The Arabidopsis ETHYLENE RESPONSE FACTOR1 Regulates Abiotic Stress-Responsive Gene ...

17 Pages·2013·1.73 MB·English

The Arabidopsis ETHYLENE RESPONSE FACTOR1
Regulates Abiotic Stress-Responsive Gene Expression
by Binding to Different cis-Acting Elements in
Response to Different Stress Signals1[W][OA]
Mei-Chun Cheng, Po-Ming Liao, Wei-Wen Kuo, and Tsan-Piao Lin*
Institute of Plant Biology, National Taiwan University, Taipei 10617, Taiwan
ORCIDIDs:0000-0003-2443-2847(M.-C.C.);0000-0002-4350-9574(T.-P.L.).
ETHYLENERESPONSEFACTOR1(ERF1)isanupstreamcomponentinbothjasmonate(JA)andethylene(ET)signalingandis
involvedinpathogenresistance.AccumulatingevidencesuggeststhatERF1mightberelatedtothesaltstressresponsethrough
ethylenesignaling.However,thespecificroleofERF1inabioticstressandthemolecularmechanismunderlyingthesignaling
cross talk still need to be elucidated. Here, we report that ERF1 was highly induced by high salinity and drought stress in
Arabidopsis(Arabidopsisthaliana).ThesaltstressinductionrequiredbothJAandETsignalingbutwasinhibitedbyabscisicacid.
ERF1-overexpressinglines(35S:ERF1)weremoretoleranttodroughtandsaltstress.Theyalsodisplayedconstitutivelysmaller
stomatal aperture and less transpirational water loss. Surprisingly, 35S:ERF1 also showed enhanced heat tolerance and up-
regulationofheattolerancegenescomparedwiththewildtype.SeveralsuitesofgenesactivatedbyJA,drought,salt,andheat
werefoundinmicroarrayanalysisof35S:ERF1.ChromatinimmunoprecipitationassaysfoundthatERF1up-regulatesspecific
suites of genes in response to different abiotic stresses by stress-specific binding to GCC or DRE/CRT. In response to biotic
stress,ERF1boundtoGCCboxesbutnotDREelements;conversely,underabioticstress,weobservedspecificbindingofERF1
to DRE elements. Furthermore, ERF1 bound preferentially to only one among several GCC box or DRE/CRT elements in the
promoterregionofitstargetgenes.ERF1playsapositiveroleinsalt,drought,andheatstresstolerancebystress-specificgene
regulation, whichintegrates JA,ET,andabscisicacidsignals.
Environmental stresses such as heat, cold, drought, minimal function; the core sequence of AGCCGCC is
and high salinity influence plant growth and produc- typically referred to as the GCC motif (Hao et al., 1998).
tivity. Plants respond and adapt to these stresses at GCC motif binding occurs through a highly conserved
physiologicalandbiochemicallevels.Abioticstresshas DNA-binding domain approximately 60 amino acids in
been shown to induce the expression of genes with length (Ohme-Takagi and Shinshi, 1995). This domain
various functions in a variety of plants (Yamaguchi- formsaninterfaceofthreeantiparallelb-strandsandone
Shinozaki and Shinozaki, 2006). Ethylene-responsive a-helix,withtheb-strandsbindingprimarilytoGCCbox.
element-binding factors (ERFs) form a plant-specific Ithasbeendemonstratedthatconstitutiveexpression
transcriptional factor superfamily of 147 members in ofERF1(AT3G23240),adownstreamcomponentofthe
Arabidopsis (Arabidopsis thaliana; Nakano et al., 2006). ethylene(ET)signalingpathway,increasesArabidopsis
ERFs influence a number of developmental processes resistance to Botrytis cinerea and Plectosphaerella cucu-
and are also important for adaptation to biotic or merina (Berrocal-Lobo et al., 2002). The expression of
abiotic stresses such as pathogen attack, wounding, ERF1 can be activated rapidly by ET or jasmonate (JA)
UV irradiation, extreme temperature, and drought and can be activated synergistically by both hormones
(Ecker, 1995; O’Donnell et al., 1996; Penninckx et al., (Lorenzo et al., 2003). Furthermore, 35S:ERF1 expres-
1996). Several Arabidopsis ERFs bind to the GCC box sion can rescue the defense response defects of corona-
consensus sequence TAAGAGCCGCC, which has a
tive insensitive1 and ethylene insensitive2 (ein2). These
results suggest that ERF1 acts downstream of the in-
1ThisworkwassupportedbytheNationalScienceCouncil,Taiwan tersection between the ET and JA pathways and that
(grantno.101–2311–B–002–013–MY2),andNationalTaiwanUniversity this transcription factor is a key element in the inte-
(grantno.101R892001toT.-P.L.). gration of both signals for the regulation of defense
*Correspondingauthor;[email protected]. response genes (Lorenzo et al., 2003). Constitutive ex-
Theauthorresponsiblefordistributionofmaterialsintegraltothe pression of ERF1 activates the transcription of down-
findings presented in this article in accordancewith the policy de-
stream effector genes, such as BASIC CHITINASE
scribed in the Instructions for Authors (www.plantphysiol.org) is:
(b-CHI)andPLANTDEFENSIN1.2(PDF1.2),topromote
Tsan-PiaoLin([email protected]).
[W]TheonlineversionofthisarticlecontainsWeb-onlydata. theETresponse(Solanoetal.,1998).Solanoetal.(1998)
[OA]OpenAccessarticlescanbeviewedonlinewithoutasubscrip- also found that EIN3 directly regulates ERF1 gene ex-
tion. pression by binding to a primary ethylene response
www.plantphysiol.org/cgi/doi/10.1104/pp.113.221911 element present in the promoter of ERF1.
1566 PlantPhysiology(cid:1), July 2013, Vol. 162,pp. 1566–1582, www.plantphysiol.org (cid:3)2013AmericanSociety ofPlant Biologists.All RightsReserved.
