The SsDREB Transcription Factor from the Succulent Halophyte Suaeda salsa Enhances Abiotic ...

14 Pages·2015·2.61 MB·English

Hindawi Publishing Corporation
International Journal of Genomics
Volume 2015, Article ID 875497, 13 pages
http://dx.doi.org/10.1155/2015/875497
Research Article
SsDREB
The Transcription Factor from
Suaeda salsa
the Succulent Halophyte Enhances
Abiotic Stress Tolerance in Transgenic Tobacco
XuZhang,1XiaoxueLiu,1,2LeiWu,1GuihongYu,1XiueWang,2andHongxiangMa1
1ProvincialKeyLaboratoryofAgrobiology,InstituteofBiotechnology,JiangsuAcademyofAgriculturalSciences,Nanjing210014,China
2NationalKeyLaboratoryofCropGeneticsandGermplasmEnhancement,CytogeneticsInstitute,NanjingAgriculturalUniversity,
Nanjing210095,China
CorrespondenceshouldbeaddressedtoHongxiangMa;[email protected]
Received19April2015;Accepted7June2015
AcademicEditor:Maria´nBresticˇ
Copyright©2015XuZhangetal.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttributionLicense,which
permitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
Dehydration-responsiveelement-binding(DREB)transcriptionfactor(TF)playsakeyroleforabioticstresstoleranceinplants.In
thisstudy,anovelcDNAencodingDREBtranscriptionfactor,designatedSsDREB,wasisolatedfromsucculenthalophyteSuaeda
salsa.ThisproteinwasclassifiedintheA-6groupofDREBsubfamilybasedonmultiplesequencealignmentsandphylogenetic
characterization.Yeastone-hybridassaysshowedthatSsDREBproteinspecificallybindstotheDREsequenceandcouldactivate
theexpressionofreportergenesinyeast,suggestingthattheSsDREBproteinwasaCBF/DREBtranscriptionfactor.Real-time
RT-PCRshowedthatSsDREBwassignificantlyinducedundersalinityanddroughtstress.OverexpressionofSsDREBcDNAin
transgenictobaccoplantsexhibitedanimprovedsaltanddroughtstresstoleranceincomparisontothenontransformedcontrols.
Thetransgenicplantsrevealedbettergrowth,higherchlorophyllcontent,andnetphotosynthesisrate,aswellashigherlevelof
prolineandsolublesugars.ThesemiquantitativePCRoftransgenicsshowedhigherexpressionofstress-responsivegenes.These
datasuggestthattheSsDREBtranscriptionfactorisinvolvedintheregulationofsaltstresstoleranceintobaccobytheactivation
ofdifferentdownstreamgeneexpression.
1.Introduction systems to engineer stress tolerance [5]. The Dehydration-
responsiveelement-bindingproteins(DREBs)aremembers
The abiotic stresses like salinity, drought, and low and high oftheAPETALA2/ethylene-responsiveelement-bindingfac-
temperaturenegativelyaffectplantgrowthandproductivity tor(AP2/ERF)familyoftranscriptionfactorsinthepromot-
[1]. They are major limiting factors for sustainable food ersofstress-induciblegenes[6].
productionastheyreduceyieldsbymorethan50%incrop GenesincludedintheDREBsubfamilyaredividedinto
plants [2]. To overcome these limitations, plants have gen- six small subgroups (A-1 to A-6) based on similarities in
eratedmechanismstotriggeracascadeofeventsleadingto the binding domain. The A-1 subgroup, which includes
changesingeneexpressionandsubsequentlytobiochemical the DREB1/CBF- (C-repeat binding factor-) like genes, are
physiological modifications that can enhance their stress mainlyinducedbylowtemperatureandactivatetheexpres-
tolerance [3]. Molecular and cellular responses to abiotic sion of many cold stress-responsive genes, whereas the A-2
stressesinvolvesignalperception,transductionofthesignal subgroup, which is comprised of the DREB2 genes, mainly
to the cytoplasm and nucleus, alteration of gene expression functionsinosmoticstress[7].Inaddition,multipleresearch
and, finally, metabolic changes that lead to stress tolerance reports indicated that the genes on the CBF/DREB family
[4]. Numerous abiotic stress-related genes and transcrip- playveryimportantrolesinregulatingabioticstressviaABA-
tion factors (TFs) have been isolated from different plant independent/dependent pathway [8–10]. It suggested that
speciesandoverexpressedinhomologousandheterologous CBF/DREBplaysdistinctiverolesinplantresponsetostress
2 InternationalJournalofGenomics
Table1:PrimersusedforRACE-PCRamplification.
