Overexpression of SlGRAS40 in Tomato Enhances Tolerance to Abiotic Stresses and Influences

17 Pages·2017·5.36 MB·English

by  Zhengguo Li

ORIGINALRESEARCH
published:26September2017
doi:10.3389/fpls.2017.01659
Overexpression of SlGRAS40 in
Tomato Enhances Tolerance to
Abiotic Stresses and Influences
Auxin and Gibberellin Signaling
YudongLiu,WeiHuang,ZhiqiangXian,NanHu,DongboLin,HuaRen,JingxuanChen,
DedingSuandZhengguoLi*
SchoolofLifeSciences,ChongqingUniversity,Chongqing,China
Abiotic stresses are major environmental factors that inhibit plant growth and
development impacting crop productivity. GRAS transcription factors play critical and
diverserolesinplantdevelopmentandabioticstress.Inthisstudy,SlGRAS40,amember
of the tomato (Solanum lycopersicum) GRAS family, was functionally characterized.
In wild-type (WT) tomato, SlGRAS40 was upregulated by abiotic stress induced by
treatment with D-mannitol, NaCl, or H O . Transgenic tomato plants overexpressing
2 2
SlGRAS40 (SlGRAS40-OE) were more tolerant of drought and salt stress than WT.
SlGRAS40-OE plants displayed pleiotropic phenotypes reminiscent of those resulting
Editedby:
Lam-SonTran, fromalteredauxinand/orgibberellinsignaling.AcomparisonofWTandSlGRAS40-OE
RIKEN,Japan transcriptomes showed that the expression of a large number of genes involved in
Reviewedby: hormonesignalingandstressresponsesweremodified.OurstudyofSlGRAS40protein
PuneetPaul,
provides evidence of how another GRAS plays roles in resisting abiotic stress and
UniversityofNebraskaLincoln,
UnitedStates regulating auxin and gibberellin signaling during vegetative and reproductive growth in
JunYan,
tomato.
PurdueUniversity,UnitedStates
*Correspondence: Keywords: abiotic stresses, auxin, gibberellin, GRAS transcription factor, SlGRAS40, tomato (Solanum
ZhengguoLi lycopersicum)
[email protected]
INTRODUCTION
Specialtysection:
Thisarticlewassubmittedto
PlantAbioticStress, Tomato(Solanumlycopersicum)issusceptibletoawiderangeofenvironmentalstresses.Drought
asectionofthejournal andhighsalinityaremajorstressfactorsleadingtodetrimentaleffects,suchasinhibitionofseed
FrontiersinPlantScience germinationandplantgrowthanddecreasedfruitproductivity.Underdroughtandsaltstress,a
Received:25June2017 series of stress related genes are induced in plants, largely regulated by a range of transcription
Accepted:11September2017 factors(HirayamaandShinozaki,2010).Todate,transcriptionfactorsfromvariousplantspecies
Published:26September2017 havebeenreportedtobeinvolvedinstressresponses(Singhetal.,2002),suchasNAC,Dof,ERF,
Citation: WRKY,bZIP,andMYC/MYB.
LiuY,HuangW,XianZ,HuN,LinD, MountingevidenceshowsthatGRAS(namedafterGAI,RGA,SCR)genesarehighlyinducible
RenH,ChenJ,SuDandLiZ(2017) by different abiotic stresses (Czikkel and Maxwell, 2007; Lee et al., 2008). GRAS family proteins
OverexpressionofSlGRAS40in are plant-specific transcription factors that play critical roles in plant development and signal
TomatoEnhancesTolerancetoAbiotic
transductionpathways,includinggametogenesis(Morohashietal.,2003),phytochromesignaling
StressesandInfluencesAuxinand
(Torres-Galea et al., 2006), lateral shoot formation (Li et al., 2003), gibberellin biosynthesis and
GibberellinSignaling.
Front.PlantSci.8:1659. signaling(Silverstoneetal.,1998;Leeetal.,2002),andauxinsignaling(Gaoetal.,2004;Sanchez
doi:10.3389/fpls.2017.01659 etal.,2007).However,someexperimentalevidencepointstocertainGRASproteinshavingroles
FrontiersinPlantScience|www.frontiersin.org 1 September2017|Volume8|Article1659
Liuetal. SlGRAS40-OEEnhancesTomatoAbioticResistance
in resisting biotic or abiotic stress. Tomato SlGRAS6 silenced gene expression analysis the following samples were collected
plants showed increased resistance to disease (Mayrose et al., fromatleastsixplants:roots,stems,andleavesfrom1-month-
2006), overexpression of OsGRAS23 improved drought and old WT plants; buds, flowers, petals, sepals, and stamens at
oxidative stress tolerance in rice (Kai et al., 2015), and ectopic anthesis; ovaries at −2, 0, and 4 dpa; and fruits at 9 dpa, 20
expression of Populus PeSCL7 gene in Arabidopsis improved dpa, mature green, breaker, breaker plus 4 day, and breaker
drought and salt tolerance (Ma et al., 2010). Overexpression of plus7daystages.Samplesfromdifferentplantsweremixedand
VaPAT1, a GRAS transcription factor from Vitis amurensis, in immediatelyfrozeninliquidnitrogen.
