Trong các tác nhân gây ra sư chín hóa sinh của thực vật, Ethylene được biết như một trong những nhân tố để bắt đầu, điều chỉnh và điều phối sự biểu hiện của hệ thống các Genes tham gia vào quá trình chín. Sự bùng nổ trong sản xuất ethylene là mốc quan trọng cho sự khởi đầu của chín của trái cây thời kỳ. Trong nghiên cứu này nhóm nghiên cứu đã cho thấy một kết quả kahr quan khi có thể kéo dài quá trình bản quản cà chua lên tớ 45 ngày mà không làm ảnh hưởng đến mùi vị và giá trị dinh dưỡng của cà chua.
Trang 1jou rn al h om ep a g e :w w w e l s e v i e r c o m / l o c a t e / j p l p h
Physiology
a Plant Polyamine, Transgenic and RNAi Research Laboratory, Department of Genetics, University of Delhi South Campus, New Delhi 110021, India
b Division of Post-Harvest Technology, Indian Agricultural Research Institute, New Delhi 110012, India
a r t i c l e i n f o
Article history:
Received 4 October 2012
Received in revised form 18 February 2013
Accepted 18 February 2013
Available online 16 March 2013
Keywords:
ACC synthase
Delayed ripening
Ethylene
Fruit quality
Polyamines
a b s t r a c t
Theripeninghormone,ethyleneisknowntoinitiate,modulateandco-ordinatetheexpressionof var-iousgenesinvolvedintheripeningprocess.Theburstinethyleneproductionisthekeyeventforthe onsetofripeninginclimactericfruits,includingtomatoes.Thereforeethyleneisheldaccountablefor thetonsofpost-harvestlossesduetoover-ripeningandsubsequentlyresultinginfruitrotting.Inthe presentinvestigation,delayedripeningtomatoesweregeneratedbysilencingthreehomologsof 1-aminocyclopropane-1-carboxylate(ACC)synthase(ACS)geneduringthecourseofripeningusingRNAi technology.ThechimericRNAi-ACSconstructdesignedtotargetACShomologs,effectivelyrepressedthe ethyleneproductionintomatofruits.Fruitsfromsuchlinesexhibiteddelayedripeningandextended shelflifefor∼45days,withimprovedjuicequality.Theethylenesuppressionbroughtabout composi-tionalchangesinthesefruitsbyenhancingpolyamine(PA)levels.Further,decreasedlevelsofethylene
inRNAi-ACSfruitshasledtothealteredlevelsofvariousripening-specifictranscripts,especiallythe up-regulationofPAbiosynthesisandascorbicacid(AsA)metabolismgenesanddown-regulationofcell wallhydrolyzingenzymegenes.Theseresultssuggestthatthedown-regulationofACShomologsusing RNAicanbeaneffectiveapproachforobtainingdelayedripeningwithlongershelflifeandanenhanced processingqualityoftomatofruits.Also,thechimericgenefusioncanbeusedasaneffectivedesignfor simultaneoussilencingofmorethanonegene.Theseobservationswouldbeusefulinbetter understand-ingoftheethyleneandPAsignalingduringfruitripeningandmolecularmechanismsunderlyingthe interactionofthesetwomoleculesinaffectingfruitqualitytraits
© 2013 Elsevier GmbH All rights reserved
Introduction
Tomatoisoneofthemostvaluablefruitcropacrosstheworld
andisrichinminerals,fibers,vitaminsandantioxidants(Rajam
etal.,2007).Italsoservesasamodelsystemforripeningrelated
studies.Tomatofruitripeningis initiatedwithclimactericburst
ofethylene,whichco-ordinates andregulatestheexpressionof
ripening-specificgenesinfruit(Osorioetal.,2011).The
biosyn-thesisofethylenestartswithS-adenosylmethionine(SAM),which
Abbreviations: ACC, 1-aminocyclopropane-1-carboxylic acid; ACS, ACC
syn-thase; ADC, arginine decarboxylase; NPT-II, neomycine phosphotransferase; ODC,
ornithine decarboxylase; PA, polyamine; PLW, physiological loss of water; Put,
putrescine; SAMDC, S-adenosylmethionine decarboxylase; Spd, spermidine; Spm,
spermine; TAs, titratable acids; TSS, total soluble solids; WT, wild-type.
∗ Corresponding author at: Department of Genetics, University of Delhi South
Campus, Benito Juarez Road, New Delhi 110021, India Tel.: +91 11 24110866;
fax: +91 11 24112437.
E-mail address: rajam.mv@gmail.com (M.V Rajam).
isconvertedinto1-aminocyclopropane-1-carboxylicacid(ACC)by ACCsynthase(ACS),andACCisfinallyconvertedintoethyleneby ACCoxidase(ACO)(AdamsandYang,1979).ACScarriesoutthe rate limiting stepin ethylene biosynthesis (Yang and Hoffman,
1984)anditintomato,is apartofmulti-genefamily, compris-ingninehomologs,whicharedifferentiallyexpressed(Caraand Giovannoni, 2008) ACS2 and ACS4 are responsible for climac-tericburstofethyleneproductionattheonsetofripening(termed System-2),whileACS1AandACS6maintainbasallevelsofethylene
ingreentissues(termedSystem-1)(Rottmannetal.,1991;Lincoln
etal.,1993).Ithasbeenreportedthatbasalethyleneisessential forprogressionofsystem2ethylene.Tomatofruitcanalso initi-atesystem2ethyleneindependentlyofsystem1,whichproves thatripening-associatedethylenebiosynthesisisregulatedbyboth auto-catalyticsystemandethylene-independentfactors(Yokotani
etal.,2009)
Anumber ofphysiologicaleffectsofethyleneinplants seem
tobeantagonizedbypolyamines[PAs:putrescine(Put), spermi-dine (Spd)and spermine (Spm)]by modulating genesinvolved 0176-1617/$ – see front matter © 2013 Elsevier GmbH All rights reserved.