󸀠 󸀠
Primername Oligonucleotides(5-3) Use
DREB-C1 TGGGGKAARTGGGTYGCHGARATYCG AP2/ERFdomain
DREB-C2 ACDGADGARTGNAGWGGYTTRTA AP2/ERFdomain
󸀠 󸀠
5AAP GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG 5 UniversalPrimer
󸀠 󸀠
5AUAP GGCCACGCGTCGACTAGTAC 5 UniversalPrimer
󸀠
5GSP1 TGACCAAACTAGACTCCCTCTAACA SsDREBa5 RACE
󸀠
5GSP2 AGTATTGCTCCGCTCCTAACTCTT SsDREBa5 RACE
󸀠 󸀠
3AUAP GGCCACGCGTCGACTAGTAC 3 UniversalPrimer
󸀠
3GSP1 GACTACCCAAGAACCGAACCCGGTT SsDREBa3 RACE
󸀠
3GSP2 GGTTATGGCTTGGATCCTTCGATAC SsDREBa3 RACE
SsDREB-G1 ATGGCAGCTACAACAATGGATATG cDNA
SsDREB-G1 TTAAGATGATGATGATAAGATAGC cDNA
[11]andthattheremightalsobeacrosstalkbetweendrought Salinity, dehydration, and ABA stress treatments were
and cold responsive genes with a DRE element [6]. DREB2 performed on S. salsa by transferring 3-week-old seedlings
homologous genes have been isolated from a variety of in Hoagland nutrient solution supplemented with 250mM
species[12].TransgenicplantsoverexpressingeitherDREB1 NaCl,20%PEG6000,and100𝜇m/LABA,respectively.Low
or DREB2A genes enhanced tolerance to abiotic stress [13– temperature treatments were performed by transferring
∘
16]. plants to a growth chamber set to 4 C under the light and
Todate,onlyfeweffortsaremadeinhalophytesinresponse thephotoperiodicconditionsdescribedabove.Sampleswere
to salt stress. The expression of AhDREB1 from Atriplex harvested at 0, 0.5, 2, 4, 8, 12, and 24h after treatment
hortensis was observed in salt stress [17], while AsDREB and immediately stored at −80∘C for further study. All
fromAtriplexhalimuswasinducedbyonlydehydration[18]. experimentswererepeatedinbiologicaltriplicates.
PpDBF1fromPhyscomitrellapatenswasinducedundersalt,
dehydrationaswellascoldstress[19],whileSbDREB2Afrom 2.2. Gene Isolation and Sequencing Analyses. Total RNA
SalicorniabrachiatawasinducedbyNaCl,drought,andheat was extracted from the leaves of S. salsa, treated with
stress[20]. 400mMNaClfor6hutilizingSVTotalRNAExtractionKit
SuaedasalsaisanativehalophyteinChinaforbothindus- (Promega,USA)accordingtotheinstruction.Theconserved
trial application and scientific research [21]. Fresh branches AP2/ERF domain of DREB genes in S. salsa was amplified
of S. salsa are highly valuable as a vegetable, and the seeds by primers DREB-C1 and DREB-C2, designed from the
can produce edible oil [21]. It can grow both in saline soils knownDREB/CBFgenesintheGenBankdatabase.Isolation
and in the intertidal zone where soil salt reaches up to 3%. of the cDNA sequences was carried out using the RNA
󸀠 󸀠
TreatmentofS.salsawith200mMNaClcouldsignificantly ligase-mediatedrapidamplificationof5 and3 ends(RLM-
increase its growth and net photosynthetic rate [22]. The RACE)method,accordingtotheGeneRacerKit(Invitrogen,
high salt tolerance might be partly the result of its efficient USA). Gene-specific nested primes 5GSP1, 5GSP2, 3GSP1,
antioxidative system [22]. For instance, Mn-SOD and Fe- and 3GSP2 were designed based on the known genomic
SODactivitiesintheleavesofS.salsaseedlingsweresignif- sequences. Sequences of all relevant primers are listed in
icantlyhigherunderNaClstressconditions(100mmolL−1) Table1.
󸀠 󸀠
thanthoseundernon-NaClstressconditions[22].However, The 5 - and 3 -RACE fragments were cloned into sep-
the mechanism of abiotic-stress-tolerance in S. salsa is still arate pGEM-T Easy plasmid vectors (Promega, USA) and
poorlyunderstood.Inthepresentstudy,wereportthecloning sequenced.ThecDNAsequencesofSsDREBwereamplified
and characterization of the SsDREB cDNA. Its expression byPCRusingtheforwardprimerSsDREB-G1andthereverse
patternwasinvestigatedinresponsetoexogenousABA,salt, primerSsDREB-G2(Table1).PCRwasperformedwitha5-
∘
cold, and drought stress treatments. Overexpression of this min94 Cdenaturationstep,followedby30cyclesof45sat
∘ ∘ ∘
cDNA in transgenic tobacco led to enhanced tolerance to 94 C,45sannealingat55 C,a1-minextensionat72 C,anda
salinityanddehydrationstresses. finalextensionperiodof10min.