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Multiple Functions of ERF1 in Stress Responses
Recent studies have shown that alterations in ET RESULTS
signaling affect plant responses to both salt and water
AbioticStressResponse,Subcellular Localization,and
stress (Cao et al., 2006, 2008; Cela et al., 2011). ET-
ExpressionPattern ofERF1
insensitivemutantsarereportedtobemoresaltsensitive,
suggesting that ET signaling reduces salt sensitivity We screened seed pools of the AtTORF-EX library
(Caoetal.,2008).AsacentralregulatorofETresponse (Weiste et al., 2007) by subjecting 2-week-old plants to
genes, ERF1 could be turned on by salt stress, and its water withholding over a 2-week period. Surviving
expression was altered in a salt-sensitive mutant, vita- plants were selected for sequencing. From this screen-
minE-deficient4(vte4;Celaetal.,2011).Inotherspecies, ing, ERF1 was found and further characterized in this
ETresponsefactorssharingsequencesimilaritytoERF1 paper. Expression of ERF1 was gradually induced by
have also been reported to be involved in various salt (150 mM NaCl), osmotic (400 mM mannitol), and
abiotic stresses. Transcription of the wheat (Triticum droughtstresstreatmentsover12handpeakedat3,6,
aestivum) TaERF1 gene was induced by drought, salin- and 1 h, respectively, for the different abiotic stress
ity, low temperature, exogenous abscisic acid (ABA),
treatments (Fig. 1A). In contrast, we observed hardly
ET,andsalicylicacidaswellasbyinfectionwithBlumeria
any induction of ERF1 under heat stress (37°C) treat-
graminis f. sp. tritici (Xu et al., 2007). Furthermore, over-
ment. According to the AtGenExpress Visualization
expression of TaERF1 activated stress-related genes, in- Tool and electronic fluorescent pictographic browser
cludingPATHOGENRESPONSEandCOLDRESPONSE/
databases, ERF1 is induced by salt stress, especially in
RESPONSIVE TO DESICCATION (COR/RD) genes,
the roots, but not by ABA treatment. Across develop-
under normal growth conditions and improved path-
mental stages, ERF1 expression is higher in dry seeds,
ogen and abiotic stress tolerance in transgenic plants.
seedlings, and senescent leaves. ERF1 subcellular lo-
These results suggested that the TaERF1 gene encodes
calizationwas determinedbytransiently expressingan
aGCCboxandCRT/DREelement-bindingfactorthat
N-terminal fusion of ERF1 to GFP in Arabidopsis pro-
might be involved in multiple stress signal transduc-
toplasts using polyethylene glycol-mediated transfor-
tionpathways(Xuetal.,2007).Ithasalsobeenshown
mation. The ERF1-GFP fusion protein was detected in
thatJERF3isolatedfromtomato(Solanumlycopersicum) nuclei(Fig.1B),andthiswasconfirmedbycomparison
could be induced by ET, JA, cold, salinity, or ABA, with 49,6-diamino-phenylindole staining of nuclei.
transcriptionally regulated the expression of genes in-
For analyzing the spatial expression of ERF1 under
volved in plant responses to osmotic and oxidative
different stress conditions,wefused a1.5-kbfragment
stresses, and enhanced the drought, salt, and freezing
oftheERF1promoterregiontotheGUSreportergene
resistanceintobacco(Nicotianatabacum),perhapsthrough
andintroducedthisconstructintoArabidopsis.Three-
reducedreactiveoxygenspeciesaccumulation(Wang
week-old T2 transgenic plants were analyzed after
et al., 2004). These results suggest that Arabidopsis
treatment with JA, salt stress, drought, or heat shock.
ERF1 might also be involved in the abiotic stress re-
There was almost no GUS expression under normal
sponse in addition to its role in the defense response.
However, the specific role of ERF1 in abiotic stress conditions (Fig. 1C). After JA treatment, GUS activity
was mainly observed in petiole, whereas after salt
and the molecular mechanism underlying signaling
stress treatment, GUS activity was observed in leaves
cross talk between biotic and abiotic stress are still
but not in petiole and the main veins of leaves. After
unclear.
drought and heat shock stress treatment, there was
WefoundtheERF1couldenhancedroughtsurvival
muchlessGUSactivity,exceptforsomeregionsofleaf
through screening of a transcription factor over-
tips or leaf margins (Fig. 1C).
expression library, AtTORF-EX (Weiste et al., 2007).
Considering that ERF1 might regulate plant re-
In further experiments, we found a dynamic role of
sponses to a variety of abiotic stresses in which the
ERF1 in both abiotic and biotic stress responses. Ex-
pressionofERF1wasrapidlyandtransientlyinduced phytohormoneABAplaysanimportantregulatoryrole,
by salt and dehydration treatments, and 35S:ERF1 we asked whether ABA would affect the induction of
transgenics were more tolerant to drought, salt, and ERF1 expression. Because ERF1 was reported to be ac-
even heat stress. Transcriptional analysis using 35S: tivated synergistically by both JA and ET, we also ex-
ERF1andERF1RNAinterference(RNAi)knockdown aminedtheeffectofdifferenthormonecombinationson
plantsfoundthatmanystress-relatedgenes,suchasCOR/ ERF1 expression. In quantitative reverse transcription
RD genes and heat shock-inducible genes, were up- (qRT)-PCR analyses, ERF1 was repressed by 30 min of
regulated in 35S:ERF1 and conversely down-regulated ABA treatment but then slowly recovered at later time
in ERF1 RNAi plants. Chromatin immunoprecipitation points (Fig. 2A). ABA-RESPONSIVE-ELEMENT BIND-
(ChIP) assays revealed a unique mechanism whereby ING FACTOR1 (AREB1) was used as a marker gene
ERF1 bound preferentially to different cis-elements of to show the effectiveness of the ABA treatment. Both
downstream genes under different stress treatments. JA and ET treatments could trigger ERF1 expression,
This novel mechanism may be a more widespread and the combination of both JA and ET resulted in a
mechanism for transcription factors to generate spe- synergetic effect on ERF1 induction. However, ERF1
cific patterns of gene expression in response to dif- inductionbyJA,ET, orcombinedJAandETtreatment
ferent environmental stimuli. wassuppressedbyABA(Fig.2B).Theseresultsindicated
Plant Physiol. Vol. 162, 2013 1567
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Copyright © 2013 American Society of Plant Biologists. All rights reserved.