transgenicArabidopsisconferredcold,droughtandhighsalinity
Plasmid Construction and Genetic
tolerance(Yuanetal.,2016).IdentifyingtherolesofotherGRAS
genesincrops,liketomato,willhelpelucidatethemechanisms Transformation
regulatingstresstoleranceandpotentiallyfacilitatethebreeding The open reading frame of SlGRAS40 without the stop codon
ofresistantvarieties. wasamplifiedfromthefulllengthtomatoleafcDNAandcloned
Inplanta,auxinandgibberellin(GA)areinextricablylinked into the expression vector under the CaMV 35S promoter.
with both developmental processes and abiotic stress responses The expression vector was transformed into Agrobacterium
through the action of GRAS proteins from the SCARECROW- tumefaciens strain GV3101, which was used to transform WT
like (SCL) and DELLA groups. AtSCL3 acts as an integrator of tomatoplantsfollowingstandardmethods(Fillattietal.,1987).
DELLAsandSHR-SCR,acomplexinwhichtwotypesofGRAS
interact, to mediate GA-promoted cell elongation in the root Gene Expression Analysis
endodermis (Heo et al., 2011; Zhang Z. L. et al., 2011). The Total RNA was extracted using an RNAeasy kit (QIAGEN,
DELLAmutantsAtGAI (Pengetal.,1997),AtRGA(Silverstone Germany) and first-strand cDNA was synthesized with
etal.,1998),andAtRGL1-3(Leeetal.,2002)allexhibitgibberellin PrimeScriptTM RT reagent Kit with gDNA Eraser (Perfect
insensitivity. It has been shown that decreased GA content Real Time) (TAKARA, Japan). Quantitative real-time PCR
enhances tolerance to drought, whereas increased GA content was performed according to the instructions provided for the
reduces it (Colebrook et al., 2014). DELLA proteins, the core Bio-Rad CFX system (Bio-Rad, USA), using SYBR(cid:13)R Premix
componentsofGAsignaling,maythereforerestraingrowthand Ex TaqTM (Tli RNaseH Plus) (TAKARA, Japan). Relative fold
enhancestresstolerancethroughacommonmechanism(Achard differences were calculated by 2−11Ct method based on the
et al., 2008). PrSCL1 (Pinus radiata SCL1) in pine and CsSCL1 comparative Ct method. For each sample, three independent
(Castanea sativa SCL1) in chestnut regulate adventitious root biological replicates were used (each with three technical
formation by regulating auxin signaling (Sanchez et al., 2007). replicates).AndalltheprimersusedforqRT-PCRwerelistedin
The SHR-SCR complex combined with auxin influx carriers SupplementaryTableS1.
LAX3andAUX1hasasynergeticeffectonprimary/lateralroot
developmentinArabidopsis(Dellaetal.,2015).Auxindirectlyor Histological Analysis
indirectly modulates the expression of several stress-responsive Stemsof1-month-oldWTandOEL3plantswerecutandsoaked
genes,andseveralauxin-responsivegenesareregulatedbyabiotic inFAAsolution(50%(v/v)ethanol,5%(v/v)glacialaceticacid,
stresses (Jain and Khurana, 2009). Besides, auxin can modulate 5% (v/v) methanal) for 24 h. Samples were dehydrated in an
ROS homeostasis indirectly by affecting the stability of DELLA ethanol gradient then embedded in paraffin. Sections were cut
proteins or directly by inducing ROS detoxification enzymes, and stained with 0.05% toluidine blue. An Olympus BX-URA2
suggesting thatauxinandGAmightcooperatewitheachother (Japan)microscopywasusedforobservations.
inresponsetostressconditions(FuandHarberd,2003;Paponov
etal.,2008). Abiotic Stress Treatments
Thereare53membersoftheGRASfamilyintomato(Huang Leaves of 1-month-old WT plants were sprayed with 200 mM
et al., 2015). In our previous study, we demonstrated that NaCl, 100 mM D-mannitol or 100 mM hydrogen peroxide for
SlGRAS24 participates in a series of developmental processes salt, osmotic, and oxidative treatments, respectively. Control
in tomato by modulating auxin and gibberellin crosstalk plants were untreated. The leaves were harvested after 1, 3, 6,
(Huang et al., 2017). Here, we found that overexpression of 12,and24h.Foreachsample,leavesfromsixplantsweremixed
SlGRAS40 (Solyc08g078800) conferred pleiotropic phenotypes andalltreatmentswereperformedthreeindependenttimes.All
andenhancedsaltanddroughttolerance.Byanalyzinghormone sampleswerefrozeninliquidnitrogenandstoredat−80◦C.