Sequence analyses were performed using the program
BLASTX (National Centre for Biotechnology Information,
2.MaterialsandMethods
USA). The ORF of SsDREB genes and the properties of
2.1. Plant Materials and Stress Treatment. Seeds of S. salsa protein encoded by them were predicted by DNAStar soft-
weregerminatedandpreculturedinpotscontainingvermi- ware. The conserved AP2 domains (Accession number:
culitewithHoaglandnutrientsolutioninagrowthchamber smart00380) were originally applied as a seed sequence to
(20/25∘C,16hlight/8hdark)under250mE⋅m−2 ⋅s−1 light search the NCBI database (http://www.ncbi.nlm.nih.gov/)
intensity. and 33 proteins were retrieved with an expected value of
InternationalJournalofGenomics 3
Table2:PrimersusedforqRT-PCRamplification.
󸀠 󸀠
Primername Oligonucleotides(5-3) Use
SsDREB-R1 AGAGGGAGTCTAGTTTGGTCATT Real-timeqRT-PCR
SsDREB-R2 TTTGGAGCCCCTACAATTTC Real-timeqRT-PCR
SsACTIN-R1 ACCGTTCCAATCTATGAGG Referencegene
SsACTIN-R2 CGTAAGCCAACTTCTCCT Referencegene
󸀠
100. Multiple alignments were prepared using ClustalW pair:forward,5 -GCCTCTAGAATGGCAGCTACAACAAT
󸀠 󸀠
[23] using default parameters (gap opening penalty = 10, GGATATG-3 (XbaI site underlined) and reverse, 5 -
󸀠
gap extension penalty = 0.2). The resulting alignments of GCCCCCGGGTTAAGATGATGATGAT AAGATAGC-3
complete protein sequences were used in MEGA (version (SmaIsiteunderlined).ThePCRproductwasfusedintothe
5) [24] for the construction of unrooted phylogenetic trees binary plant transformation vector pCAMBIA2301 under
usingtheneighbor-joining(NJ)methodaccordingtoJones- thecontroloftheCaMV35Spromoter.Theconstructswere
Taylor-Thorntonmodelwithuniformratesamongsitesand mobilizedtoAgrobacteriumtumefaciensstrainEHA105.This
completedeletionofgapsdata.Thereliabilityoftheobtained Agrobacteriumstrainwasusedfortransformationintobacco
treeswastestedusingbootstrappingwith500replicates. leafdiscsfollowingthestandardprotocol[27].Theputative
transgeniclinesselectedonmediumcontaininghygromycin
2.3. DRE Binding and Transcriptional Activity of FeDREB1 wereconfirmedbyPCRwithgene-specificprimers.
inYeast. TheDNA-bindingactivityofSsDREBproteinwas The seeding of transgenic tobacco plants was selected
measured using a yeast one-hybrid system. Three tandem on solid 1/2 MS medium containing 100𝜇g/mL kanamycin
∘
repeatsofthecoresequenceoftheDRE(TACCGACAT)and under long-day condition (16h light/8h dark) at 25 C. The
its mutant (mDRE) sequence (TATTTTCAT) were cloned transgenic lines of tobacco plants were confirmed by qRT-
into the Sac I/Spe I restriction sites of the plasmid pHIS2.1 PCRanalysis.
cloning reporter vector upstream to the HIS3 minimal
promoter according to the protocol described by Clontech 2.6.SalinityandDroughtStressToleranceEvaluationinTrans-
(Clontech, Mountain View, CA, USA). The entire coding genic Plants. Independent homozygous transgenic plants
region of SsDREB was cloned into the Sma I site of the lines and homozygous wild-type transgenic with pCAM-
YepGAP expression vector containing no GAL4 activation BIA2301empty vector(WT)were preculturedinMS liquid
∘
domain (AD) [25]. The recombinant YepGAP expression mediumfor4daysingrowthchamber(20/25 C,16hlight/8h
vector containing SsDREB cDNA and the pHIS2.1 vector dark) under 250mE/m2/s light intensity. Then, both plants
containingthreetandemrepeatsoftheDREormDREwere were transferred in an aqueous MS medium supplemented
cotransformedintotheyeaststrainY187.Thegrowthstatusof withPEG6000(0,5,1015,and20%)orNaCl(0,50,100,150,
thetransformedyeast wascomparedonSD/-Leu-Ura-His+ 200,250,and300mM)for2days.Leaveswithandwithout
10mM3-ATplatestotesttheexpressionoftheHISreporter stresstreatmentsweresampledforphysiologicalparameters.
gene.EmptyYepGAPwasusedasanegativecontrol.