Cheng et al.
Figure1. ExpressionprofileofERF1.A,qRT-PCRanalysesofERF1inductionbyabioticstresses.TotalRNAwasextractedfrom
plantsharvestedattheindicatedtimesaftereachtreatment.Two-week-oldseedlingsweredriedonWhatman3MMpaper(Drought),
treatedwith150mMNaCl(NaCl),treatedwith400mMmannitol(Mannitol),orincubatedat37˚C(Heat).Theamplificationof
ACTIN2wasusedasaninternalcontroltonormalizealldata.Thelevelofthetranscriptbeforestresstreatmentswassetto1.0.Three
independent experimentswereperformedwith similar results. Error bars indicate SE (ANOVA; *P ,0.05). B, Fluorescence mi-
croscopyimagesofArabidopsisprotoplast.Constructsof35S:GFPor35S:ERF1-GFPweretranslocatedintoArabidopsisprotoplastby
polyethylene glycol transfection. The expression of the introduced genes was detected after 16 h. Nuclei are shown with
49,6-diamino-phenylindole(DAPI)staining.Bars=20mm.C,GUSstainingofERF1promoter:GUStransgenicplants.Three-week-old
homozygousplants(G3andG6)wereeithermocktreatedortreatedwith50mMJA,150mMNaCl,30minofdroughtstress,or1hof
heatshockstress(45˚C).HistochemicalGUSstainingwasperformedovernighton10seedlingsforeachexperiment.
that that ABA-negative regulation of ERF1 could over- and RNAi plants had (Fig. 3D). 35S:ERF1 plants also
ride JA or ET induction. Consistent with this, GUS had higher germination rates on medium containing
staining also showed that ABA treatment could sup- 100 to200 mM NaCl (Fig. 3E).Bystatistical calculation,
pressERF1promoterinductionbyJAandET(Fig.2C). in the control and 100 mM NaCl treatments, the ger-
minationrateswereaboutthesame.Butinthe150mM
NaCl condition, germination of the wild type was
Overexpression ofERF1EnhancedDroughtandSalt inhibitedandonlyabout4%ofseedsgerminated,while
ToleranceinArabidopsis the germination rate of 35S:ERF1 plants still reached
100%. Even in the 200 mM NaCl treatment after 5 d,
We produced both transgenic plants overexpressing
almost no wild-type plants germinated, but the 35S:
the ERF1 gene under the control of the cauliflower
ERF1 plants had more than 60% of seeds germinated
mosaicvirus35Spromoter(35S:ERF1)aswellasRNAi
(Fig. 3E). In contrast, ERF1 RNAi lines had reduced
knockdown lines using a specific 50-bp fragment of
germination at 150 or 200 mM NaCl.
the ERF1 coding sequence. Expression of ERF1 in the
Asanadditionalassayofsalttolerance,wemeasured
35S:ERF1transgenicplantsandknockdownplantswas
root elongation under high salinity. The seeds were in-
verified by qRT-PCR assays (Fig. 3A). 35S:ERF1 plants
cubatedinone-half-strengthMurashigeandSkoog(MS)
had a greater resistance to water deficit (Fig. 3B). After
medium for 3 d and then transferred to the plates con-
12dwithoutwatering,35S:ERF1plantsremainednearly
taining 150 mMNaCl, and the root length increaseover
turgid without manifesting major macroscopic symp- 5dofsalttreatmentwasmeasured.Significantlygreater
toms of drought-related stress, whereas wild-type and
rootelongationwasobservedinthe35S:ERF1seedlings,
RNAiplantswerevisiblydamaged.Thesurvivalratefor
while ERF1 RNAi lines were similar to the wild type
35S:ERF1plantsafterresumptionofwateringwasabout
(Fig. 3F).
90%, compared with only about 33% for wild-type and
RNAi plants (Fig. 3D). Similarly, 35S:ERF1 plants also
hadgreatersalttolerance(Fig.3C).Plantsweregrownin ERF1Overexpression ReducedLeafWaterLossand
normal conditions for about 3 weeks and then were
Stomatal ApertureButIncreasedProandABA Content
wateredwith 100 mM saline for 4 d, then 200 mMsaline
foranother4d,andthen300mMsalinefortherestofthe To investigate the underlying mechanisms of the
time.Afterthissaltstresstreatment,nearlyall35S:ERF1 drought resistance phenotype of 35S:ERF1, we per-
plants had survived, while only 44% of the wild-type formed water loss and stomatal aperture assays. As
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Copyright © 2013 American Society of Plant Biologists. All rights reserved.
Multiple Functions of ERF1 in Stress Responses
Figure2. ABA inhibition effecton ERF1expression. In qRT-PCRandGUS staininganalyses, 2-week-oldwild-typeor ERF1
promoter:GUStransgenicplantswereeithermocktreatedortreatedwith50mMJA,50mMET,bothJA+ET,ortogetherwith
50mMABA.A,qRT-PCRanalysesofERF1andAREB1underABAtreatment.TotalRNAwasextractedfrom2-week-oldplants
harvestedattheindicatedtimesafter50mMABAtreatment.Threeindependentexperimentswereperformedwithsimilarre-
sults. B, qRT-PCR analyses of ERF1 under different combinations of hormone treatments. Three independent experiments
wereperformedwithsimilarresults.ErrorbarsindicateSE.C,HormoneapplicationsonGUSstainingofERF1promoter:GUS
transgenicplants.Sampleswerecollectedafter1hofeachtreatment.