responsivenessandgeneexpressionweshowthatalteredauxin For drought tolerance assay, every plant was planted in an
and gibberellin signaling are likely to be responsible for the independentsquarebasin(samebasinswereused),each12plants
defectivegrowthofSlGRAS40-OE. ofWTandSlGRAS40-OEL2,L3,L4wereplacedinabigpot,and
wateredintothebigpottwiceaweektomakesurethesoilwater
MATERIALS AND METHODS ineverybasinwasuniform,andalltheplantsweregrownunder
some illumination and temperature conditions. After 1 month,
Plant Materials and Growth Conditions WTandSlGRAS40-OEplantsinthebigpotwereaveragedivided
Tomato plants (Solanum lycopersicum cv. Micro-Tom) were intotwogroups,onegroupascontrolwaswaterednormally,and
growninthegreenhouseincontrolledconditionswith18hlight theothergroupasdroughttreatmentwasdeprivedofwaterfor
(25◦C)/6 h dark (18◦C) cycles and 60% relative humidity. For upto17days.Similarly,thegroupassalttreatmentwaswatered
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Liuetal. SlGRAS40-OEEnhancesTomatoAbioticResistance
with200mMNaClevery48h(200mlperplant)forupto23days. werewashedinwaterthreetimes,andstainedwith0.05%aniline
Duringthetreatmentprocess,alltheplantsineachgroupswere blue for 4 h in the dark. An Olympus BX-URA2 microscope
grownundersameilluminationandtemperatureconditions,and (Japan)withultravioletlightexcitationwasusedforobservations
threeindependenttreatmentswereperformed.Totalchlorophyll ofpollentubes.
contentandrelativewatercontent(RWC)(Panetal.,2012)were
Hormone Treatment for Plant Development
measuredduringtreatment.Afterdroughtorsalttreatment,leaf
samplesatthesamedevelopmentalstagewereharvested,frozen Analysis
inliquidnitrogenimmediately,andstoredat−80◦C. Ten-day-old WT and SlGRAS40-OE L3 plants were sprayed
Forosmoticandsalttoleranceexperiments,seedsofWTand with 20 µM GA every 3 days for 4 weeks. Control plants
3
SlGRAS40-OEL2,L3,andL4weresterilizedandsownon½× were sprayed with water. Plant height was measured every
MSaloneor½×MScontaining150mMD-mannitolor75mM weekandfloweringtimewasrecorded.Theauxindose-response
NaCl(Huangetal.,2016).Seedswereincubatedinacontrolled experimentwasperformedwith8-mmlonghypocotylsegments
growth chamberwith18hlight(25◦C)/6hdark(18◦C)cycles. excisedjustbelowthecotyledonnodesfrom7-day-oldWTand
Thegerminationratewascountedafter7days,andthelengthsof SlGRAS40-OE L3 seedlings. Hypocotyl segments were floated
primaryrootsandhypocotylsweremeasuredafter2weeks. onto sucrose/MES buffer [1% (w/v) sucrose, 5 mM MES/KOH,
pH6.0] and pre-incubated for 1–2 h. The hypocotyl segments
Stoma Morphology and Stomatal werethenrandomlydistributedtofreshbuffersolutionswithor
Conductance Assay withoutNAAandmeasuredafter23hofincubationwithgentle
Animprintmethodwasusedfortheassay.Colorlessnailpolish agitationatroomtemperature(Wangetal.,2005).
wasappliedtotheundersideofleavesofWTandOEL3plants. TheseedsofWTandSlGRAS40-OEL2andL3weresterilized
After5min,thenailpolishwascarefullytornoffandtheimprint and soaked in sterilized water with gentle agitation at room
viewed with an Olympus BX-URA2 (Japan) microscope. The temperature, then seeds were transferred to ½ × MS medium
lengthandwidthofstomaweremeasuredwithImageJsoftware. with 1 µM IAA and/or 50 µM GA3, or on ½ × MS medium
Stomatalconductancewasestimatedasthestomalength/width. without hormone for controls. The seedlings were grown in a
growthchamberwithcontrolledconditions,18hlight(25◦C)/6
Analysis of Antioxidant Enzyme Activities hdark(18◦C)cycles.After15days,thenumberoflateralroots,
and MDA, Proline, and Soluble Sugar andthelengthsofprimaryrootsandhypocotylsweremeasured.
Everytreatmentwasdoneonatleast10plantsor30seedsandall
Content
thetreatmentswereperformedthreeindependenttimes.
After drought and salt treatment, leaves at the same
developmental stage were harvested for measuring enzyme Hormone Treatment for Gene Expression
activities and Malondialdehyde (MDA), proline and soluble Analysis
sugar content. MDA content was measured according to the
Fifteen-day-old WT seedlings were soaked in liquid ½ × MS
method by Heath and Packer (1968), proline content with the
mediumcontaining20µMIAAor20µMGA for0,1,3,6,12,
3
method described by Bates et al. (1973), and soluble sugar
or24h,thenwholeseedlingswerefrozeninliquidnitrogenand
content by the method described by Fukao et al. (2006). The storedat−80◦C.
activity of superoxide dismutase (SOD), catalase (CAT) and
Fifteen-day-oldWTandOEL3seedlingsweresoakedinliquid
peroxidase (POD) were determined according to the methods
½×MSmediumcontaining20µMIAAor20µMGA for3h,
3
described by Mittova et al. (2000) and by Morohashi (Mittova
then whole seedlings were frozen in liquid nitrogen and stored
etal.,2000;Morohashi,2002).H2O2contentandthesuperoxide at −80◦C. Seedlings were soaked in liquid ½ × MS medium
anion radical (O−) content were determined according to
2 without hormones as controls. Each treatment was performed
the instructions provided in kits available from Jiancheng
threeindependenttimes.