Trang 2Fig 1. T-DNA map of RNAi-ACS binary vector Antisense chimera was designed to be 50 bp shorter than the sense chimera, such that after transcription the antisense RNA folds back and complements with sense RNA to form dsRNA molecules with loop in between.
intheethylenesignalingand biosyntheticpathways(Apelbaum
etal.,1981;Handaetal.,2011).IncreasedlevelsofPAshaveshown
toresultindelayedfruitripening,increasedfruitfirmness,
pro-longedshelflife,reducedethyleneandrespirationrateemissions
(Bregolietal.,2002;Nambeesanetal.,2010).Inplants,Putiseither
synthesized directly from ornithine via the action of ornithine
decarboxylase(ODC)orindirectlyfromarginineviaarginine
decar-boxylase(ADC).HigherPAs(SpdandSpm)aresynthesizedbythe
sequentialadditionofaminopropylgroupstoPutbySpdsynthase
(SPDSYN)andSpmsynthase(SPMSYN).Theaminopropylgroups
areprovidedfromdecarboxylatedS-adenosylmethionine(dcSAM)
whichisformedbydecarboxylationofSAMbySAM
decarboxyl-ase(SAMDC).SAMactsasthecommonprecursorforbothPAand
ethylenebiosynthesispathways,suggestingittobeaconstraintfor
eitherofthebiosyntheticpathway(Tiburcioetal.,1990)
Thegreentomatotransformsintoripefruitwhileit
accumu-latescarotenoidpigmentsandturnsred,developsflavorandaroma
withalterationinsugars,acidsandvolatileprofilewithsoft
tex-ture(Giovannoni,2001).Ripening-associatedsofteningisamajor
factor limiting fruitshelf life and storageand thus, contributes
tothetonsof lossesofthis crop.In fact,severalattempts have
beenmadetocontrolripeningprocess.Fordelayedripening,major
focushasbeenonthemanipulationofethyleneproduction,
per-ceptionandactionemployingeithersenseorantisensetechnology
(Hamiltonetal.,1990;Oelleretal.,1991;Theologisetal.,1993;
Yeetal.,1996;Wilkinsonetal.,1997;Xiongetal.,2003,2005)
Alltheseresearchershavebeensuccessfulinobtaininglow
ethyl-eneproducingtomatoes,displayinganextendedshelflifebutwith
compromisedfruitqualitytraits.However,selectivenatureofRNAi
canbemore specificfor suppressionand ismoreeffectivethan
eithersenseoranantisenseRNA(Fireetal.,1998).Additionally,
suppressionofmorethanonehomologwouldbemoreeffective
overshut-downofsinglehomolog.Fromtheperusalofliterature,it
isapparentthatPA-ethylenenexusplaysacrucialroleinfruit
ripen-ing.Thus,lookingatthemultifunctionalandregulatoryaspectsof
PAandethylene,itispossiblethatcontrolledmanipulationofthese
keyregulatorsratherthanstructuralorregulatorygeneoperating
ina singlebranchofbiosynthesis pathwaymayresultin better
improvementoffruitshelf lifeandqualitytraits.Inthepresent
study,inordertomodulatePA-ethylenelevels,wehavesuppressed
theexpressionofthreehomologsofACSgene(ACS6,ACS1Aand
ACS2),therebytargetingsystem1and 2productionofethylene
simultaneouslyattheonsetoffruitripening,usingRNAiapproach
Fruit-specificdown-regulationofthesegeneswasaccomplishedby
2A11promoterfromtomato
Materials and methods
Insilicoanalysis
Thenucleotidesequencesoffruit-specifichomologsofACS,viz.,
ACS6(GenBankID,AF179249),ACS1A(GenBankID,U18056.2)and
ACS2(GenBankID,NM001247249.1)fromSolanumlycopersicum
werecomparedbypair-wisealignment usingNCBIBLAST2Seq
ToavoidunintendedtargetingbyRNAiintransformedtomato,the selectedACShomologswereevaluatedfortheirprobableoff-target
insilico.Thecomplete mRNAsequences forallthree ACSgenes wereusedforhomologysearchingwithalltheavailablesequences
innrdatabaseusingNCBIBLAST.TheindividualmRNAsequences werealsocheckedforprobableoff-targetsbysplittingitinto21bp fragmentimitatingthe siRNA.The off-targetfree partialmRNA sequenceofACS6,ACS1AandACS2werechosenforpreparingRNAi construct.Theprimersequencesdesignedfortheamplificationof ACShomologs,2A11promoterandantisenseACS(Chimeric)are giveninTableS1
GenerationoftomatotransformantswithRNAi-ACS(chimera) construct
ThepartialcDNAofthreeACShomologsACS6(201bp),ACS1A (286bp) and ACS2 (506bp) were amplified from tomato (fruit pericarp) total RNA by semi-quantitative RT-PCR using specific set of primers designed with restrictionsites ACS1A and ACS2 wereclonedintopBSKvectorsequentiallyusingKpnI–EcoRIand EcoRI–BamHI restriction sites respectively The 2A11 promoter (1.35kb–GenBankID,DQ453963)wasisolatedfromtomatoand clonedintopGEM-Teasy.Followingsequencing,2A11promoter wasexcisedbyEcoRIandSacIandligatedintopCAMBIA2300binary vector.ACS6wasclonedin pCAMBIA2300downstream to2A11 promoter usingSacIand KpnIrestrictionsitesfollowedby liga-tionofACS1A+2(frompBSK)withKpnIandBamHIrestrictionsites SenseACS6+ACS1A+ACS2wasusedasatemplateinPCRreaction
toamplifyanantisensechimera,whichis50bpshorterthanthe senseone.ItwasclonedintopCAMBIA2300binaryvector harbor-ing2A11promoter,polyAsignalandsenseACS6+1A+2byusing BamHIandXbaIrestrictionsites.ThepolyAsignalfrompRT101 vec-torwasalsoclonedintopCAMBIA2300vectorusingXbaIandPstI restrictionsites(Fig.1).RNAi-ACSconstructwasmobilizedinto AgrobacteriumtumefaciensLBA4404strainbychemical transforma-tion,andwasthenutilizedtotransformcotyledonsoftomato(S lycopersicumMill.cv.PusaEarlyDwarf)byaproceduredescribed
byMadhulathaetal.(2007) GenomicDNAisolationandtransgeneintegrationintomato transformants
GenomicDNAwasisolatedfromtheleavesofwild-type(WT) andtransformantsusingCTABmethod(Doyleand Doyle,1990) The transformants were screened by PCR for the presence of transgene.Primerpairsfortheamplificationof750bpfragment
ofNPT-IIgeneareF,5-TCAGAAGAACTCGTCAAGAA-3 and R,5 -ATGGGGATTGAACAAGATGG-3
GenomicDNA(10g)wasdigestedwithEcoRIenzymeand sub-jectedtoDNAhybridizationtoconfirmtheintegrationandcopy numberoftransgeneusingradiolabeledNPT-IIgeneprobe.Blots werepreparedbystandardprotocol(Sambrooketal.,1989)using
Trang 3primerkitfollowing themanufacturer’sguidelines(BRIT,India)
Pre-hybridizationandhybridizationwerecarriedoutasdescribed
bySambrooketal.(1989)
AtotalofthirtyRNAi-ACS(chimeric)transformants,confirmed
fortransgeneintegrationbyPCRandDNAhybridization(Fig.S2)
weremaintainedbothintransgenicgreen-house/net-house
condi-tions
Expressionanalysis
Pericarp tissue (50mg) of mature green (MG), breaker red