2.7. Measurement of Photosynthetic and Chlorophyll Fluo-
2.4. Gene Expression Assay by Quantitative Real-Time RT- rescence Parameters. Leaf net photosynthetic rate (𝑃𝑛) was
PCR. Total RNA was extracted from the roots, stem, and measuredusingaportableinfraredgasanalyzer(LI6400XT
leaves using SV Total RNA Extraction Kit (Promega, USA) portablephotosyntheticsystem,Lincoln,USA).Chlorophyll
according to the instruction. First-strand cDNA was pro- indexwasmeasuredusingchlorophyllcontentmeter(FMS-2
duced from 1𝜇g of RNA using PrimeScript RT reagent Kit PulseModulatedFluorometer,HansatechInc.,UK).
(Takara,Dalian,China),accordingtothemanufacturer’spro-
tocol.Eachsamplewasamplifiedinbiologicalandtechnical 2.8. Measurement of Free Proline and Soluble Sugars Con-
triplicate by quantitative real-time RT-PCR using a Roche tent. Fresh leaf material (0.3g) was extracted with 5mL
2.0Real-TimePCRDetectionSystemwiththeSYBRGreen of deionized water at 100∘C for 10min, and shaken with
Supermix (Takara, Dalian, China). The reaction mixture 0.03g of permutit for 5min. The extract was separated by
∘
was cycled as follows: 30s denaturation at 95 C, then 40 centrifugationat3,000rpmfor10min,andthentheproline
∘ ∘ ∘
cycles of 5s at 95 C, 10s at 60 C, and 20s at 72 C. The content of the aqueous extract was determined using the
amplificationofS.salsaActingene(FJ587488)wasusedasthe acidninhydrinmethod.Theorganicphasewasdeterminedat
normalizationcontrol.ThemRNAfolddifferencewasrelative 515nm.Theresultingvalueswerecomparedwithastandard
to that of untreated samples used as calibrator. The relative curveconstructedusingknownamountsofproline(Sigma).
−ΔΔCT
quantificationvalueforSsDREBwascalculatedbythe2 Fresh leaf material (0.2g) was extracted with 80% (v/v)
∘
method[26].Allrelevantprimersusedinthisworkarelisted ethanol at 70 C for 30min. The extract was separated by
inTable2. centrifugation at 12,000rpm for 10min and diluted with
water to 10mL. Then, the soluble sugar content of the
2.5.GenerationofTransgenicTobacco. Togeneratetransgenic aqueousextractwasdeterminedusingsulfuricacidanthrone
plants,SsDREBcDNAwasamplifiedusingaspecificprimer colorimetric method. The resulting values were compared
4 InternationalJournalofGenomics
Table3:PrimersofdownstreamgenesofSsDREBforsemiquantitativeRT-PCR.
󸀠 󸀠
Gene(GeneBankID) Oligonucleotides(5-3)
𝛼-tublin(AJ421412) TAACCATCATAGAAGAGGGTTC
GCAATCCTTCTTGACAATGAGG
TTGGCCTTCTACTTCCATCC
GlutathioneS-transferase(D10524)
TGTCAACTGCAACCATGAGAG
TGTCACGGGACCACATTAC
Cu/ZnSOD(EU123521)
CACCAGCATTTCCAGTAGC
GTGCCAGGTGGAGTGAGAGG
Lea5(AF053076)
GGGACGTGGTATGGTAACCA
AATAGCTGGGAAAATTGCATG
lipidtransferase(ltp1)(X62395)
CAGTGGAAGGGCTGATCTTG
TCTTCACCAGTCCAGCCTGAC
H+-ATPaseBsubunit(AF220611)
GAAGGAACATCTGGAATTGAC
TCAGCAGGAATGATGTCTCC
H+-ATPase(X66737)
TCATGGAAGCTGCTGCTGTC
AGGGGAAATGTTATTGTCTCC
Peroxidase(AY032675)
CACATTGGGAAGTACCACTAG
GAAATCCTGGCTCCGCTCTG
TOBPXD(D11396)
TGGAGTTGCCTTGGTAAGAG
withastandardcurveconstructedusingknownamountsof protein and DRE element, the recombinant plasmid pAD-
sugar. SsDREB was separately transformed into yeast strain Y187
containing the reporter genes HIS3 under the control of
DRE.Asnegativecontrols,pAD-SsDREBwasalsoseparately
2.9. Semiquantitative RT-PCR for Expression Analysis of
transformed into Y187 harboring the reporter genes HIS3
Downstream Genes of SsDREB. Semiquantitative RT-PCR
under the control of a mutant DRE (mDRE) (Figure2(a)).
amplification was performed with selected gene primers
These results suggested that the DRE::pAD-SsDREB trans-
(Table3),usingthefirststrandcDNA,synthesizedfromRNA
genicyeastcellsgrewwellonSD/-His10mM3-AT,whereas
samplescollectedfromWTandtransgenictobaccoseedlings.