reductioninstomatalapertureisacriticalaspectofthe with the wild type under normal conditions. However,
drought response, we hypothesizedthat the enhanced whentreatedwith0.4Mmannitol,therewasnosignificant
drought resistance of the 35S:ERF1 plants might be difference among overexpression, RNAi, and wild-type
related to reduced leaf water loss. Indeed, the tran- plants (Fig. 5A). We also found that (D1-PYRROLINE-5-
spiration rate from 35S:ERF1 was reduced relative to CARBOXYLATESYNTHETASE1(P5CS1),whichencodes
the wild type in detached leaf assays (Fig. 4A). Con- thekeyenzymeinstress-inducedProsynthesis,wasmore
sistent with this, 35S:ERF1 plants had a constitutively highlyexpressedin35S:ERF1plantsbutlessexpressedin
reduced stomatal aperture compared with wild-type theRNAiplants(SupplementalFig.S1).Thesedataindi-
plants (Fig. 4, B and C). ABA treatment reduced sto- catedthat ERF1 positivelyregulatesPro accumulation.
matal aperture to a similar extent in transgenic and ABA is believed to play an important part in plant
responses to environmental stress. Moreover, P5CS1
wild-type plants when the smaller initial aperture of
expression and Pro accumulation are partially ABA
35S:ERF1 was taken into account. The stomatal aper-
dependent (Szabados and Savouré, 2010; Verslues and
ture response to ABA treatment in the ERF1 RNAi
Sharma, 2010). Therefore, we suspected that ABA con-
mutant was similar to that of the wild type (Fig. 4C).
tent might be altered in ERF1 transgenic plants. Inter-
Also consistent with the detached leaf assays and re-
duced stomatal aperture, 35S:ERF1 plants had signifi- estingly,theABAcontentwasupto2-foldhigherin35:
ERF1plantsthaninwild-typeplantsbutlowerinERF1
cantly higher leaf temperature than wild-type plants
RNAiplants(Fig. 5B).OneoftheRNAilineshadABA
(Fig. 4A). All these assays indicated that reduced leaf contentjustaslowastheABA-deficientmutantaba2-1,
water loss was one factor, although possibly not the
and this was used as a comparison.
onlyfactor,intheenhanceddroughtresistanceof35S:
ERF1 plants.
ERF1InductionRequiredBothETandJASignalingunder
Pro is a compatible osmolyte that contributes to
SaltStressandWasNegativelyRegulated byABA
drought tolerance through the protection of cellular
structure and the role of Pro metabolism in redox We then examined the role of ABA in ERF1 expres-
buffering (Szabados and Savouré, 2010, Verslues and sion.Interestingly,salt-inducedexpressionofERF1was
Sharma, 2010, Sharma et al., 2011). To determine notimpairedinaba2-1butwasreducedsignificantlyin
whether ERF1 overexpression affected Pro accumula- ABA-hypersensitive abi1 and abi2 knockout mutants
tion, Pro contents in 35S:ERF1 and ERF1 RNAi mu- (Merlot et al., 2001; Fig. 6A). This indicated that ERF1
tants were measured. As seen in Figure 5A, 35S:ERF1 was connected to ABA both through its effect on ABA
plants accumulated higher Pro levels, whereas ERF1 contentandthenegativeregulationofitsexpressionby
RNAi lines accumulated lower Pro levels, compared ABA signaling.
Plant Physiol. Vol. 162, 2013 1569
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Cheng et al.
Figure3. Drought-andsalt-tolerantphenotypesof35S:ERF1transgenicArabidopsis.A,ExpressionlevelsofERF1mRNAin
35S:ERF1 (OE5 and OE6) and ERF1 RNAi (RNAi7 and RNAi15) transgenic plants. B, Drought tolerance of wild-type (WT),
35S:ERF1,andERF1RNAitransgenicplantsafterwithholdingwaterfor12to16dandrehydrationfor4d(Recover).C,Three-
week-oldplantswereirrigatedwithdifferentconcentrationsofNaClsolution(100mMfor4d,200mMforanother4d,and300
mMfortherestofthetime).Theseexperimentswererepeatedthreetimeswithsimilarresults.D,Survivalratesofwild-type,35S:
ERF1,andERF1RNAitransgenicplantsunderdroughtandsaltstress.ErrorbarsindicateSE(Student’sttest;*P,0.001).E,Seed
germinationratesof35S:ERF1andERF1RNAitransgenicplantsundersaltstresstreatment.ERF1overexpressedandRNAiseeds
weregerminatedunderdifferentconcentrationsofNaCl.Thegerminationrateswerecalculatedafter3d(toppanel)and5d
(bottompanel).Resultsareaveragesofthreereplicates.ErrorbarsindicateSE(Student’sttest;*P,0.05,**P,0.01).F,Root
elongationassays.Three-day-oldseedlingsweretransferredtoanMSagarplatewith150mMNaClandincubatedverticallyfor
7dbeforerootlengthsweremeasured.Resultsareaveragesofthreereplicates.ErrorbarsindicateSE(Student’sttest;*P,0.05).
To know whether the salt stress induction of ERF1 thatERF1inductionwassuppressedinjar1-1(Fig.6D).
requires ET signaling, we further examined ERF1 ex- These data indicated that the salt induction of ERF1
pression in ET-insensitive mutants, etr1-1 and ein2-5 required both ET and JA signaling and that increased
(Fig. 6B). ERF1 induction was blocked in etr1-1 and ETresponsecouldoverridethenegativeeffectofABA.
ein2-5 under either salt or drought stress. We also
tested ABA inhibition of ERF1 induction in the ET-
Transcriptome AnalysisofTransgenicArabidopsis
hypersensitivemutantctr1andfoundthatctrlhadhigher Overexpressing ERF1
than wild-type ERF1 expression even in the presence of
ABA(Fig.6C).Also,totestwhethersaltstressinduction To investigate the involvement of ERF1 in the reg-
of ERF1 requires JA signaling, we examined ERF1 ex- ulation of the expression of abiotic stress-responsive
pression in the JA-insensitive mutant jar1-1 and found genes,andtofurtherunderstandwhy35S:ERF1transgenic
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Copyright © 2013 American Society of Plant Biologists. All rights reserved.