(BR), pink red (PR) and red ripe (RR) fruit was pulverized to
homogenate powder The homogenate was used for isolation
of total RNA according to the manufacturer’s guidelines with
TriZol reagent (Invitrogen, USA) with subsequent RNase free
DNase(Fermentas,Canada)treatmenttoremoveanyDNA
con-tamination Semi-quantitative RT-PCR was performed to check
transcriptlevelsofthetargetgeneandvariousgenesinvolvedin
fruitripening,inRNAi-ACSandWTtomatoplants.DNA-freeRNA
(200ng)wasthenusedforone-stepRT-PCRreactionbyfollowing
themanufacturer’sguidelines(Taurus-Scientific,India).The
prod-ucts wereanalyzed on ethidiumbromide stained 1.5% agarose
gel.Primersusedinthestudyweredesigned withreferenceto
nucleotide sequences registered in the Genbank database The
reactionconditionswereasfollows:45◦Cfor50min,followedby
94◦Cfor5min,ncyclesof94◦Cfor30s,annealingtemperature
for30sand72◦Cfor30s,finalextensionat72◦Cfor15min.All
semi-quantitative-RT-PCR experiments were carried out thrice
in three independentexperiments by using the pulverized
tis-sue of three fruits (at same stage) from same plant (pooled)
The results presented are relative The accession numbers for
ripening-associated genes selected for transcript analysis are:
ornithine decarboxylase (SlODC, NM001247687.1), arginine
decarboxylase(SlADC1,NM001247135.1),S-adenosylmethionine
decarboxylase(SlSAMDC1,EU196515.1;SlSAMDC2,EU196516.1;
SlSAMDC3, EU196517.1), spermidine synthase (SlSPDSYN,
NM001247564.1), ACS6 (AF179249), SlACS1A (U18056.2),
SlACS2 (NM001247249.1), SlACS4 (NM001247351), SlACO1
(NM001247095), SlE8 (X13437.1),
1-deoxy-d-xylulose-5-phosphate synthase (SlDXS1, AF143812), (SlPSY1, EF157835.1),
lycopene-epsiloncyclase(SlLES,Y14387),SlTAGL1(AY098735.2),
polygalacturonase (SlPG, X05656), expansin (SlEXP1, U82123),
-galactosidase4(SlTBG4,AF020390),␣-xyloglucan
endotransglu-cosylase/hydrolase (SlXTH5, AY497475), l-galactono-1,4-lactone
dehydrogenase (SlGLDH, NM001247674.1), ascorbate oxidase
(SlAO, AY971876.1), dehydroascorbate reductases (SLDHAR1,
AY971873.1;SLDHAR2,AY971874;SlMDHAR,NM001247084.1)
andactin(SlActin,BT012695).Thedetailsofprimersareprovided
inTableS2
IsolationofsmallRNAsandRNAhybridizationfordetectionof
siRNAsinRNAi-ACStomatolines
TotalRNA (200–400g)fromBR fruitwasenriched for low
molecularweightRNAasperthemethoddescribedbyLuetal
(2007).About90gofthelowmolecularweightRNAwas
frac-tionatedtoseparatesmallRNAs(20–25nt)using15%denaturing
polyacrylamidegel accordingto Luet al.(2007) RNAfractions
wereelectroblottedontothepositivelychargednylonmembrane
(MDI,India)usingsemi-dryblot(BenchtopLabsystems,India)and
the membrane was incubated for overnight at 42◦C in a
pre-hybridization buffer(Sambrook etal., 1989).Hybridization was
carriedoutat36◦Cfor20husingradiolabeledACS1A,ACS2and
ACS6DNAasprobe
Estimationofrateofethyleneproductionintomatofruits Threeredfruitswereenclosedin100mLofanair-tightcontainer for1hatroomtemperature.Theheadspaceatmosphere(3mL)of thecontainerwaswithdrawnandinjectedintogaschromatograph (HP5890,HewlettPackard,USA)forethyleneestimation(Singh andPal,2008).Theexperimentwascarriedoutthricewithtwenty replicatesineach
Measurementofrespiratoryactivityintomatofruits Theindividualfruitsweresealedinanair-tightcontainerfor
1h.Respiratoryactivityoftheharvestedfruitswasdeterminedby headspacegasanalysisofjarusingCO2/O2 analyzer(Checkmate
9900O2/CO2,PBIDansensor,Denmark)(SinghandPal,2008).The experimentwascarriedoutthricewithtwentyreplicateseach Determinationofphysiologicallossofwater(PLW)
PLWinRRfruitsduringfirsttendaysoftheirstorage(atroom temperature)wasestimatedby subtractingthesampleweights fromtheirpreviousrecordedweightsandwasrepresentedas% PLW/day comparedtotheinitialweight.Data wasrecordedfor twentybiologicalreplicateseachinthreeindependentsets Polyamineanalysis
About100mgofpericarptissue(fromdifferentstagesoffruit ripening,viz.,BRandRRstages)washomogenizedin1mLof10% perchloricacid Thehomogenateofthreefruits(withsameage, fromsameplant)waspooled.Theextractwasfractionated, dan-sylated,chromatographedandquantifiedbythemethoddescribed
byBajajandRajam(1996)usingdualwavelengthfluorometer (Bio-Rad,VersaFluor,USA)withanexcitationwavelengthof350nmand
anemission wavelengthof495nm.ThePolyaminecontentwas estimatedinthreefruitseachinthreeindependentexperiments Determinationofon-vineripeningperiod
Flowersweretaggedatanthesisanddayswerenotedforfruit formationinthreeindependentsetswithtwentyreplicatesineach
MGfruitsdisplayingfirstsignofcolorchangewereidentifiedas
BRstage.DaysforBRtoreachRRwerenotedforRNAi-ACS,WT andunrelated(UR)controlfruitstodeterminetheon-vineripening period
Determinationoffruitshelflife
RR fruits(twentyreplicates) ofRNAi-ACS,WTandURplants werekeptatroomtemperatureandtimewasnotedforvisualsigns
ofshriveling.Theexperimentwascarriedoutthrice
Measurementoftotalsolublesolids(TSS)content Tomatofruit(RRstage)homogenatewasutilizedformeasuring TSScontentbyahandrefractrometer(model:Fisher,Japan)(Singh andPal,2008).Thehomogenateofthreefruits(fromsameplant) waspooled.TSScontentwasmeasuredinninefruitseachinthree independentexperiments
Estimationoftitratableacidity The homogenate of three fruits(RR stage)from same plant werepooled.Totaltitratableacidity(TA)wasthendeterminedby titratingthehomogenateagainst0.1NNaOHsolutionusing phe-nolphthaleinasanindicatortotheendpointatpH8.1(Singhand
Trang 4experiments
Determinationofascorbicacid(AsA)content
AsAcontentintomatofruitsatRRstagewasestimated
titrime-tricallyusing2,6-dichlorophenolindophenolasanindicatordye
AsAstandardwaspreparedbydissolving100mgofl-AsAin100mL
of1%HPO3(SinghandPal,2008).Thepooledhomogenateofthree
fruits(fromsameplant)wasutilizedfortheestimation.AsAcontent
wasmeasuredinninefruitsinthreeindependentexperiments
Quantificationoflycopenecontent
Lycopene fractions of the homogenized pericarp tissue (RR
stage) were determined using spectrophotometric method as
describedbyAOAC(2000).Thepooledhomogenateofthreefruits
(from same plant) were utilized in each experiment Lycopene