the yeast cells harboring mDRE::pAD-SsDREB transgenic
Thereactionmixturewascycledasfollows:3mdenaturation
∘ ∘ ∘ yeastcellscouldnotgrowonthesamemedium(Figure2(b)).
at95 C,then35cyclesof45sat94 C,45sat55 C,and1m
at72∘C.TheamplificationofS.salsa𝛼-tublingenewasused These results strongly indicated that the SsDREB can bind
the normal DRE element exclusively to drive target gene
as the normalization control. PCR-amplified products were
expressioninvivo.
visualizedonethidiumbromide-stained1.5%agarosegels.
3.3. Expression of SsDREB in Response to Various Abiotic
3.Results Stresses. The expression pattern of SsDREB in different
organs of S. salsa was examined under normal conditions.
3.1. Isolation and Phylogenetic Analysis of SsDREB cDNA.
TheexpressionlevelofMsDREB2Cwashighestinleavesfol-
A full length-cDNA sequence, designated as SsDREB, was
lowedbyrootsandstem(Figure3(a)).Therefore,expression
isolatedfromS.salsa.ThiscDNAis1095-bplongcorrespond-
of SsDREB in leaf was investigated under different abiotic
ing to a protein of 364 amino acids. SsDREB possesses two
stresses.Quantitativereversetranscription-PCR(qRT-PCR)
regionsrichinserine,oneregionrichinglutamine,andan
revealed that the transcript of SsDREB was induced by salt
acidic C-terminal sequence, PSXEIDW, which is known to
and drought stress. SsDREB expression was induced by salt
function in transcriptional activation activity [25, 28]. The
treatmentat0.5hoursaftertreatmentandpeakedat4h,with
putativeaminoacidsequenceshowedthatthe SsDREBhad
thehighestabundanceofabout16-foldincrease(Figure3(b)).
a conserved EREBP/AP2 domain of 64 amino acids with
The expression increased slowly from 0.5h but rapidly
valine (V) and leucine (L) at the 14th and 19th residues,
peaked at 8h and then decreased gradually under mimic
respectively (Figure1(a)). Phylogenic tree analysis of DREB ∘
dehydration stress (Figure3(b)). Under cold stress (4 C)
proteins showed that SsDREB, together with Arabidopsis
treatment, SsDREB expression was gradually declined and
RAP2.4,ZmDREB1,OsDBF1,andChDREB2,isattributable
thenslightlyrecoveredafter4haftertreatment(Figure3(c)).
totheDREB(A-6)lineage(Figure1(b)).
Similarly, there was no significant expression change of
SsDREB after exogenous ABA application, indicating that
3.2. SsDREB Protein Specifically Binds to the DRE Element. StDREB1 may function in an ABA-independent signaling
To verify the possible binding function between SsDREB pathway(Figure3(c)).
InternationalJournalofGenomics 5
(1) 1 10 20 30 40 58
AP2 (1) HYRGVRQRPWGKFVAEIRE---KGARVWLGTFETAEDAALAYDRAAFRMRGSRALLNF
AtDREB2A (1) SFRGVRQRIWGKWVAEIREP-NRGSRLWLGTFPTAQEAASAYDEAAKAMYGPLARLNF
GhDREB1 (1) PFRGIRMRKWGKWVAEIREP-NKRSRIWLGSYTTPVAAARAYDTAVFYLRGPSARLNF
OsDREB1A (1) VFRGVRRRNAGRWVCEVRVPGRRGCRLWLGTFDTAEGAARAHDAAMLAIN-AACCLNF
SiABI4 (1) RYRGVRQRSWGKWVAEIREP-RKRSRKWLGTFATAEDAARAYDRAALLLYGPRAHLNL
SsDREB (1) LYRGVRQRHWGKWVAEIRLP-KNRTRLWLGTFDTAEEAALAYDKAAYKLRGDFARPNF
ZmDREB2 (1) TFRGVRMRAWGKWVSEIREP-RKKSRIWLGTFPTAEMAARAHDVAALAIKGRAAHLNF
Consensus (1) FRGVRQR WGKWVAEIREP RKRSRLWLGTF TAEDAARAYD AA LRG A LNF
(a)
AtCBF1
76 AtCRT/DREB2
AtCRT/DREB3
91
100NtDREB1A
100 CbCBF
BnCBF-like
97 97 BnCBF16
GhDREB1-like
PaDREB1
DREB A-1
99 SlCBF1
66 85 SlCBF2
95 CaCBF1B
92 SlCBF3
99
OsDREB1B
100 OsDREB1A
100 95 ZmDREB-like
OsDREB1F
AtTINY
63 DREB A-4
87 ZmDREB2
100 SiABI4 DREB A-3
BdABI4-like
97 GmDREB
AtDREB2B
99
EsDREB2A DREB A-2
100
