;Maharajetal.,

1999;Liuetal.,2009).Liuetal.(2011)reportedachangeinthe

profilecarotenoidsgenesexpressionsintomatoestreatedwith

UV-C.Achangeincolorisoneofthemostobvioustransformationsthat

takesplaceduringtomatofruitripeningandinvolvesthe

ethylene-dependenttransitionofchloroplaststochromoplasts(Opiyo;Ying,

2005;Barsanetal.,2010).Moreover,UV-Ctreatmentmaycause

changesinotherantioxidantpathways,suchastheproductionof

antioxidantenzymes(Erkanetal.,2008)andsynthesisofphenolic

compounds (Charles et al., 2008b) and/or bioactive amines

(Stevens et al., 1998; Maharaj et al., 1999; González-Aguilar etal.,2004;Tiecheretal.,2013).Thesecompoundsmayprevent

thedegradationofchlorophylland/orslowcarotenoiddegradation

(Maharajetal.,1999;Liuetal.,2009;Tiecheretal.,2013).

The effects of UV-C treatment on the levels of compounds

derivedfromplantsecondarymetabolism,previouslyreportedby

several authors (Charles et al., 2008a,b; Erkan et al., 2008;

González-Aguilar et al., 2004; Liu et al., 2009; Pombo et al., 2011;Tiecheretal.,2013)werepartiallyconfirmedbythiswork

(Table 1). The UV-C slows the accumulation of lycopene and

b

-carotene in fruit, which explains the lower intensity of the

characteristicredcolor(Fig.2B).Whenapplying1-MCPpriorto

UV-C, this physiological response was strengthened (Fig. 2B).

Variations inthe fruitprofile of thesecompounds accordingto

differences in variety, ripening stage, growth, and postharvest

conditionsiswidelyreported(Charlesetal.,2008a,b;Erkanetal.,

2008;Liuetal.,2009;Pomboetal.,2011).UV-Cradiationinduced

the accumulationof at least three of the phenolic compounds

investigated(Table1),which isinagreementwithCharlesetal.

(2008b), who also found higher concentrations of phenolic

compounds,anacceleratedlignificationprocessandtheformation

ofsuberinintomatoestreatedwithUV-C.

Thefactthat UV-Cradiationstimulatedtheaccumulationof

thesecompoundsisinterestingnotonlyforprolongingshelf-life,

but also for increasing plant defenses, and for increasing

potentially bioactive compound levels (Stevens et al., 1998;

González-Aguilaret al.,2004;Charles etal., 2008;Erkanetal., 2008;Tiecheretal.,2013).ItisplausiblethattheERFsinfluenced

by UV-C (Fig.1)control the biosynthesis of these compounds

becausethetomatoisaclimactericfruit,andethyleneisinvolved

inthecontrolof severalof itsbiosyntheticpathways(Cara and

Giovannoni,2008).

Thedelayofsenescencesignals(Fig.2)maybecorrelatetothe

levelsofpolyamines(Table1).Theseresultssupportwhathasbeen

reportedinthepeachbyGonzález-Aguilaretal.,(2004) andby

Maharajetal.(1999)andTiecheretal.(2013)intomatoes.These

authorssuggestedthatbyactingasastressor,UV-Cinitiatesthe

synthesisofpolyaminesthatmaybeinvolvedintheregulationof

ripening.

Becauseethylene can activatedifferenttranscription factors,

including regulators of metabolic pathways involved in fruit

ripening and those related to the stress response, the

T able 1 Levels of caro tenoids, d -to copherol, phenolics, and pol y amines fr om e x ocarps of “ Micr o T om ” tomato fruit 6 d after the UV-C tr eatment. Caro tenoids T ocophero l Phenolics Pol y amines Ly copene (m g k g  1) b -caro tene (m g k g  1) Lut ein + zeaxanthin (mg kg  1) d -to copherol (m g k g  1) Chlor og enic acid (m g k g  1) r -coumaric acid (m g k g  1) q uerce tin (mg kg  1) Putr escine (m g k g  1) Spermidine (mg kg  1) Contr ol 69. 1a * 1 2a 3.6a 1 4.64a 1 92.8c 7 .8a 6.8b 5. 1c 17 .3b 1-MCP + UV-C 20c 6.3b 2.4b 7 .43b 256.9b 9.7b 7 .9b 5 7 .1b 40.2a UV-C 26.6b 8.2b 2.5b 9.67b 36 7 .6a 1 0. 1b 1 9.8a 94.2a 43. 1a * Means with the same lo w er case lett er in the column ar e no t statisticall y differ ent according to the Tuke y’ s test (p # 0.05).