fractionswereestimatedinninefruitsinthreeindependent
exper-iments
Dataanalysis
Allresultswereobtainedfromatleastthreeindependent
exper-iments.Datapresentedareaverage(mean)withthestandarderror
fromalltheexperimentsandsignificantdifferenceswere
deter-minedbyStudent’st-test(P<0.05)
Results and discussion
ThreehomologsofACS,viz.,ACS1A,ACS2,andACS6were
consid-eredintandemforRNAi-mediateddown-regulationtoeliminate
anyredundancyassociatedwithgenefunctionandtoachieve
maxi-mumsuppressionoftheautocatalyticburstinethyleneproduction
However,ACS4wasnotconsideredforpresentstudyduetothe
unavailabilityofanyoff-targetfreeregionanditshomologywith
othertomato geneslikeexpansin(datanotshown).Toalleviate
thepleiotropiceffectswhichmightariseduetoconstitutive
down-regulationofethylene,fruit-specificexpressionofRNAi-ACSwas
achievedby2A11promoter(Fig.1).Thispromotershowslow
lev-elsofactivitythroughoutfruitdevelopmentbutgetsinducedto
highactivityattheonsetofripening(Pearetal.,1989).Previous
experimentsinourlabhaveshownthattomatoplantstransformed
with2A11-GUSconstructexhibitedGUSexpressionexclusivelyin
ripeningfruitwithpronouncedactivityinpericarp,vascular
bun-dles,placentaltissue andseedtegument(Fig.S1) Theseresults
werealsosupportedbydataobtainedbyLinetal.(2006)
EthylenesuppressioninRNAi-ACSfruits
FruitsfromallthirtyRNAi-ACStomatolinesalongwith
unre-latedcontrol(UR,otherRNAilinesfromlab)andWTplantswere
analyzedforrateofethyleneevolution.ResultspresentedinFig.2
showthatfruitsfromRNAi-ACSlinesliberatedreducedlevelsof
ethylene.EthyleneliberationwasfoundtobeleastinRNAi-ACS60
andRNAi-ACS81,releasingonly4–5%whencomparedwith
con-trols(WTandUR).FruitsofotherRNAi-ACSlinesshowed10–70%
ofethyleneevolutiontothat ofcontrolfruits.Thisdifferencein
ethyleneevolutionamongRNAi-ACSlinescouldbeattributedto
variablesuppressionofthetargetgene.Thisinturnisascribableto
differentialabundanceoftheintroduceddsRNA,asinfluencedby
siteofintegrationanddosageeffectoftheintroducedgenes(Fig
S2B)(Kohlietal.,2003;Kerschenetal.,2004)
Transcriptlevelsofthetargetedhomologs,viz.,ACS1A,ACS2and
ACS6,werecheckedbysemi-quantitativeRT-PCR.Resultsshowed
thatlevelsofACS2transcriptsinWTfruitswerehighthroughMG
toPRstageand declineddramaticallyatRRstage(Fig.3A).The observeddeclineinACStranscriptsiscollinearwiththeonsetof 2A11promoteractivity.RNAi-ACSlinesreleasinglowlevelsof eth-ylene,displayeddrasticreductioninACS2transcriptlevels.This declineintranscriptlevelswasmorepronouncedinRNAi-ACS81 lineatallripeningstagesandsubtledinRNAi-ACS123and RNAi-ACS125 In WT tomato, ACS1A and ACS6 transcript levels were substantiallylowerthanACS2mRNAlevels(asweredetectedinWT tomatoeswith30cyclesofamplificationincontrastto25cyclesfor ACS2).Previously,itwasreportedthattheexpressionofACS6and ACS1Agenesisrestrictedtotheearlyfruitdevelopmentalstages (Nakatsukaetal.,1998;Barryetal.,2000),butourresultsrevealed thattheACS1AandACS6transcriptspersistedthroughMGtoRR stageinWTtomatoes.InRNAi-ACStomatolines,theexpression
ofACS6andACS1AgenesfollowedthesametrendasthatofACS2 Theseresultsshowedacorrelationbetweentranscriptabundance andrateofethyleneevolutioninRNAi-ACSfruits
siRNAsspecifictoACS1A,ACS6andACS2weredetectedatBR stageinfruitsofRNAi-ACSlines.RNAi-ACS81linehasexhibited highlevelsofsiRNAswithundetectablelevelsoftranscriptsofall thetargetedgenes(Fig.3B).Hence,declineinACStranscriptsin RNAi-ACSfruitsexpressingdsRNAisanRNAimanifestation
OnthebasisofRT-PCRandsiRNAdetectionresults,RNAi-ACS lineswerecategorizedintohighlysilencedlines(RNAi-ACS60and RNAi-ACS81) and moderately silenced lines (RNAi-ACS123 and RNAi-ACS125).RNAi-ACS81andRNAi-ACS123displayinghighand moderatesilencing,alongwithWTwereutilizedforfurtherwork
ontheexpressionanalysisofripening-relatedgenes.Declinein transcriptslevelsofACS4,ACO1andE8wasnotedincaseofboth theRNAi-ACSlinesstudied,overWTfruits(Fig.3C).Aplausible explanationtothisobservationisthepositivefeedbackregulation
ofthesegenesbyethylene(Barryetal.,1996;Nakatsukaetal.,
1998).DeclineinACS4geneexpressionmaynotbesolelyduetothe ethyleneregulation,itmightalsobeduetothesharedhomology betweenACS2andACS4genesequenceswhichcouldhavecaused theoff-targetingofACS4gene.Moreover,conversionofSAMtoACC
byACCsynthasemarkstheratelimitingstepforethylene biosyn-thesiswhichpossiblyexplainsthatACSgenerepressionhasleadto lowlevelsofACO1transcriptsinRNAi-ACS81line.Transcriptionof E8infruithasbeenshowntoberegulatedandstimulatedby eth-ylene,whichiswellevidentbythepresenceofethyleneregulated sequencesinE8genepromoter(Deikmanetal.,1998).TAGL1codes fortranscriptionfactor,whichactivatesethylenebiosynthesisby bindingtoACS2promoter(Vrebalovetal.,2009).Theexpressionof thisgenewasanalyzedandwecouldnotseeanysignificant differ-enceinTAGL1transcriptaccumulationinfruitsofRNAi-ACSlines overWT,suggestingthatalterationsinethylenelevelsinRNAifruits didnotaffecttheexpressionofthisgene(Fig.3C)
PolyamineaccumulationinRNAi-ACSlinesoftomatowith reducedethylenelevels
InWTtomatofruitsatBRandRRstagesofripening,Putlevels werefoundtobehighestamongthreePAsfollowedbySpdandSpm titers.PutandSpmweremainlypresentinfreefractionsfollowed
byconjugatedandboundforms.ButSpdpoolsweremaintained
byalmostequalfractionsoffreeandconjugatedformsfollowedby boundform.AsthefruitattainsRRstage,PAlevelswerereducedto almosthalfoftheinitiallevelsatBRstage(Fig.4).RNAifruitsatBR stageexhibited13–25%increaseinPut,15–40%increaseinSpdand 15–45%increaseinSpmoverWT.IncontrasttoWTRRtomatoes, RNAi-ACS81tomatoshowedhigherlevelsofPAsatRRthanatBR, with100%increaseinPutand150%increaseinSpdandSpmlevels ExpressionpatternofODCandADCgenesdidnotalter,while SAMDC1andSPDSYNmRNAlevelswereincreasedinboth RNAi-ACS81andRNAi-ACS123fruitsoverWT(Fig.5).Therewasalso
Trang 5Fig 2. Ethylene levels in fruits of RNAi-ACS lines and controls Bars represent the means of twenty biological replicates and three independent experiments, with standard error values as error bars * Significant at P < 0.05 between controls and RNAi lines.