59 AtDREB2A
50 BrDREB2A-like
97 GhDREB1
68 GmDREB2 DREB A-5
AtRAP2.1
94 ZmDREB1
OsDBF1
100 SsDREB DREB A-6
100 AtRAP2.4
68 GhDREB2
0.2
(b)
Figure 1: Conserved domain and phylogenetic analysis of SsDREB protein. (a) SsDREB protein has the same AP2 domain compared
with other DREB proteins from Arabidopsis thaliana, Zea mays, Gossypium hirsutum, Setaria italica, and Oryza sativa. The 14th
valine and the 19th leucine acid inside the AP2/ERF domain are presented in boxes. (b) Phylogenetic analysis of proteins from
DREB subfamily. The number was the bootstrap value of the clade and low bootstrap values (<50) were removed from the tree. The
accessionnumberofeachappendedproteinisasfollows:AtCBF1(AAC49662),AtCRT/DREB2(AAD15976),AtCRT/DREB3(AAD15977),
NtDREB1A (ABD65969), CbCBF (AAR26658), BnCBF-like (AAL38242), BnCBF16 (AAM18960), GhDREB1-like (ABD65473), SlCBF3
(AAS77819), SlCBF2 (AAS77821), PaDREB1 (BAD27123), CaCBF1B (AAQ88400), OsDREB 1B (AAN02488), OsDREB 1A (AAN02486),
ZmDREB2 (AAM80485), ZmDREB-like (AAN76804), AtRAP2.1 (AAC49767), AtRAP2.4 (AAC49770), SlCBF1 (AAK57551), AtTINY
(CAA64359),AtDREB2B(BAA33795),AtDREB2A(BAA36705),ZmDREB1(AAM80486),AtAP2(AAC39489),ZmERF/AP2(BAE96012),
GmDREB (AAP83131), EsDREB2A (AAS58438), OsDBF1 (AAP56252), GhDREB1 (AAO43165.1), CbCBF25 (AAR35030), GmDREBa
(AAT12423), BdABI4-like (XP 003568646), SiABI4-like (XP 004963859), BrDREB2A-like (XP 009125600), BpDREB (ABB89755.1),
GmDREB(ABB36645),OsDREB1F(AAX23723),andGhDREB2(AAT39542).
6 InternationalJournalofGenomics
SsDREB
PAGP TADH1
DRE DRE DRE PminHis3 His3
mDRE mDRE mDRE PminHis3 His3
(a)
DRE yeast cell mDRE yeast cell
mPAD wPAD
mDRE wDRE
(b)
Figure2:AnalysisofSsDREBbindingtotheDREelementintheyeastone-hybridsystem.(a)ConstructionoftheYepGAP-SsDREBplasmid.
TheentireSsDREBcodingregionwasfusedtotheactivationdomainofGAL4.RecombinantYepGAP-SsDREBorplasmidwastransformed
into yeast cells that are harboring two reporter genes under the control of either wild-type or mutant DRE motifs. PGAP and TADH1
indicatethepromoterandterminatorofADH1gene,respectively.(b)Transformedyeastcellswereexaminedforgrowthonselectivemedium
(SD−His+10mM3-AT)at30∘C(left).Leftpanelshowsthepositionofeachtransformedyeastcell.TheemptyYepGAP(PAD)wasusedasa
control.wDREandwPADindicateyeastcellsharboringDREBproteinsandDRE-controlledreportergenes,whilemDREandmPADindicate
yeastcellsharboringDREBproteinsandmDRE-controlledreportergenes.
3.4. Confirmation of Putative Transgenic Tobacco Plants decrease in leaf size was observed in both transgenic and
Expressing SsDREB. The putative transgenic lines selected WT plants. The salt stress proved more detrimental in the
on medium containing hygromycin were confirmed by WTplantsascomparedtotransgenicseedlings.At300mM
PCR with gene-specific primers using primers the pCAM- NaCl,thetransgenicplantsshowedbettergrowthundersalt
BIA2301 binary vector corresponding to sequences flank- stress with larger leaf area and higher turgor maintenance
ing the SsDREB cDNA. As expected, a PCR product of pressure(Figure5(b))ascomparedtoWT.At20%PEG,the
1095bpwasobtained(Figure4(a)).PCR-positiveplantswere transgenic plants showed significantly better growth under
successfully transferred to green house for further analysis. stress with larger leaf area and higher turgor maintenance
Positivetransgenic lines also showed expression of SsDREB pressure(Figure5(c))ascomparedtoWT.