Multiple Functions of ERF1 in Stress Responses
Figure4. WaterlossindetachedleavesandtheinfluenceofERF1overexpressiononABA-mediatedstomatalclosure.A,Water
lossfromdetachedleavesasafunctionoftimeinCol-0and35S:ERF1plants(OE3andOE6).Thisexperimentwasrepeated
three times with similar results. Values are means of the percentage of leaf water loss 6 SE (n = 15). Error bars indicate SE
(ANOVA; *P , 0.05).B, Micrographs representing thedynamics of ABA-mediated stomatal closureinCol-0and 35S:ERF1
plants.C,Stomatalaperturesweremeasuredonepidermalpeelsofwild-type(WT),35S:ERF1(OE5andOE6),andERF1RNAi
(RNAi7 andRNAi15) transgenicplants.Stomatawerepreopened under light for 2.5 h andthen incubatedinthe indicated
concentrationsofABAfor2.5hunderlight.Thisexperimentwasrepeatedthreetimeswiththesametrend.Valuesaremeans6
SE(n.60).ErrorbarsindicateSE(ANOVA;*P,0.05).D,Infraredthermalimagesof3-week-old35S:ERF1(OE3,OE5,and
OE6)andwild-type(Col)plants.
plantsareresistanttodroughtandsalt,atranscriptome resistance.TheseresultssuggestthatERF1functionsnot
analysis of 35S:ERF1 plants was performed using an only in drought- and salt stress-responsive gene ex-
Agilent Arabidopsis 2 Oligo Microarray (Agilent Tech- pression but also in heat shock. To understand if ERF1
nologies) covering about 21,000 genes. Relative to vec- directlyactivatedthesegenes,wesearchedforGCCboxes
tor control plants, 1,156 genes were expressed at least in the 1-kb promoter regions of these genes using Plant-
2-fold higher in 35S:ERF1. Genes involved in abiotic CARE (http://bioinformatics.psb.ugent.be/webtools/
stress responses (drought, salt, and heat stress) were plantcare/html/). However, most of the downstream
selected from Gene Ontology analysis by GeneSpring genes did not have GCC boxes but had the DRE ele-
11 software and are listed in Table I. This analysis ment in their promoters (Table I), indicating that ERF1
confirmed that many drought stress- or salt stress- may bind to the DRE element.
inducible genes are potentially downstream of ERF1, Wewerenonethelessinterestedtodeterminewhether
including RD29B, COR47, LEA4-5, RD20, and many 35S:ERF1 plants had improved heat tolerance. Seven-
others. Among the 1,156 ERF1 up-regulated genes, 46 day-old vector control and transgenic plants were ger-
and 61 genes showed drought- and salt-responsive minatedat22°Confilterpaperpremoistenedbyliquid
gene expression, respectively (Fig. 7A). Eighteen of germination medium and then subjected to heat stress
the46droughtstress-induciblegenesarealsoinvolved treatmentat45°C.Only 7%ofthevectorcontrolplants
in the salt stress response. A limitation of this data is survived 3 d after recovery from heat stress, whereas
that ectopic expression of ERF1 may lead to the up- half of the 35S:ERF1 plants survived. These results
regulationofgenesthatarenotnormallyinfluencedby clearly indicate augmented thermotolerance of the
ERF1. To address this concern, we tested the expres- 35S:ERF1 plants (Fig. 8).
sion of several ERF1 up-regulated genes in the ERF1
RNAi mutants. Six ERF1 up-regulated genes, LEA4-5,
RD20,RD29B,COR47,HSP17.6A,andHSP23.6-MITO, ERF1BindstoSpecificGCCBoxandDREElementsof
showed clear reduction of their stress-inducible ex- SubsetsofStress-Responsive GenesUp-Regulatedin
pression in ERF1 RNAi plants (Fig. 7B). ResponsetoDifferentStressSignals
To investigate whether ERF1 directly regulates abi-
35S:ERF1ActivatesHeatShockGenesandExhibitsHeat otic stress-responsive genes, we searched for a com-
mon cis-acting element presented in the promoters of
ShockStressTolerance
ERF1 up-regulated genes. The DRE contains the core
Surprisingly,32heatstress-relatedgeneswereamong sequenceA/GCCGACandhasbeenidentifiedasacis-
the ERF1 up-regulated genes. These included AtHsfA3, actingpromoterelementregulatinggeneexpressionin
which encodes a transcription factor involved in heat response to drought, salt, and cold stresses in Arabi-
shock-induciblegeneexpression,mitochondria-localized dopsis (Hao et al.,2002;Sakumaet al.,2002). Many of
small HSP23.6 (HSP23.6-M; At5g51440), DnaJ-like pro- the ERF1 up-regulated genes contained DRE elements
tein (At1g72070), HSP70 (At3g12580), and HSP17.6A in their promoter regions. Electrophoretic mobility
(At5g12030), all of which likely function in heat shock shift assays using ERF1-GFP fusion protein purified
Plant Physiol. Vol. 162, 2013 1571
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Copyright © 2013 American Society of Plant Biologists. All rights reserved.
Cheng et al.
SRO5, GLP9, and ATOSM34; and the heat shock-
responsive genes ATHSFA3, HSP101, HSP70, and
HSP23.6-M,accordingtoSupplementalFigureS5(Fig.
9A). ChIP assays demonstrated stress-specific ERF1
binding to DRE elements in the promoters of drought
stress-responsive genes (LEA4-5, KIN2, GEA6, and
Figure5. ProandABAcontentsinERF1transgenicplants.TotalProor
ABAwas prepared from 3-week-old Arabidopsis grown on MS agar
plates. Pro contents were also measured after treating with 0.4 M
mannitolfor24h.DataarepresentedasmeansandSEofthreerepli-
cations.A,ProcontentsinERF1transgenicplants.ErrorbarsindicateSE
(ANOVA;*P,0.01).DW,Dryweight;WT,wildtype.B,ABAcon-
tentsofERF1transgenicplants.ErrorbarsindicateSE(Student’sttest;
*P,0.01).FW,Freshweight.
from Arabidopsis transgenic plants showed that the
ERF1proteinboundspecificallytotheDREelementof
the RD29B gene promoter (Supplemental Fig. S2). The
GFP protein alone did not bind. ERF1 binding was
significantly reduced in the presence of excess unla-
beledprobes,butDNAfragmentswithamutatedDRE
were less efficient in competing for ERF1 binding,
consistent with specific binding of ERF1 to the DRE
element.