transcriptionalexpressionofERFswasalsoevaluated.UV-Cwas

foundtohavedifferenteffectsontheexpressionofthesegenes

(Fig. 1B–O).Ohme-TakagiandShinshi(1995)characterizedthefirst

fourERFsintobaccodemonstratingthattheyresponddifferently

toethylene.Chenetal.(2008)reportedthatintomatoesERFsmay

be differentially regulated during ripening and in response to

stress.

MostoftheERFsstudiedhere(Sl-ERFA.1,Sl-ERFA.3,Sl-ERFB.1,

Sl-ERFB.2,Sl-ERFB.3,Sl-ERFC.6,Sl-ERFD.1,Sl-ERFD.3,Sl-ERFE.1,

Sl-ERFF.5,Sl-ERFG.2)showedhighertranscriptaccumulationwhen

thetomatoesweretreatedwithUV-Csuggeststhatthesegenesare

strong candidates for explaining the UV-C response, and its

relationshiptoethylene.Thedelayintheripeningprocess,despite

the increase in ethylene production, ACO1 level, and ERFs

transcription level, could be due to activation of metabolic

pathways of antioxidant protection for these ERFs (Liu et al.,

2011;Erkanetal.,2008;Tiecheretal.,2013).Ingeneral,when

1-MCPwas applied prior toUV-C,reduced accumulation of ERFs

transcriptswasobserved,thusconfirmingthattheexpressionof

thesetranscriptionfactorscanberegulated byethylene(Zhang

etal.,2004;Pirrelloetal.,2006;Wangetal.,2007).Moreoverthis

datasuggeststhatregulationofERFtranscriptsbyUV-Cisethylene

dependent.

Inthiswork,theclassificationproposedbyPirrelloetal.(2012),

whoclassifiedtomatoERFsinto8sub-classes(A,B,C,D,E,F,G,H)

wasused;however,membersofsub-classHwerenotevaluated.Of

the14ERFsassessedinthepresentstudy,6(Sl-ERFA.1,Sl-ERFB.1,

Sl-ERFB.2,Sl-ERFD.1, Sl-ERFD.3andSl-ERFE.4) wereisolated and

characterizedbyPirrelloetal.(2012),whowasthefirsttorelate

theseERFstoothertypesofplantstress.