bysemiquantitativeRT-PCR,whereasexpressionofSbDREB
wasnotobservedinWTplants(Figure4(b)).Nophenotypic 3.5.2. Photosynthesis and Chlorophyll Fluorescence Parame-
modificationsuchasdwarfismwasnoticedintheseSsDREB ters. Netphotosynthesisrate(𝑃𝑛)andstomatalconductance
transgenicplantlines. (𝐺𝑠) in WT and transgenic plants were similar under con-
trol condition. Net photosynthesis rate (𝑃𝑛) was 13.8 and
3.5.TobaccoPlantsOverexpressingSsDREBEnhance 14.0𝜇mol CO2 ⋅ m−2 ⋅ s−1 in WT and transgenic plants
SalinityandDehydrationTolerance while stomatal conductance was 0.37 and 0.38𝜇mol CO2 ⋅
m−2 ⋅ s−1 under control condition. Under salinity stress
3.5.1.MorphologicalFeaturesofPlants. Allofthetransgenic net photosynthesis rate and stomatal conductance reduced
linesandWTtobaccoplantsgrewwellundernormalcondi- drastically as compared to control conditions in WT and
tion(Figure5(a)).Afterdehydrationandsalinitytreatment, transgeniclines.Transgenicsshowedsignificantlyhighernet
InternationalJournalofGenomics 7
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(a)
18 1.6
16 1.4
vel 14 vel 1.2
on le 12 on le 1.0
xpressi 180 xpressi 0.8
Relative e 46 Relative e 00..46
2 0.2
0 0.0
0 0.5 2 4 8 12 24 0 0.5 2 4 8 12 24
(h) (h)
NaCl 4∘C
PEG ABA
(b) (c)
Figure3:Quantitativereal-timePT-PCRanalysisofSsDREB.(a)TranscriptlevelsofSsDREBintheroots,stems,andleavesofuntreated
plants.(b),(c)TherelativeexpressionlevelofSsDREBinS.salsaleavesatindicatedtimepointsexposedtosalinitystress(250mMNaCl),
dehydrationstress(20%PEG),lowtemperature(4∘C),and100𝜇MABA,respectively.ColumnsindicaterelativeexpressionlevelsofSsDREB
normalizedagainstlevelsofSsActinascalculatedbyreal-timeqRT-PCR(mean±SEofthreebiologicalreplicates).
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
2000
p) 1000
(b 750
500
(a)
M 1 2 3 5 6 8 10 11 12 14 15 16 18 22 25 27
2000
p) 1000
(b 750
500
(b)
Figure4:(a)PCRamplificationofthespecificSsDREBgenefromgenomicDNAofthetransgeniclines.(b)RT-PCRanalysisofSsDREB
expressionintransgeniclines.(MmarkerDL2000,1CK+,2WT,3–27transgeniclines).
8 InternationalJournalofGenomics
(a) (b) (c)
Figure5:Phenotypeoftransgenictobaccoplantsundernormalandstresscondition.(a)Undernormalcondition.(b)Treatedwith300mM
for2days.(c)Treatedwith20%PEGfor2days.Left:WTplant;right:transgenicplan.
photosyntheticrateandstomatalconductanceatallfiveNaCl thanthatoftheWTplantswithallsalinityanddehydration
concentrationgradients,comparedtoWTplantsindicating. stress,demonstratingthattheoverexpressionofSsDREBgene
Net photosynthesis rate was reduced to 12.6, 11.1, 8.7, 6.3, could enhance plant salinity and dehydration tolerance in
and 2.2𝜇mol CO2 ⋅ m−2 ⋅ s−1 in WT plants 2 days after 50, transgenictobacco(Figures6(e)-6(f),6(k)-6(l)).
100,150,200,and250mMNaCltreatment,respectively.But
transgenicplantsmaintainednetphotosynthesisrateat13.6, 3.6. Overexpression of StDREB1 Activates the Expression of
12.5, 11.6, 10.3, and 8.9𝜇mol CO2 ⋅ m−2 ⋅ s−1 in the same Stress-Responsive Genes. Given that the SsDREB transgenic
treatment (Figure6(a)). In addition, stomatal conductance plants showed enhanced tolerance to salinity and drought
was reduced to 0.34 and 0.04mol H2O ⋅ m−2 ⋅ s−1 in WT and freezing stress, we decided to quantify the molecular
plants, while transgenic plants maintained around 0.37 and responses of eight stress-responsive genes in the transgenic
0.21H2O⋅m−2⋅s−1daysafter50and250mMNaCltreatment, lines to see the level of expression under stress conditions.