no change in transcript levels of SAMDC2 and SAMDC3 genes
Thesetwo homologsexhibited differentialtranscript
accumula-tion,with SAMDC2 mRNAlevelsbeing higher in BRstage than
RR,whileSAMDC3transcripts,althoughdetectedinBRstage
van-ishedatRRstage.Interestingly,transcriptlevelsofSAMDC2and
SAMDC3werefoundtobelowerthanSAMDC1mRNAlevels.Thus,
SAMDC1seemstobepredominantlyresponsibleforSAMDC
activ-ityinripeningfruits.TheincreasedaccumulationofSAMDC1and
SPDSYNtranscriptswasthereforeaccountableforenhanced Spd
andSpmcontentinRNAi-ACSfruits.ElevatedSAMDCandSPDSYN
transcriptsaccumulationinturnmaybebecauseofdiversionof SAMpoolstowardPAbiosyntheticpathway(duetoreductionin ethylenebiosynthesis).Henceforth,wehypothesizeapositive feed-backregulationofSAMDCandSPDSYNgenetranscriptionbytheir upstreamprecursor,i.e.,SAM,whichisalsosupportedbythe exper-imentscarriedoutinourlab,i.e.,asignificantdecreaseinethylene productionwasobservedwithover-expressionofPAbiosynthesis genesduringfruitripening(unpublishedresults).Thus,inresponse
tohigher availabilityof precursor(SAM), PA biosynthesis path-wayisactivatedcausinghigheraccumulationofSpdandSpm.The
Fig 3.Expression pattern of ethylene biosynthesis and related genes in tomato fruit during ripening (A) Semi-quantitative RT-PCR analysis of ACS transcript levels in WT and RNAi-ACS tomato lines at different stages of fruit ripening; (B) detection of ACS specific siRNAs in RNAi-ACS transformants by modified RNA hybridization; (C) semi-quantitative RT-PCR analysis for expression of ethylene biosynthesis and related genes, at BR and RR stages of fruit ripening ‘n’ denote no of cycles in semi-quantitative RT-PCR analysis.
Trang 6100
200
300
400
500
600
*†‡
*†‡ *†‡
*†‡
*†‡
*†‡
*† *†
*† *†
*†
*†
RNAi-ACS li nes
Free Con j Boun d
Fig 4.The levels of free, conjugated and bound PAs in WT and RNAi-ACS tomato
fruits Bars represent the means of three biological replicates and three independent
experiments, with standard error values as error bars *,†, ‡as Significant differences
in free, conjugated and bound PAs, respectively at P < 0.05 as compared to their
respective controls.
increaseinPutamountinfruitsofRNAi-ACSlinescanbedueto
theenhancedinter-conversionofexcesspoolsofhigherPAs,viz.,
Spd/SpmtoPutthroughacetylationmechanismbySpd/Spm
N1-acetyltransferase‘SSAT’(Seiler,2004;HazarikaandRajam,2011)
Ourdataindicatesthatinterferencewithethylenebiosynthesis
intomatofruitsofRNAi-ACSlineshadresultedintheaccumulation
ofPAsinsuchfruits,whichsupportsthecompetitiveinteraction
betweenethyleneandPAbiosyntheticpathways
DelayedripeningandenhancedshelflifeoffruitsinRNAi-ACS
linesoftomato
Reductioninethylenelevelshasledtosignificantreductionin
CO2 evolution(markerforrespiration)inRNAi-ACStomatolines
overcontrols(WTandUR).Amongthevariouslinesanalyzedfor
rateofrespiration,RNAi-ACS60 andRNAi-ACS81 werefoundto
exhibit∼50%reductioninrespiratoryactivityinharvestedfruits
overcontrolfruits,whilerestoftheRNAi-ACSlinesshowedupto
30%reductioninrateofrespiration(Table1).Thevariationin
respi-ratoryactivityofdifferentfruitscorrespondstothedifferentlevels
ofethyleneliberatedbysuchfruits.Theresultsareinaccordance
withtheobservationsmadebyDefilippietal.(2004)inappleand
Wangetal.(2010)intomato.Resultsonphysiologicallossofwater
(PLW)showedalmostsimilartrendsofreducedPLWpercentage
amongtheRNAi-ACStomatoesoverWTfruits(Table1).The
reduc-tionwasprominentwithupto40%reductionintherateofPLWin
RNAi-ACStomatoesovercontrols.Suchareductioninrespiratory
activity,PLWandthusslowermetabolicratehasdelayedripening
andextendedtheshelflifeofRNAi-ACStomatofruits
AsignificantdelayofonvineripeningforRNAi-ACStomatoes
wasrecordedascomparedtocontrolfruits.Onvineripeningperiod
(BRtoRR)wasdelayedfor∼45daysinRNAi-ACS60 and
RNAi-ACS81overcontrols(Table1).RRfruitsharvestedfromcontrols
andRNAi-ACSlinesshowedsignificantdifferenceintheirshelflife
understoragecondition(roomtemperature).Controlfruitskept
atroomtemperaturestartedrotting after8–10days ofharvest,
whilemostpromisingRNAi-ACSlinesshowedextendedshelflife
ofabout45daysbeyondnormalshelflifebeforedecaying(Table1;
Fig.6A).Guillénetal.(2007)havedemonstratedasimilar
corre-lationbetweendegreeofethyleneinhibitionandrateofripening
withdose-andtime-dependentapplicationof1-MCPintomato
Fig 5.Semi-quantitative RT-PCR analysis for expression of PA biosynthesis genes
in WT and RNAi-ACS81 and RNAi-ACS123 fruits ‘n’ represents no of cycles in semi-quantitative RT-PCR analysis.