BioengineeringCompany(Nanjing,China).
GA (2000 ng per ovary, Sigma, USA) or 2,4-D (200 ng per
3
ovary, Sigma, USA) was applied to emasculated ovaries on the
Pollen Germination and Pollen Tube
day equivalent to anthesis (0 dpa) in 10 µl of 5% ethanol and
Growth Assays
0.1%Tween80(Sigma,USA)solution.Ovariesweretreatedwith
PollenfromWTandOEL3plantsatfloweringtimewastested thesamevolumeofsolutionwithouthormonesascontrol.Eight
in pollen germination assay. A 30 µl drop of medium (20 ovaries (from four plants) were used per treatment, and each
mM MES buffer, pH6.0, 2% sucrose, 15% PEG4000, 1 mM treatmentwasperformedthreeindependenttimes.Thesamples
KNO3,3mMCa(NO3)2·4H2O,0.8mMMgSO4·7H2O,1.6mM werefrozeninliquidnitrogenandstoredat−80◦CbeforeRNA
H3BO3) was placed on a glass slide in a moist petri dish. A extraction and gene expression analysis. Flower emasculation
flowerundergoinganthesiswasshakentoletpollenfallontothe was carried out 2 days before anthesis (−2 dpa) to prevent
medium.Thepetridishwassealedandkeptinthedarkat25◦C self-pollination(Serranietal.,2007a).
for5–6h.ObservationsweremadewithanOlympusBX-URA2
microscope(Japan).Forpollentubegrowthassays,1-dpaovaries RNA Sequencing
were soaked in buffer (95% ethanol-glacial acetic acid, v/v, 3:1) Total RNA (RNeasy Plant Mini Kit, Qiagen, USA) was
for 24 h, then transferred to 8 M NaOH for 2 days. Samples extracted from shoot apical meristems from 1-month-old
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Liuetal. SlGRAS40-OEEnhancesTomatoAbioticResistance
WT and SlGRAS40-OE L3 plants, two individuals per RESULTS
sample. A cDNA library was constructed for sequencing
Phenotypic Characterization of Transgenic
on the IlluminaHiSeq2000TM system (BGI Inc.). Bowtie2
Plants Overexpressing SlGRAS40
was used to map clean reads to the reference genome of S.
lycopersicum in the Tomato Sol Genomic Network database WegeneratedtransgenictomatoplantswiththeSlGRAS40cDNA
(http://solgenomics.net/), and the homogenized data used underthecontroloftheCaMV35SpromoterbyAgrobacterium
to calculate gene expression levels with RSEM. Differentially tumefaciens-mediated transformation. Three independent lines
expressed genes (DEGs) were detected with NOIseq with the L2, L3, L4 were obtained overexpressing the gene 8.6-fold,
following parameters: fold change ≥ 2.00 and probability ≥ 30.5-fold,and11.7-fold,respectively(Figures1A,C).SlGRAS40
0.8. Gene ontology (GO) functional enrichment and Kyoto overexpressingplantsexhibitedpleiotropicphenotypesincluding
EncyclopediaofGenesandGenomes(KEGG)pathwayanalysis dwarfism(Figure1B),delayedfloweringtime,decreasedfruit-set
wereperformedbetweenWTandSlGRAS40-OEsamples. ratio, and arrested fruit and seed development. More details of
thephenotypesareshowninSupplementaryTableS2.SlGRAS40
was expressed in all WT tissues tested, and expressed at much
Statistical Analysis higherlevelsinleavesandflowersundergoinganthesiscompared
All the experiments included three independent repeats, and toroots(Figure1D).