Nucleotide sequence analysis revealed that the pro-
moters of the ERF1 downstream genes contained GCC
box and DRE sequence motifs (Supplemental Fig. S3).
ChIP assays were employed to examine whether the
ERF1 protein binds to the gene promoters using 35S:
ERF1-GFP transgenic plants in which a GFP-coding se-
quencewasfusedinframetothe39endoftheERF1gene.
ThechromatinsolutionwassonicatedtosheartheDNA
intoapproximately500-bpfragments(SupplementalFig.
S4). Quantitative real-time ChIP-PCR assays using an Figure 6. Effects of ABA, ET, and JA on ERF1 gene expression. The
anti-GFPantibodyshowedthatERF1bindstotheGCC relativemRNAamountsofERF1wereanalyzedbyqRT-PCR(theex-
boxorDREinthegenepromotersinnormalunstressed pression level of Col-0 was set to 1). Total RNAwas prepared from
conditions (Supplemental Fig. S5). We also performed 3-week-oldArabidopsisgrown onMSagar platestreatedwith0.4 M
ChIP assays to examine ERF1 promoter binding under mannitolfor24hor150mMNaClfor1h.DatarepresentmeansandSE
different abiotic stress conditions, including drought, of three replications. Error bars indicate SE (ANOVA; *P , 0.01). A,
EffectsofhighsalinityonERF1geneexpressioninaba2,abi1,andabi2
salt, and heat shock. Primers to amplify GCC box- or
knockoutmutants.B,EffectsofhighsalinityanddroughtstressonERF1
DRE-containingpromoterfragmentsweredesignedfor
gene expression in etr1-1 and ein2-5 mutants. WT, Wild type. C,
the JA-responsive genes b-CHI, PDF1.2, ELI3-2, and EffectsofhighsalinityandABAonERF1geneexpressioninctr1mu-
GSTF7; the drought-responsive genes LEA4-5, KIN2, tants. D, Effects of high salinity and JA on ERF1 gene expression in
GEA6,andAt3g02480;thesalt-responsivegenesP5CS1, jar1-1mutants.
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Multiple Functions of ERF1 in Stress Responses
TableI. Abioticstress-relatedgenesup-regulatedin35S:ERF1
Abioticstress-relatedgenesconstitutivelyexpressedin35S:ERF1transgenicplantswerecomparedwithwild-typeplants.Genesinthistableused
intheChIPassayarehighlightedinboldfaceletters.Includedinthistablearegeneswithadirectedroleinabioticstressorthatareinvolvedindirectly
inabioticstressresponses(drought,salt,andheatstress).Thenumberofplussignsindicatesthenumberofsequences.GenesthathavenoDREor
GCCintheirpromoterregionsareindicatedbyminussigns.
GeneSymbol GeneName ProductDescription FoldChange DREa GCCa
Alldrought,salt,andheatstress
At3g23240 ERF1 Ethyleneresponsefactor1 201.76
At1g43160 RAP2.6 EthyleneresponsefactorsubfamilyB-4 72.62 ++ 2
At2g38340 DREB19 Dehydrationresponseelement-bindingprotein19 21.19 2 +
At1g12610 DDF1 Dwarfanddelayedflowering1 4.60 ++ 2
At2g38470 WRKY33 WRKYDNA-bindingprotein33 2.79 +++ +
Bothdroughtandsaltstress
At1g08930 ERD6 Earlyresponsetodehydration6 7.13 2 2
At1g05680 UGT74E2 UDP-glucosyltransferase74E2 5.42 ++++ 2
At5g13330 RAP2.6L RelatedtoAP26L 4.82 2 2
At1g02930 GSTF6 GlutathioneS-transferasef6 3.94 2 2
At2g17840 ERD7 Earlyresponsetodehydration7 3.82 2 2
At1g27730 STZ Salttolerancezincfinger 3.15 2 2
At2g17290 CPK6 Calcium-dependentproteinkinase6 2.83 +++ 2
At3g19580 ZF2 Zincfingerprotein2 2.80 2 ++
At2g41010 CAMBP25 Calmodulinbindingproteinof25kD 2.75 2 2
At5g62470 MYB96 R2R3-typeMybtranscriptionfactor96 2.73 2 2
At5g52300 RD29B Responsivetodesiccation29B 2.54 ++ 2
At2g47190 MYB2 MYBtranscriptionfactor2 2.44 2 2
At2g33380 RD20 Responsivetodesiccation20 2.25 ++ 2
At1g69270 RPK1 Receptor-likekinase1 2.21 2 2
Bothdroughtandheatstress
At1g20440 COR47 Cold-regulated47 2.90 +++ 2
At5g05410 DREB2A DRE-bindingprotein2A 2.80 ++ 2
At3g24500 MBF1C Multiproteinbridgingfactor1C 2.64 2 ++
Bothsaltandheatstress
At3g08720 S6K2 Ser/Thrproteinkinase2 6.05 2 2
At2g30250 WRKY25 WRKYDNA-bindingprotein25 5.88 2 2
At1g59860 –b HSP20-likechaperonesuperfamilyprotein 2.95 + +
At5g59820 RHL41 Responsivetohighlight41 2.25 2 2
Droughtstressonly
At2g40170 GEA6 Lateembryogenesisabundant6 61.94 +++ +
At5g59220 HAI1 HighlyABA-inducedPP2Cgene1 9.93 2 +
At5g06760 LEA4-5 Lateembryogenesisabundant4-5 7.00 ++ +
At2g35930 PUB23 CytoplasmicallylocalizedU-boxdomain-containingE3ubiquitinligase 5.43 2 2
At4g02380 SAG21 Senescence-associatedgene21 5.37 + 2
At1g52890 ANAC019 NACdomain-containingprotein19 5.17 ++ +
At3g02480 – Lateembryogenesisabundantproteinfamilyprotein 4.11 + 2
At3g30775 ERD5 Earlyresponsetodehydration5 3.58 + 2
At2g41430 ERD15 Earlyresponsetodehydration15 3.36 + 2
At2g35300 LEA18 Lateembryogenesisabundant18 2.61 + 2
At1g32560 LEA4-1 Lateembryogenesisabundant4-1 2.46 + 2
At2g02800 KIN2 Kinase2 2.38 + 2
At2g30870 GSTF10 GlutathioneS-transferasef10 2.37 ++ ++
At4g25490 CBF1 C-repeat/DRE-bindingfactor1 2.35 ++ 2
At2g18050 HIS1-3 HistoneH1-3 2.31 + 2
At1g22190 RAP2.4 RelatedtoAP24 2.27 + 2
At4g02200 – Drought-responsivefamilyprotein 2.27 2 2
At1g33560 ADR1 Activateddiseaseresistance1 2.25 +++ 2
At5g45340 CYP707A3 CytochromeP450 2.21 + 2
At1g20450 ERD10 Earlyresponsetodehydration10 2.20 ++ 2
At1g76180 ERD14 Earlyresponsetodehydration14 2.19 + 2
At3g15500 ANAC055 NACdomain-containingprotein55 2.17 + +
(Tablecontinuesonfollowingpage.)