Cell wall componentsincluding cellulose, hemicelluloseand pectin arethe majorcontributors for flesh firmnessand hence the shelf life During ripening, cell wall undergoes substan-tialdisassembly caused by increasedexpression of various cell walldegradingenzymeslikepolysaccharidehydrolases/glycoside hydrolase, transglycosylases, lyases and expansins (Brummell,
2006).EXP1,TBG4,PG and XTH5geneshave been shownto be specificallyexpressedduringfruitripeningandplaymajorrolein fruitsoftening(Pirrelloetal.,2009).Here,comparativeexpression analysisofgenesinvolvedincellwallhydrolysisindicated consid-erablereductionintranscriptsofEXP1,TBG4,PGandXTH5genes duringfruitripeninginRNAi-ACSlinesover WTfruits(Fig.6B) Thiscouldbeduetothereducedethylenelevelsinthese toma-toesasexpressionofthesegeneshasbeenreportedtobeethylene responsive(MaclachlanandBrady,1994;SmithandGross,2000; Zhaohuietal.,2009).InRNAi-ACSlines,theinhibitionofEXP1,TBG,
PGandXTHexpressionisconsistentwiththedelayoffruitripening andprolongedshelflifeoffruitsinfluencedbyreductionin ethyl-enelevels.Thus,declineinrespiratoryactivityandlowertranscript abundanceofcellwalldegradinggeneshasledtothedelayed ripen-ingandenhancedshelflifeinRNAi-ACStomatolines.Inaddition, increasedPAaccumulationinfruitsofRNAi-ACSlinesmayhavealso influencedtheenhancedshelflifepossiblybystabilizingthe mem-branes.PAshavebeenreportedasmodulatorofsupra-molecular conformationofpectin.EvidencesareavailablesupportingthatPA canboundcovalentlywithcellwallandinhibitionofPA biosyn-thesisinterfereswithcellwallformationmakingitamorphousand itsexogenousapplicationreversesthechanges(Bertaetal.,1997; Messiaenetal.,1997)
ImprovedfruitqualityinRNAi-ACStomatolines Totalsolublesolids(TSS)andtitratableacids(TAs)areofspecial significanceforprocessingindustry.TSSofaproducecomprisesof sugar,mineralandacidcontentsandreflectsitsspecificgravityor density.ItwashighlyencouragingtoobservethatRNAi-ACS toma-toesreleasingtracesofethylenerecordedveryhighlevelofTSS with∼40–45%increase(Table2).Althoughpreviousreports(Opiyo andYing,2005)havesuggestedTSStobeethyleneindependentbut ourresultsindicateacorrelationbetweenaccumulationofTSSand ethylenereduction,andtheunderlyingmechanismforthisstillto
beworkedout
Trang 7Table 1
Storage attributes of fruits from control and RNAi-ACS lines of tomato.
RNAi lines Respiration rate/CO 2 (nL/g fresh wt./h) PLW (%/day) On vine ripening (days) Shelf life (days)
RNAi-ACS60 4.12 ± 0.31 * 0.48 ± 0.09 * 56.66 ± 0.72 * 53.72 ± 0.93 *
Data is the mean ± standard error, based on at least three independent experiments with twenty replicates in each experiment.
* Significant at P < 0.05 between controls and RNAi lines.
RNAi-ACSfruitswerefoundtobear∼1.5–2.0foldincreasein
TAsover controls(Table2).Lower rateof respiration in
RNAi-ACS fruits explains higher accumulation of TAs in these fruits,
since organic acids (e.g citric acid) have been established as
substrates of respiration, and is an ethylene-dependent factor
(Defilippietal.,2004).ThehigherlevelsofTSSwithsimultaneous
increaseinTAsmightprovideacharacteristicflavorforRNAi-ACS
fruits
Inplantcells,ascorbicacid(AsA)iscontinuouslyoxidizedand
reducedwhereascorbicfreeradicalsanddehydroascorbate(DHA)
are the oxidation products, which can be reconverted back to
AsA.Ascorbatehasbeenestablishedasanimportantco-factorfor
invitroactivityofACO(Smithetal.,1992).Asageneraltrend,it
hasbeenseenthatAsAlevelsdeclineduringripeningand
senes-cence,andhasbeencorrelatedwithitsconsumptioninethylene
biosynthesispathway.Althoughinourcase comparablelevelof
AsAwasnotedinallRNAi-ACSlinesscreened(Table2 inspiteof
theblockageofethylenebiosynthesispathway.GLDHoxidizes
L-galactono-1,4-lactonetoAsA.DHAR1,DHAR2,MDHARandAOare
involvedinAsAoxidationandrecycling(Stevensetal.,2007).When
transcript profile of genes involved in AsA biosynthesis and recyclingpathwaywasanalyzed,GLDH1andAOgenesshowedan enhancedexpressionatBRstagewhiletheothergenes,viz.,DHAR1, DHAR2,MDHARwereunalteredintheirexpressionpattern(Fig.7) Theup-regulatedexpressionofbothbiosyntheticaswellas oxida-tiongenes,maintainsAsApoolsinRNAi-ACStomatofruitsandalso pointstowardtheirregulationbyethylene
DecreaseinlycopenecontentinRNAi-ACSlinesoftomato Lycopenecontentshowedsignificantreductioninitslevelsin
RRfruitsofRNAi-ACSlinesovercontrols,rangingfrom10to40% reduction (Table 3) Fruitsfrom RNAi-ACS lines – RNAi-ACS60, RNAi-ACS81andRNAi-ACS71.2,releasingtracesofethylene exhib-itedlightredcolorevenafter∼70daysoftheirharvest(atBRstage) Tomatoesfromtheselinesdisplayed∼40%reducedlycopene con-tentoverthecontrols.Expressionpatternoflycopenebiosynthesis gene,PSY1showedsignificantreductionintranscriptlevelinBRand
RRstagesoffruitripeningwhileDXS1andLESwhichisinvolvedin catabolismoflycopene,showednosignificantdifferencefromthat
Fig 6.Storage attributes of tomato fruits from controls and RNAi-ACS lines (A) Demonstration of extended shelf-life of RNAi-ACS tomatoes at room temperature after 70
Trang 8Table 2
Fruit quality traits in control and RNAi-ACS lines of tomato.
RNAi lines TA (g/100 g) AsA (mg/100 g) TSS ( ◦ BRIX)
Wild-type 0.42 ± 0.08 27.97 ± 0.84 5.62 ± 0.13
Unrelated control 0.39 ± 0.19 27.13 ± 0.76 5.44 ± 0.50
RNAi-ACS60 0.97 ± 0.13 * 26.35 ± 1.32 8.03 ± 0.19 *
RNAi-ACS71.2 0.94 ± 0.10 * 27.21 ± 1.32 7.06 ± 0.18 *
RNAi-ACS123 0.81 ± 0.13 * 30.26 ± 1.60 7.77 ± 0.19 *
RNAi-ACS128 0.73 ± 0.11 * 26.86 ± 1.04 6.98 ± 0.59 *
RNAi-ACS109 0.72 ± 0.10 * 25.88 ± 0.75 7.31 ± 0.63 *
RNAi-ACS124 0.73 ± 0.09 * 29.13 ± 0.86 7.08 ± 0.51 *
Data is the mean ± standard error, based on at least three independent experiments
with nine replicates in each experiment.