significantdifferencesweredeterminedbytheStudent’st-testat Thesixthnodestemsof1-month-oldWTandSlGRAS40-OE
significancelevelsofP<0.05(∗)andP<0.01(∗∗). L3 plants were used for histological analysis (Figure1E). In
FIGURE1|Phenotypiccharacterizationofwild-typeandtransgenicplants.(A)One-month-oldplantsofWTandSlGRAS40-overexpressinglines.L2,L3,L4,three
independentSlGRAS40-overexpressinglines.(B)Heightofplantsshownin(A).Errorbarsshowthestandarderrorbetweenthreebiologicalreplicates(n=3)with
morethan10plantsforeachreplicateperformed.(C)ExpressionlevelsofSlGRAS40inplantsofWTandSlGRAS40-OElines.Expressiondatawerenormalizedwith
SlGRAS40expressioninWTas1.Errorbarsshowthestandarderrorbetweenthreebiologicalreplicates(n=3).(D)TissueprofilinganalysisofSlGRAS40indifferent
organsofwild-typetomato.ExpressiondatawerenormalizedwithSlGRAS40expressioninrootsetas1.(E)Histologicalanalysisof1-month-oldstemsfromWT(i,iii)
andSlGRAS40-OEL3(ii,iv)showninlongitudinalsection(i,ii)andtransversesection(iii,iv).(F)Analysisofhistologicaldatafrom(E).(i)and(ii)aredatafrom
longitudinalsections;(iii)and(iv)aredatafromtransversesections.Alldataaremeasurementsunder40×microscopicfield.Asterisksindicatesignificantdifferences
usingStudent’st-test(*P<0.05,**P<0.01).
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Liuetal. SlGRAS40-OEEnhancesTomatoAbioticResistance
longitudinalsections,therewerefewercelllayersandfewercells soluble sugar in WT plants after salt treatment, whereas OE
in OE plants compared with WT, and the average area of OE plants accumulated more soluble sugar after drought and salt
cellswasbiggerthanWTcells.Intransversesection,OEplants stress.
had fewer and smaller cells compared to WT (Figure1F). The Theactivitiesofthreeantioxidantenzymes,POD,SOD,and
differenceincellularmakeupexplainswhySlGRAS40-OEstems CAT, were measured in this study. There were no significant
arethinnerandshorterthanWTstems. differencesineitherPODorSODactivitiesinWTandOEplants
under normal conditions. Under drought stress both POD and
Overexpression of SlGRAS40 Enhances SOD activities were up-regulated in OE plants, but not in WT
plants.Bycomparison,thesetwoactivitiesweresignificantlyup-
Tolerance to Drought and Salt Stress
regulated in WT plants after salt treatment (Figure4A). Salt-
WT plants were treated with D-mannitol, NaCl, or H O
2 2 stressedOEhadlowerlevelsofPODandSODthanWTinthe
to produce the effects of osmotic, saline or oxidative stress,
same conditions. CAT activity was higher in OE than in WT
respectively.SlGRAS40wasup-regulatedinresponseto100mM
plantsunderbothcontrolanddroughtstressconditions,butafter
D-mannitol and to 100 mM H O , but in response to 200
2 2 salttreatmenttheCATactivitywasup-regulatedinWTbutnot
mMNaClSlGRAS40wasfirstdown-regulatedthenup-regulated
in OE (Figure4A). These data indicate that overexpression of
(Figure2A).SlGRAS40maythereforebeinvolvedinabioticand
SlGRAS40 can enhance ROS scavenging ability under salt and
oxidativestressresponsesintomato.
droughtstress.
To evaluate the role of SlGRAS40 in tolerance to drought
andsalt,SlGRAS40-OEL2,L3,L4andWTplantsweredeprived
of water for up to 17 days as a drought stress treatment, or Expression Analysis of Stress-Related
watered with 200 mM NaCl solution every 48 h for up to Genes in SlGRAS40-OE and WT Plants
23 days as a salt stress treatment. Under saline or drought
under Drought and Salt Stress
stresses,allOEplantsgrewbetterthanWT(Figure2B).Under
ToinvestigatethemolecularmechanismsunderlyingSlGRAS40-
drought stress, desiccation symptoms, such as wilting of lower
enhanced tolerance to drought and salt stress, expression of
leaveswerereadilyobservedinWT,whereasOEplantsexhibited
plant stress response biomarkers was checked by quantitative
only slight damage (Figure2C). Under salt stress, WT plants
reverse transcription (qRT)-PCR (Figure4B). The transcript
mostlyexhibitedincreasedchlorosisandnecrosisafter23days,
levels of several genes involved in ROS generation and
but there was no obvious damage in OE plants (Figure2C).
scavenging, including ascorbate peroxidase (APX), CAT, SOD,
Relativewatercontent(RWC)andtotalchlorophyllcontentboth
POD,lipoxygenase(LOX),andglutathioneS-transferase(GST),
declinedinWTandOEplantsduringstresstreatments,butlevels
weremeasuredinWTandtransgenicplantsunderbothnormal
were much higher in OE than in WT (Figures2D–G). After
and stress conditions. The transcript abundance of all these
drought stress, the stomatal conductance of WT had decreased
genes,exceptSlGST,decreasedinWTandOEplantsafterstress
(Figures3A,B),indicatingthatstomatalporeswerewideropen.
treatment,buttheexpressionlevelsinOEplantswerehigherthan
The effect would be to increase transpiration leading to lower
thoseinWTinbothcontrolandstressconditions(Figure4B).
RWC (Figure2D). On the contrary, after drought stress the
SlGST wasup-regulatedinWTafterdroughtorsaltstress,and
stomatalconductanceofSlGRAS40-OEL3leavesincreasedand
up-regulated in OE plants after salt stress, but after drought
was significantly higher than that of WT leaves (Figure3B).
stress SlGST expression level was lower in OE than in WT.