Zhouetal.(1997),whostudiedERFsSl-ERFA.3,Sl-ERFC.6and

Sl-ERFG.2,(describedinhisworkaspti4,pti5andpti6,respectively),

reportedtheabilityoftheseERFstobindspecificregionsofEREBR’s

(ethylene-responsiveelement-bindingproteins),alsoknownasthe

GCC-boxof PR-genes,increasingthetolerance ofplantstobiotic

stress.TheregulationoftheseERFgenesthroughphosphorylation

mayalsoinfluencetheinteractionofthesetranscriptionfactors

withtheGCC-boxregionsofPR-genes(Guetal.,2000;Xuetal.,

2008, 2011). In the present study, these ERFs were strongly

influenced by the abiotic stress generated by UV-C treatment,

showingasignificantincreaseintheaccumulationoftranscripts,

especially Sl-ERFC.6,which showed anapproximately 250-fold

increaseinexpressionrelativetocontrolfruit.Thisindicatesthat

theinductionofERFsmaycontributetotheacquisitionoftolerance

toadverseconditions(Heetal.,2001;Guetal.,2002;Chenetal.,

2008).Liuetal.(2011)alsoreportedasignificantincreaseinthe

expression of these three genes, especially Sl-ERF C.6, which

correspondstopti5.Byover-expressingSl-ERFC.6intomatoes,He

etal.(2001)reportedincreasedlevelsofGluBandcatalasegene

transcripts,whichareassociatedwithresistancetodiseasessuch

asPseudomonassyringae pv. Tomato.Likewise,Guet al. (2002),

observedthatinArabidopsisthalianaplantstheERFsSl-ERFA.3,

Sl-ERFC.6andSl-ERFG.2interact withtheGCC-boxregions of

PR-genes,resultinginpathogendefense.Chenetal.(2008)reported

thatwaterstressandlowtemperaturesreducethelevelsofSl-ERF

A.3transcripts.However,mechanicaldamagealsoincreasedthe

expressionofthisgene,whichsuggeststhattheremaybedifferent

regulatorymechanismsdependingonthestimulus.

The increase of transcript accumulation ofSl-ERF B.3agrees

withtheresultspublishedbyLiuetal.(2011),whichshowedthe

relationshipofthisgenetotheripeningtomatoesprocessand,Liu

etal.(2014)thatalsoreporteddelayoftheonsetofripeningcaused

forover-expression ofSl-ERF B.3-SRDX(a climactericdominant

repressorreversion).

Results from the present study on Sl-ERF E.1, previously

characterized by Tournier et al. (2003) as LeERF2, reveal the

strongimpactofethyleneonERFexpression,inagreementwith

theresultsofPirrelloetal.(2006),Liuetal.(2011),Zhangetal.

(2009) and Zhang and Huang (2010) who also related the

expression of this transcriptionfactor tothe hormoneethylene

intomatoandtobaccoplants.

Sl-ERF E.2 and Sl-ERF E.4 showed significantly reduced

accumulation of transcripts after UV-C treatment. Zhang et al.

(2004), who described Sl-ERF E.2 as JERF1, demonstrated that

expressionofthis ERFintomatoeswasinducedbyanumberof

factors: ethylene,methyl jasmonate(MeJA), abscisic acid(ABA)

and salt treatment.In rice, plants over-expressing JERF1 show

increaseddroughttolerance(Zhangetal.,2010).Incontrast,the

resultspresentedhereinsuggestthatSl-ERFE.3isnotsignificantly

involvedin theresponse toUV-C,although Wang etal. (2004)

reportedthatSl-ERFE.3respondstojasmonicacid,ethylene,cold,

saltstressandabscisicacidbybindingtoGCC-boxandDREregions

oftargetgenes.

Inthisstudy,Sl-ERFF.5alsoshowedincreasedtranscriptionasa

resultofUV-Ctreatment.Chenetal.(2008),studyingSl-ERFF.5

(whichtheyrefertoasLeERF3b),relatedtheexpressionofthisERF

tostressgenerated bydroughtand low temperatures.This ERF

possessesaamphiphilicrepressorbindingdomain(EAR)(Xuetal.,

2008;Panetal.,2010;Pirrelloetal.,2012).Panetal.(2010)deleted

theEARofSl-ERFF.5(referredtoasSl-ERF3in theirstudy)and

observed the induction of PR-gene expression, with increased

tolerancetosaltstressandreducedlipidperoxidationinRalstonia

solanacearum.

Herein, arelationshipbetweenUV-Ctreatment andripening

delay was established, and correlated with changes in 13 ERF

transcriptsevaluatedduringpostharvesttreatment.Theethylene

actioninresponsetoUV-Ctreatmentwasconfirmedwith1-MCP

applicationbeforeUV-C.ItisclearthatalthoughUV-Cpromotesan

increaseinethyleneproduction,theconcomitantincreasesinthe

ACO1expressionprofile, andvirtually allof theERFsevaluated,

resultinextendedfruitpreservation.Thedelayinfruitripening

maybecausedbytheactivationofERFsthatcouldactasregulators

ofmetabolicpathwaysduringripening.However,thishypothesis

needstobebettertested.

Acknowledgements

WethankCapes-Cofebubforthestudyscholarshipandresearch

funding.ResearchfundingwasalsoprovidedbyCNPqandFapergs.

We alsothankDr. RachelKopec forvaluable help withEnglish

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