* Significant at P < 0.05 between controls and RNAi lines.
Fig 7.Expression profile analysis of ascorbic acid biosynthesis and recycling genes.
‘n’ represents no of cycles in semi-quantitative RT-PCR analysis.
ofWTintermsoftheirmRNAlevels(Fig.8).Reductioninlycopene
contentinfruitsofRNAi-ACSlinesovercontrolscanbeattributed
toreducedPSY1mRNAlevels,whichispossiblyamanifestationof
adropinethylenereleaseshownbytheseRNAi-ACSlines
Fig 8.Expression profile of lycopene metabolic genes ‘n’ represents no of cycles
Lycopene content in control and RNAi-ACS fruits.
RNAi lines Lycopene (mg/100 g)
Unrelated control 10.04 ± 1.34 RNAi-ACS1A 4.80 ± 0.13 *
Data is the mean ± standard error, based on at least three independent experiments with nine replicates in each experiment.
* Significant at P < 0.05 between controls and RNAi lines.
Acknowledgements
Thisworkwasgenerouslysupportedbyagrantfrom Depart-mentofBiotechnology(BT/PR8657/PBD/16/738/2007),NewDelhi SeniorResearchFellowshiptoAartiGuptabytheCouncilof Sci-entificandIndustrial Research,NewDelhiisacknowledged.We alsothankUniversityGrantsCommissionforSpecialAssistant Pro-gramandDepartmentofScienceandTechnology,NewDelhifor FISTprogram
Appendix A Supplementary data
Supplementary data associated with this article can be found, intheonlineversion,at http://dx.doi.org/10.1016/j.jplph 2013.02.003
References
Adams DO, Yang SF Ethylene biosynthesis: Identification of 1-aminocyclopropane-1-carboxylic acid as an intermediate in the conversion of methionine to ethylene Proc Natl Acad Sci USA 1979;76:170–4.
AOAC.Gaithersburg MD, Artés F, Marín JG, Martínez J, editors Official methods of analysis 17th ed University of Wisconsin, Madison: Association of Official Ana-lytical Chemists; 2000.
Apelbaum A, Burgoon AC, Anderson JD, Lieberman M, Ben-Arie R, Mattoo AK Polyamines inhibit biosynthesis of ethylene in higher plant tissue and fruit protoplasts Plant Physiol 1981;68:453–6.
Bajaj S, Rajam MV Polyamine accumulation and near loss of morphogenesis in long term Callus cultures of rice: restoration of plant regeneration by manipulation
of cellular polyamine levels Plant Physiol 1996;112:1343–8.
Barry CS, Blume B, Bouzayen M, Cooper W, Hamilton AJ, Grierson D Differential expression of the 1-aminocyclopropane-1-carboxylate oxidase gene family of tomato Plant J 1996;9:525–35.
Barry CS, Llop-Tous MI, Grierson D The regulation of 1-aminocyclopropane-1-carboxylic acid synthase gene expression during the transition from system-1
to system-2 ethylene synthesis in tomato Plant Physiol 2000;123:979–86 Berta G, Altamura MM, Fuskoni A, Ceruti F, Capitani F, Bagni N The plant cell wall is altered by inhibition of polyamine biosynthesis utilizing the principle of protein dye binding Anal Biochem 1997;72:248–50.
Bregoli AM, Scaramagli S, Costa G, Sabatini E, Ziosi V, Biondi S, et al Peach (Prunus per-sica) fruit ripening: aminoethoxyvinylglycine (AVG) and exogenous polyamines affect ethylene emission and flesh firmness Physiol Plant 2002;114:472–81 Brummell DA Cell wall disassembly in ripening fruit Funct Plant Biol 2006;33:103–19.
Cara B, Giovannoni JJ Molecular biology of ethylene during tomato fruit develop-ment and maturation Plant Sci 2008;175:106–13.
Trang 9Defilippi BG, Dandekar AM, Kader AA Impact of suppression of ethylene action or
biosynthesis on flavour metabolites in apple (Malus domestica Borkh) fruits J
Agric Food Chem 2004;52:5694–701.
Deikman J, Xu RL, Kneissl ML, Ciardi JA, Kim KN, Pelah D Separation of cis elements
responsive to ethylene, fruit development, and ripening in the 5-flanking region
of the ripening-related E8 gene Plant Mol Biol 1998;37:1001–11.
Doyle JJ, Doyle JL Isolation of plant DNA from fresh tissue Focus 1990;12:13–5.
Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC Potent and specific
genetic interference by double stranded RNA in Caenorhabditis elegans Nature
1998;391:806–11.
Giovannoni JJ Molecular biology of fruit maturation and ripening Annu Rev Plant
Physiol Plant Mol Biol 2001;52:725–49.
Guillén F, Castillo S, Zapata PJ, Martínez-Romero D, Serrano M, Valero D Efficacy
of 1-MCP treatment in tomato fruit 1 Duration and concentration of 1-MCP
treatment to gain an effective delay of postharvest ripening Postharvest Biol
Technol 2007;43:23–7.
Hamilton AJ, Lycett GW, Grierson D Antisense gene that inhibits synthesis of the
hormone ethylene in transgenic plants Nature 1990;346:284–7.
Handa AK, Nambeesan S, Mengiste T, Laluk K, Abuqamar S, Mattoo AK Polyamine
spermidine is an upstream negator of ethylene-regulated pathogenesis of
Botry-tis cinerea in tomato leaf Acta Hortic 2011;914:109–12.
Hazarika P, Rajam MV Biotic and abiotic stress tolerance in transgenic tomatoes by
constitutive expression of S-adenosylmethionine decarboxylase gene Physiol
Mol Biol Plants 2011;17:115–28.
Kerschen A, Napoli CA, Jorgensen RA, Müller AE Effectiveness of RNA interference
in transgenic plants FEBS Lett 2004;566:223–8.
Kohli A, Twyman RM, Abranches R, Wegel E, Stoger E, Christou P Transgene
integration, organization and interaction in plants Plant Mol Biol 2003;52:
247–58.
Lin BY, Li M, Lin DQ, Jin ZQ Cloning and functional analysis on promoter of
tomato fruit-specific 2A11 from Lycopersicon esculentum Chin Agric Sci Bull
2006;22:62–6.