This indicated that under drought stress the stomatal pores of
A key proline synthetase gene SlP5CS was up-regulated after
SlGRAS40-OEleaveswerenarrowedsomorewaterwasretained
stresstreatmentinWTplants,whileOEplantshadhigherlevels
(Figure2D).
than WT under control and salt conditions (Figure4B). An
ascorbicacidsynthetase(SlGME2),anethylene-responsiveLEA
Antioxidant Status of SlGRAS40-OE Plants
protein (SlER5), an ethylene-responsive factor (SlERF1) and a
under Drought and Salt Stress heatshockprotein(SlHsp90-1)allhavehigherexpressionlevels
Abiotic stresses induce oxidative stress in plants which leads to inOEplantsthaninWTafterdroughtandsaltstress(Figure4B).
the accumulation of abundant ROS. We investigated whether TheseresultsindicatedthatSlGRAS40maybeinvolvedinstress
H2O2andO−2 accumulateinWTandOEL2,L3,L4plantsafter signalingpathwaysbymodulatingthesegenesintomato.
drought or salt treatment. Results show that drought and salt
stressinducedmoreH O andO−toaccumulateinWTplants,
2 2 2 Overexpression of SlGRAS40 Improves
but there was no obvious change in OE plants (Figure4A).
Seed Germination Rate under Osmotic and
MDA, proline, and soluble sugar contents were measured in
treatedandcontrolplants(Figure4A),asaccumulationofthese Salt Stress
compoundsischaracteristicofphysiologicalstress.MDAcontent The osmotic and salt tolerance of seed germination was tested
increased significantly in WT plants under drought and salt on seeds harvested from WT and OE L2, L3, L4 plants
stress,andslightlyincreasedinOEplantsbutnotasmuchasin (Figures5A,B).Thegerminationrateofbothgenotypesdeclined
WT. The proline content of both WT and OE plants increased under 150 mM D-mannitol and 75 mM NaCl, respectively
after drought and salt stress, but more proline accumulated in (Figure5C).ThegerminationrateofOEseedswassignificantly
OE plants under salt stress. There was an obvious decrease in higher than that of WT seeds under either stress condition, on
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Liuetal. SlGRAS40-OEEnhancesTomatoAbioticResistance
FIGURE2|OverexpressionofSlGRAS40enhancestolerancetodroughtandsaltstress.(A)QuantitativeRT-PCRanalysisofSlGRAS40mRNAfromleavesof
1-month-oldWTplantssprayedwith100mMD-mannitol,200mMNaCl,or100mMH2O2.ExpressiondatawasnormalizedwithexpressionofSlGRAS40intreated
plantsat0hsetas1.AsterisksindicatesignificantdifferencesusingStudent’st-test(*P<0.05,**P<0.01).(B)Photographsofrepresentativeplantsafter17daysof
droughttreatmentor23daysofsalttreatmentcomparedtocontrolplants.(C)Phenotypesofthefifthleavesofplantsshownin(B).RWC(D,E)andtotalchlorophyll
content(F,G)weremeasuredafterdrought(D,F)andsalt(E,G)stresstreatment.Errorbarsshowthestandarderrorofdatafromthreereplicates.
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Liuetal. SlGRAS40-OEEnhancesTomatoAbioticResistance
FIGURE3|OverexpressionofSlGRAS40reducesstomaopeningunderdroughtstress.(A)StomamorphologyofWTandSlGRAS40-OEL3leavesundermockand
droughtconditions.Redindicatesporelength,blackindicatesporewidth.(B)StomatalconductanceofWTandSlGRAS40-OEL3leavesundermockanddrought
conditions.Stomatalconductance=porelength/porewidth.Errorbarsshowthestandarderrorbetweenthreebiologicalreplicates(n=3)withmorethanfiveleaves
perreplicate.
average 76.3 vs. 66.7% after osmotic treatment and 76 vs. 58% showed that overexpression of SlGRAS40 induced GA-deficient
aftersalttreatment. phenotypes,soSlGRAS40maybeinvolvedinGAbiosynthesisor
Rootelongationwassignificantlydelayedunderosmoticand signaling.