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Copyright © 2013 American Society of Plant Biologists. All rights reserved.
Cheng et al.
TableI.(Continuedfrompreviouspage.)
GeneSymbol GeneName ProductDescription FoldChange DREa GCCa
At1g56280 DI19 Drought-induced19 2.13 +++ +
At3g06760 – Drought-responsivefamilyprotein 2.03 + +
At1g32640 MYC2 MYCtranscriptionfactor2 2.01 2 2
Saltstressonly
At1g73260 KTI1 Kunitztrypsininhibitor1 23.14 + 2
At4g11650 OSM34 Osmotin34 21.71 + 2
At4g16260 – Glycosylhydrolasesuperfamilyprotein 20.22 2 2
At4g12720 NUDT7 Nudixhydrolasehomolog7 12.29 2 2
At4g14630 GLP9 Germin-likeprotein9 9.03 + 2
At3g23250 MYB15 MYBtranscriptionfactor15 8.38 2 2
At5g54230 MYB49 MYBtranscriptionfactor49 7.96 2 2
At5g62520 SRO5 SimilartoRCDOne5 7.61 + 2
At1g25220 ASB1 Anthranilatesynthaseb-subunit1 5.04 2 2
At1g02920 GSTF7 GlutathioneS-transferasef7 4.89 + 2
At3g02140 TMAC2 TwoormoreABREs-containinggene2 4.69 2 2
At4g05100 MYB74 MYBtranscriptionfactor74 4.22 2 2
At5g39610 NAC6 NACdomaintranscriptionfactor6 4.10 2 +
At5g07440 GDH2 Gludehydrogenase2 3.96 2 2
At1g48000 MYB112 MYBtranscriptionfactor112 3.90 + +
At1g03220 – Eukaryoticaspartylproteasefamilyprotein 3.60 2 +
At1g28380 NSL1 Necroticspottedlesions1 3.39 + +
At1g55450 – S-Adenosyl-L-Met-dependentmethyltransferasesuperfamilyprotein 3.36 + 2
At3g25780 AOC3 Alleneoxidecyclase3 3.18 + 2
At3g57530 CPK32 Calcium-dependentproteinkinase32 3.17 + +
At3g44540 FAR4 Fattyacidreductase4 3.16 + 2
At4g21440 MYB102 MYBtranscriptionfactor102 3.14 + 2
At1g03230 – Eukaryoticaspartylproteasefamilyprotein 3.11 2 2
At5g02020 SIS Salt-inducedSerrich 3.07 2 2
At5g67480 BT4 BTBandTAZdomainprotein4 3.07 +++ 2
At4g08500 MEKK1 MAPK/ERKkinasekinase1 2.82 +++ 2
At3g48360 BT2 BTBandTAZdomainprotein2 2.78 ++ 2
At3g21780 UGT71B6 UDP-glucosyltransferase71B6 2.77 ++ 2
At2g47730 GSTF8 GlutathioneS-transferasef8 2.68 ++ +
At5g44610 MAP18 Microtubule-associatedprotein18 2.53 +++ 2
At5g43170 ZF3 Zincfingerprotein3 2.35 +++ 2
At4g37530 – Peroxidasesuperfamilyprotein 2.26 ++ +
At4g02520 GSTF2 GlutathioneS-transferasef2 2.23 +++ 2
At2g38380 – Peroxidasesuperfamilyprotein 2.20 ++ +
At1g18570 MYB51 MYBtranscriptionfactor51 2.17 ++ +
At1g01140 CIPK9 CBL-interactingproteinkinase9 2.15 +++ 2
At5g67450 ZF1 Zincfingerprotein1 2.10 2 2
At1g50460 HKL1 Hexokinase-like1 2.09 + 2
At5g24470 VSP2 Vegetativestorageprotein2 2.03 + 2
Heatstressonly
At5g52640 HSP90.1 Heatshockprotein90.1 8.61 +++ 2
At5g51440 – HSP20-likechaperonessuperfamilyprotein 8.49 ++ 2
At5g57560 XTH22 Xyloglucanendotransglucosylase/hydrolase22 7.76 2 +
At3g12580 HSP70 Heatshockprotein70 7.47 + +
At5g03720 HSFA3 HeatshockfactorA3 6.19 + 2
At4g12400 HOP3 Carboxylateclamp-tetratricopeptiderepeatproteins 5.80 ++ 2
At4g25200 HSP23.6-MITO Mitochondrion-localizedsmallheatshockprotein23.6 5.12 + 2
At1g74310 HSP101 Heatshockprotein101 4.99 ++ 2
At1g07400 – HSP20-likechaperonesuperfamilyprotein 4.65 + 2
At5g07100 WRKY26 WRKYDNA-bindingprotein26 3.92 + +
At3g63350 ATHSFA7B HeatshockfactorA7B 3.81 + 2
At5g47910 RBOHD NADPH/respiratoryburstoxidaseproteinD 3.41 2 +
At1g21910 DREB26 Dehydrationresponseelement-bindingprotein26 3.29 + 2
At3g08970 TMS1 Thermosensitivemalesterile1 2.93 2 ++
At1g53540 – HSP20-likechaperonesuperfamilyprotein 2.78 + +
(Tablecontinuesonfollowingpage.)