Lincoln JE, Campbell AD, Oetiker J, Rottmann WH, Oeller PW, Shen NF,
et al Le-ACS4, a fruit ripening and wound-induced
1-aminocyclopropane-1-carboxylate synthase gene of tomato (Lycopersicon esculentum) J Biol Chem
1993;268:19422–30.
Lu C, Meyers BC, Green PJ Construction of small RNA cDNA libraries for deep
sequencing Methods 2007;43:110–7.
Maclachlan G, Brady C Endo-1,4--glucanase, xyloglucanase and xyloglucan
endo-transglycosylase activities versus potential substrates in ripening tomatoes.
Plant Physiol 1994;105:965–74.
Madhulatha P, Pandey R, Hazarika P, Rajam MV High transformation
fre-quency in Agrobacterium mediated genetic transformation of tomato by using
polyamines and maltose in shoot regeneration medium Physiol Mol Biol Plants
2007;13:191–8.
Messiaen J, Cambier P, Van Cutsem P Polyamines and pectins I Ion exchange and
selectivity Plant Physiol 1997;113:387–95.
Nakatsuka A, Murachi S, Okunishi H, Shiomi S, Nakano R, Kubo Y, et al Differential
expression and internal feedback regulation of
1-aminocyclopropane-1-carboxylate synthase, 1-aminocyclopropane-1-carboxylate oxidase and
ethyl-ene receptor genes in tomato fruit during development and ripening Plant
Physiol 1998;118:1295–305.
Nambeesan S, Datsenka T, Ferruzzi MG, Malladi A, Mattoo AK, Handa AK
Over-expression of yeast spermidine synthase impacts ripening, senescence and
decay symptoms in tomato Plant J 2010;63:836–47.
Oeller PW, Lu MW, Taylor LP, Pike DA, Theologis A Reversible inhibition of tomato
fruit senescence by antisense RNA Science 1991;254:437–9.
Opiyo AM, Ying TJ The effects of 1-methylcyclopropene treatment on the shelf life
and quality of cherry tomato (Lycopersicon esculentum var cerasiforme) fruit.
Intern J Food Sci Technol 2005;40:665–73.
Osorio S, Alba R, Damasceno CMB, Lopez-Casado G, Lohse M, Zanor MI, et al
Sys-tems biology of tomato fruit development: combined transcript, protein, and
metabolite analysis of tomato transcription factor (nor, rin) and ethylene receptor (Nr) mutants reveals novel regulatory interactions Plant Physiol 2011;157:405–25.
Pear JR, Ridge N, Rasmussen R, Rose RE, Houck CM Isolation and characterization of
a fruit-specific cDNA and the corresponding clone from tomato Plant Mol Biol 1989;13:639–51.
Pirrello J, Regad F, Latché A, Pech JC, Bouzayen M Regulation of tomato fruit ripen-ing In: Perspectives in agriculture, veterinary science, nutrition and natural resources CAB Reviews 2009;4:1–14.
Rajam MV, Madhulatha P, Pandey R, Hazarika PJ, Razdan MK Applications of genetic engineering in tomato In: Razdan MK, Mattoo AK, editors Genetic improve-ment of Solanaceae crops: tomato, vol 2 Enfield: Science Publishers; 2007 p 285–311.
Rottmann WH, Peter GF, Oeller PW, Keller JA, Shen NF, Nagy BP, et al 1-Aminocyclopropane-1-carboxylate synthase in tomato is encoded by a multi-gene family whose transcription is induced during fruit and floral senescence J Mol Biol 1991;222:937–62.
Sambrook J, Fritsch EF, Maniatis T Molecular cloning: a laboratory manual 2nd ed Cold Spring Harbour, New York: Cold Spring Harbor Laboratory Press; 1989 Seiler N Catabolism of polyamines Amino Acids 2004;26:217–33.
Singh SP, Pal RK Controlled atmosphere storage of guava (Psidium guajava L.) fruit Postharvest Biol Technol 2008;47:296–306.
Smith DL, Gross KC A family of at least seven -galactosidase genes is expressed during tomato fruit development Plant Physiol 2000;123:1173–83.
Smith JJ, Ververidis P, John P Characterization of the ethylene-forming enzyme partially purified from melon Phytochemistry 1992;31:1485–94.
Stevens R, Buret M, Duffé P, Garchery C, Baldet P, Rothan C, et al Candidate genes and quantitative trait loci affecting fruit ascorbic acid content in three tomato populations Plant Physiol 2007;143:1943–53.
Theologis A, Oeller PW, Wong L-M, Rottmann WH, Gantz DM Use of a tomato mutant constructed with reverse genetics to study fruit ripening, a complex developmental process Dev Genet 1993;14:282–95.
Tiburcio AF, Kaur-Sawhney R, Galston AW Polyamine metabolism In: Miflin BJ, Lea
PJ, editors Intermedatory nitrogen metabolism 16 The biochemistry of plants San Diego: Academic Press; 1990 p 283–325.
Vrebalov J, Pan IL, Arroyo AJ, McQuinn R, Chung M, Poole M, et al Fleshy fruit expan-sion and ripening are regulated by the tomato SHATTERPROOF gene TAGL1 Plant Cell 2009;21:3041–62.
Wang M, Cao J, Sun LLJ, Jiang W Effect of 1-methylcyclopropene on nutritional quality and antioxidant activity of tomato fruit (Solanum lycopersicon L.) during storage J Food Qual 2010;33:150–64.
Wilkinson JQ, Lanahan MB, Clark DG, Bleecker AB, Chang C, Meyerowitz EM, et al.
A dominant mutant receptor from Arabidopsis confers ethylene insensitivity in heterologous plants Nat Biotechnol 1997;15:444–7.
Xiong AS, Yao QH, Li X, Fan HQ, Peng RH Double antisense ACC oxidase and ACC synthase fusion gene introduced into tomato by agrobacterium-mediated trans-formation and analysis the ethylene production of transgenic plants Acta Biol Exp Sin 2003;36:35–41.
Xiong AS, Yao QH, Peng RH, Li X, Han PL, Fan HQ Different effects on ACC oxidase gene silencing triggered by RNA interference in transgenic tomato Plant Cell Rep 2005;23:639–46.
Yang SF, Hoffman NE Ethylene biosynthesis and its regulation in higher plants Annu Rev Plant Physiol 1984;35:155–89.
Ye ZB, Li HX, Zheng YL, Liu HL Inhibition of introducing antisense ACC oxidase gene into tomato genome on expression of its endogeous gene J Huazhong Agric Univ 1996;15:305–9.
Yokotani N, Nakano R, Imanishi S, Nagata M, Inaba A, Kubo Y Ripening-associated ethylene biosynthesis in tomato fruit is autocatalytically and developmentally regulated J Exp Bot 2009;60:3433–42.
Zhaohui X, Xiaohong K, Yunbo L, Benzhong Z, Wentao X Effect of ethylene on poly-galacturonase, lipoxygenase and expansin in ripening of tomato fruits Trans Tianjin Univ 2009;15:173–7.