salt stress, but OE root length was longer than WT root length
under the corresponding stress treatments (Figure5D). OE
Overexpression of SlGRAS40 Influences
hypocotyls were longer than WT after D-mannitol treatment,
Fruit Size and Disturbs Fertilization
butshorterthanWTundersaltstress(Figure5E).Theseresults
showedthatSlGRAS40-OEseedsandseedlingsresistdroughtand In plants overexpressing SlGRAS40, the fruit size was smaller
saltstressbetterthanWT. than WT (Figures7A,D). The normal fertilization process was
also disrupted as the fruit set ratio and the number of seeds
were significantly lower than in WT, and fresh weight and
Overexpression of SlGRAS40 Alters
fruit production also decreased (Figures7B,E–H). Studying
Responsiveness to IAA and GA3 the expression pattern at different stages of WT ovary and
The expression level of SlGRAS40 fell after IAA and GA fruit development showed that SlGRAS40 was up-regulated by
3
treatment in WT (Figure6A), indicating that SlGRAS40 is pollination and fertilization, but has relatively low expression
responsivetoauxinandgibberellin.Toexploretherelationship levels in fruits (Figure7C). Final fruit size is controlled by
betweenSlGRAS40andthetwophytohormones,WTandOEL3 genesrelatedtocelldivisionandcellexpansion(Gillaspyetal.,
seedlingsweretreatedwith1µMIAAor50µMGA .SlGRAS40- 1993),sowecheckedtheexpressionlevelsofsuchgenesinWT
3
OE seedlings had fewer lateral roots and longer primary roots and OE L3 fruits harvested at 4, 9, and 20 dpa (Figure7I).
thanWTafterIAAtreatment(dataofL3inFigure6B,dataof Genes related to cell division SlCyCB1.1 (Solyc06g073610) and
L2inSupplementaryFigureS1),indicatingthatoverexpression SlCyCD3.1 (Solyc02g092980) were significantly down-regulated
of SlGRAS40 weakens the responsiveness to IAA. The auxin in4dpa,9dpaand20dpaOEfruitscomparedwithWT.Thegene
sensitivityofSlGRAS40-OEplantswasexploredbydetermining SlEXP18 (Solyc06g076220), which regulates cell expansion, was
the auxin dose-response of elongation of hypocotyl segments. significantly down-regulated in 20 dpa OE fruits, and the gene
Maximumhypocotylsegmentelongationwasobtainedwith10−5 SlPec(Solyc06g083580)wasremarkablydown-regulatedin4dpa
MNAAbothinOEL3andWT,butthehypocotylelongationof and9dpaOEfruit(Figure7I).
OEL3waslessthanWTateachauxinconcentration(Figure6I). As the fruit-set ratio and seed number decreased, a cross-
These data indicated that overexpression of SlGRAS40 reduced fertilizationassaywasperformedtoexplorewhetherthestamens
hypocotylauxinresponsiveness. or pistils in SlGRAS40-OE plants were defective (Figure8A).
Inresponseto50µMGA ,hypocotylsofbothWTandOEL3 The fruit-set ratio was 95, 30, and 35% for WT, OE-L3 and
3
seedlingselongatedmorethancontrolswithoutGA .Outgrowth OE-L4 self-pollination, respectively, and when WT was the
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of the first true leaves from the shoot apex was suppressed in female recipient the fruit-set ratio of WT fell to 77 and 72%
OE L3 seedlings compared with WT, and WT seedlings had with OE L3 and OE L4 pollen, respectively, while the seed
more lateral roots than OE L3 seedlings (Figure6B, and data number, fruit size and fresh weight all decreased. When OE
of L2 in Supplementary Figure S1). The dwarf phenotype and L3 and OE L4 (♀) stigmas received WT pollen (♂), the fruit-
delayed flowering time could be rescued to a level similar to set ratio increased to 60 and 61%, respectively, while the
WT by spraying with 20 µM GA (Figure6F). These results seed number, fruit size and fresh weight all increased slightly
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FrontiersinPlantScience|www.frontiersin.org 7 September2017|Volume8|Article1659
Liuetal. SlGRAS40-OEEnhancesTomatoAbioticResistance
FIGURE4|OverexpressionofSlGRAS40elevatesROSscavengingability.(A)H2O2,O2−,MDA,proline,andsolublesugarcontentsandPOD,CAT,andSOD
activitiesinleavesofWTandSlGRAS40-OEplantsundernormalandstressconditions.LeafsampleswerefromtheplantsshowninFigure2B.(B)Transcriptlevels
ofstressrelatedgenesinwild-typeandtransgenicplantsundernormalandstressconditions.LeafsamplesfromtheplantsshowninFigure2BwereusedforRNA
extraction.Errorbarsshowthestandarderrorbetweenthreereplicatesperformed.AsterisksindicatesignificantdifferencesusingStudent’st-test(*P<0.05,
**P<0.01).
(Figure8B). In pollen germination and pollen tube growth of fertilization caused by SlGRAS40 overexpression is related
assays, the pollen from transgenic plants (OE L3) germinated to altered auxin and gibberellin responses in SlGRAS40-OE
normally and the pollen tube grew like WT. In OE L3 plants pollinatedovaries.
pollen tubes could grow through the stigma and style into the
Differential Expression of Genes Related to
ovary, and then spread out toward the ovules as occurs in
WT plants (Figure8C). When emasculated ovaries (L3) were Auxin and GA in SlGRAS40-OE Plants
treated with 2,4-D or GA3 on the day equivalent to anthesis The expression of a range of genes related to auxin and GA
(0 dpa), fruit-set ratio and fruit size increased significantly was assessed in WT and OE L3 seedlings treated with IAA
(Figure8D,SupplementaryFigureS2),indicatingthatdisruption and GA for 3 h (Supplementary Figure S3). Expression of the
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FrontiersinPlantScience|www.frontiersin.org 8 September2017|Volume8|Article1659
Liuetal. SlGRAS40-OEEnhancesTomatoAbioticResistance
FIGURE5|ComparativeanalysisofWTandSlGRAS40-OElinesunderdroughtandsaltstress.(A,B)SeedgerminationofWTandSlGRAS40-OElinesafter2
weeksinmediumcontaining150mMD-mannitolor75mMNaCl.(C)GerminationrateofWTandSlGRAS40-OElinesundercontrolanddroughtorsalttreatments.