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Multiple Functions of ERF1 in Stress Responses
TableI.(Continuedfrompreviouspage.)
GeneSymbol GeneName ProductDescription FoldChange DREa GCCa
At3g51910 HSFA7A HeatshockfactorA7A 2.74 + 2
At2g26150 HSFA2 HeatshockfactorA2 2.71 + 2
At1g56410 ERD2 Earlyresponsetodehydration2 2.67 + 2
At5g12030 HSP17.6A Heatshockprotein17.6A 2.43 2 +
At4g17250 – Heatacclimation 2.33 ++ 2
At5g37770 CML24 Calmodulin-like24 2.07 + 2
aDRE (G/ACCGAC) or GCC (GCCGCC) in the 3,000-bp upstream region from the 59 end of the longest complementary DNA. b–, Not
applicable.
At3g02480) under drought stress treatment (Fig. 9B). bound the DRE second closest to the start site
Moreover,ERF1alsoboundtotheDREelementinthe (Supplemental Fig. S9).
promotersofsalt-responsive(P5CS1,SRO5,GLP9,and
ATOSM34) and heat shock-responsive (ATHSFA3,
HSP101, HSP70, and HSP23.6-M) genes in a salt- or DISCUSSION
heatshock-specificmanner(Fig. 9B).ERF1alsobound
ERF1 has been proposed to regulate Arabidopsis
the GCC box in the promoters of JA-responsive genes
resistance to the necrotrophic fungi B. cinerea and
(b-CHI, PDF1.2, ELI3-2, and GSTF7; Fig. 9B). ChIP
P. cucumerina by integrating ET and JA defense re-
assays showing a lack of ERF1 binding to other
sponses(Berrocal-Loboetal.,2002;Lorenzoetal.,2003).
promoter regions not containing the GCC box or
DRE demonstrated the specificity of the ChIP assays Although ERF1 has been reportedto beinducedby salt
stress and its expression was alteredin a salt-sensitive
(Supplemental Fig. S6). For the genes that were up-
mutant (Cela et al., 2011), no significant evidence has
regulated in both drought and high salinity, such as
been provided for its role in abiotic stress. Emerging
RD29B,ERD7,andRD20,weobservedERF1bindingto
evidence suggests that hormone signaling pathways
DRE elements of their promoters under both stresses
regulatedbyABA,salicylicacid,JA,andET,aswellas
(SupplementalFig.S7).Interestingly,ERF1boundtothe
reactive oxygen species signaling pathways, play key
promoters of nearly all of these genes under normal
rolesin thecrosstalk betweenbioticandabioticstress
growth conditions (Fig. 9B). This indicated that there
was stress-specific recruitment or blocking of some signaling. Several factors, including transcription fac-
tors and kinases, may be common players that are
genesthatledtodifferentpatternsofERF1bindingin
involved in cross talk between stress signaling path-
different stresses.
ways. Several novel observations presented here shed
Among the ERF1-regulated genes, some have both
new light on the role of ERF1. We discovered a novel
GCCboxandDREelementsintheirpromoter,andsome
functionofERF1positiveregulationofbothbioticand
of them possess more than one DRE element. To inves-
abiotic stress responses, such as drought, salinity, and
tigate the ERF1 binding preferences of GCC boxes and
heat shock stress, by binding to different cis-elements
DRE elements present in the same promoter, primers
(DRE element or GCC box) in response to different
were designed to specifically amplify GCC boxes and
stress signals. Along with the expression pattern of
DRE elements from the promoters of b-CHI, PDF1.2,
ERF1 and stress resistance phenotypes of ERF1 over-
ELI3-2, GEA6, LEA4-5, and HSP70 (Supplemental Fig.
expression plants, these data suggest that ERF1 may
S8A). These assays showed that ERF1 preferentially
act as a master integrator between biotic and abiotic
binds to the GCC box in promoters of JA-responsive
stress signals.
genes (b-CHI, PDF1.2 , andELI3-2; Supplemental Fig.
S8B). Conversely, ERF1 preferentially bound DRE ele-
Stress-SpecificBinding ofERF1toGCCBoxandDRE
ments in the promoters of drought-responsive genes
PromoterElementsIsaMechanismtoControltheCross
(GEA6andLEA4-5)aswellastheheatshock-responsive
TalkofDifferentStress Signals
gene HSP70 (Supplemental Fig. S8, C and D). These
resultsindicatedthatERF1preferentiallyboundtoGCC There are 147 AP2/ERF transcription factors in the
boxesinthepromotersofbioticstress-responsivegenes Arabidopsis genome, which can be divided into four
and preferentiallybound toDRE elementpromoters of subfamilies, of which DREB and ERF members ac-
abioticstress-responsivegenes.Incaseswheremultiple countforover85%ofthewholefamily(Sakumaetal.,
DRE elements were present in the same promoter 2002; Feng et al., 2005; Nakano et al., 2006). Members
(RD20, RD29B, COR15B, COR47, and HSP101), ERF1 of different subfamilies were reported to display dis-
bound specifically to only one of the DRE elements tinctDNA-bindingactivities.Forexample,severalERF
(SupplementalFig. S9, B and C). ERF1 binding usually proteins bind to the GCC box AGCCGCC (Ohme-
occurred at the DRE element nearest to the 59 tran- Takagi and Shinshi, 1995; Hao et al., 1998; Fujimoto
scriptional initiation site of the target gene. An excep- et al., 2000; Hao et al., 2002), some proteins of the
tiontothistrendwasCOR15B,whereERF1specifically DREBsubfamilybindtheDREortheC-repeatelement
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