(D)PrimaryrootlengthofWTandSlGRAS40-OEseedlingsundercontrolanddroughtorsalttreatments.(E)ShootlengthofWTandSlGRAS40-OEseedlingsunder
controlanddroughtorsalttreatments.Errorbarsshowthestandarderrorsbetweenthreereplicates.AsterisksindicatesignificantdifferencesusingStudent’st-test
(*P<0.05,**P<0.01).
following genes were tested: SlIAAs, SlARFs, SlPINs, SlTIR1s, in OE seedlings compared with untreated seedlings. SlGA2ox3
SlAFBs,andSlGH3,allinvolvedinauxinsignaling;SlGA20ox1, was strongly up-regulated in WT, but significantly down-
3 and 4, SlGA3ox1 and 2, and SlGA2ox1, 2, 3, 4, and 5; regulatedinOEafterGA treatment.Theseresultsshowedthat
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genesencodinggibberellinreceptorsSlGAST andSlGID1;genes overexpressionofSlGRAS40alteredtheexpressionlevelsofgenes
encoding gibberellin synthetases SlKS, SlKAO, SlKO, SlCPS, involvedinauxinandGAsignaling,suggestingthatSlGRAS40is
SlGPS; and SlDELLA, which is an inhibitor of gibberellin involvedinregulatingthebiosynthesisand/orsignalingofauxin
signaling.Allthesegenesdifferedinhowtheywereexpressedin andGAintomato.
WTandOEseedlings.SlPINs,SlTIR1s,SlAFBs,SlKO,andSlGPS
werealldown-regulatedinOEseedlingscomparedtoWT.Also
Expression Levels of Genes Related to
some genes responded differently after treatment with IAA or
Fruit Set Were Altered in SlGRAS40-OE
GA between OE and WT seedlings. SlIAA4, SlIAA7, SlIAA9,
3
SlARF5, and SlKS were all up-regulated after GA treatment in Plants
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WT, but were down-regulated in OE seedlings (Supplementary Innormal fruit development, theinitiationof fruit setdepends
Figure S3). SlARF6 was down-regulated in WT after IAA on the successful completion of pollination and fertilization,
treatment,andup-regulatedinOEseedlings.SlAFB4wasdown- correspondingwithariseinlevelsofauxinandGAinpollinated
regulatedinWTafterGA andIAAtreatment,butup-regulated ovaries (Serrani et al., 2007b). We compared gene expression
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inOEseedlings.SlDELLAwasup-regulatedafterGA andIAA of auxin and GA signaling genes related to fruit set in −2,
3
treatmentinWT,butdown-regulatedinOEseedlingsafterIAA 0, and 4 dpa ovaries, and ovaries treated with 2,4-D, or GA3
treatment(SupplementaryFigureS3).SlGID1andSlGAST were for 4 days (Supplementary Figure S4). All the auxin related
up-regulated after IAA treatment in WT, but down-regulated genes have lower expression in 0 dpa OE ovaries compared
FrontiersinPlantScience|www.frontiersin.org 9 September2017|Volume8|Article1659
Liuetal. SlGRAS40-OEEnhancesTomatoAbioticResistance
FIGURE6|OverexpressionofSlGRAS40altersresponsivenesstoIAAandGA3.(A)QuantitativeRT-PCRanalysisofSlGRAS40mRNAfromleavesof15-day-oldWT
seedlingstreatedwith20µMIAAand20µMGA3.(B)Phenotypesof15-day-oldWTandSlGRAS40-OEL3seedlingsgrownon½×MSmediumcontaining1µM
IAAand/or50µMGA3.(C)NumberoflateralrootsofWTandSlGRAS40-OEL3seedlingstreatedwithIAA,GA3orIAA+GA3.(D)PrimaryrootlengthofWTand
SlGRAS40-OEL3seedlingsshownin(B).(E)HypocotyllengthofWTandSlGRAS40-OEL3seedlingsshownin(B).(F)RescueofSlGRAS40-OEL3dwarfismby
exogenousGA3application.(G)Plantheightand(H)PhasetransitiontimeofGA3treatedplantsshownin(F).(I)HypocotylelongationofWTandSlGRAS40-OEL3
afterNAAtreatment.AsterisksindicatesignificantdifferencesusingStudent’st-test(*P<0.05,**P<0.01).
FrontiersinPlantScience|www.frontiersin.org 10 September2017|Volume8|Article1659