respectively(Figure6(b)).Similarly,transgenicsshowedsig- Semiquantitative RT-PCR analyses of these target genes
nificantly higher net photosynthetic rate and stomatal con- wereperformedfortheWTandfortheSsDREBtransgenic
ductanceatallfourPEGconcentrationgradients,compared tobacco plants. An increase in transcription level of these
to WT plants (Figures 6(g)-6(h)). The results showed that genes was noticed in almost all transgenic plants cultivated
SsDREB transgenic plants showed higher tolerance to salt under standard growth conditions in comparison to those
stress. in WT ones (Figure7). This most significant increase was
Chlorophyll fluorescence parameters were also inves- in expression of ltp1, Lea5, and H+-ATPase genes, while
tigated. Maximal PS II quantum efficiency (Fv/Fm) and expression of Cu/Zn SOD, TOBPXD, and GST was slightly
effective PS II quantum yield (YII) in WT and transgenic higherintransgenicplantsunderthesamesituation.Allthese
plants were similar under control condition. Fv/Fm was findingsstronglysuggestedthatSsDREBmightupregulatethe
0.84 and 0.83 in WT and transgenic plants while Y (II) expressionofstress-relatedfunctionalgenes.
was 0.78 and 0.77 under control condition.The transgenics
showedhigherFv/FmandY(II)atallfiveNaClconcentration 4.Discussion
gradients,comparedtoWTplants(Figures6(c)-6(d)).Fv/Fm
This study describes the isolation and characterization of a
was reduced to 0.82, 0.81, 0.78, 0.75, and 0.71 in WT plants
DREBfactorfromhalophyteSuaedasalsa,termedSsDREB.
2 days after 50, 100, 150, 200, and 250mM NaCl treatment,
Todate,onlyfeweffortsaremadeinhalophytesinresponseto
respectively.ButtransgenicplantsmaintainedFv/Fmat0.83
saltstress.TheAhDREB1fromAtriplexhortensisexpression
under 50 and 100mM NaCl stress and then drop slightly
wasobservedinsaltstress[17],whileAsDREBfromAtriplex
to 0.82, 0.81, and 0.80 under 150, 200, and 250mM NaCl
halimus was induced by dehydration but not in salt stress
treatment (Figure6(c)). In addition, Y(II) was reduced to
[18].PpDBF1fromPhyscomitrellapatenswasinducedunder
0.74 and 0.38 in WT plants, while transgenic plants were
salt,dehydrationaswellascoldstress[19].DREB2-typeTFs
maintainedaround0.76and0.592daysafter50and250mM
SbDREB2AfromhalophyticplantsSalicorniabrachiatawas
NaCl treatment, respectively (Figure6(d)). Similarly, the inducedbyNaCl,droughtandheatstress[20].
transgenics showed higher chlorophyll fluorescence param- ThesequenceanalysisofSsDREBidentifiedanAP2/ERF
eters at all four PEG gradients, compared to WT plants domain of 64 amino acids that is predicted to fold into
(Figures 6(i)-6(j)), indicating that expression of SsDREB in a structure containing three anti-parallel 𝛽-sheets and one
transgenictobaccoenhancedabiotictolerance. 𝛼-helix. SsDREB possessed two regions rich of serine, one
regionrichofglutamine,andanacidicC-terminalsequence,
3.5.3.ProlineandSolubleSugarContent. Prolineandsoluble PSXEIDW, which is known to function in transcriptional
sugarcontentaccumulateinplantssubjectedtosalinityand activationactivity[25,28].Thisstructureisthoughttoplay
dehydration stress conditions to confer stress tolerance in akeyroleinrecognizingandbindingtospecificcis-elements
bothtransgenicandWTplants.Thecontentsofsolublesugar [7].Sequencealignmentandphylogeneticanalysesrevealed
and free proline in transgenic plants were slightly richer thattheSsDREBgroupedwiththeDREB(A-6)lineage.Inthis
InternationalJournalofGenomics 9
16.0 16.0
14.0 14.0
) )
1 1
−s12.0 −s12.0
2· 2·
− −
m10.0 m10.0
·O2 8.0 ·O2 8.0
C C
ol 6.0 ol 6.0
m m
(𝜇 4.0 (𝜇 4.0
n n
P 2.0 P 2.0
0.0 0.0
0 50 100 150 200 250 0 5 10 15 20
NaCl content (mM) PEG content (%)
DREB DREB
CK CK
(a) (g)
0.5 0.5
) )
−1 0.4 −1 0.4
s s
2· 2·
− −
m 0.3 m 0.3
·O ·O
H2 0.2 H2 0.2
ol ol
m m
( 0.1 ( 0.1
Gs Gs
0.0 0.0
0 50 100 150 200 250 0 5 10 15 20
NaCl content (mM) PEG content (%)
DREB DREB
CK CK
(b) (h)
0.9 0.9
0.8 0.8
m m
F 0.7 F 0.7
v/ v/
F F
0.6 0.6
0.5 0.5
0 50 100 150 200 250 0 5 10 15 20
NaCl content (mM) PEG content (%)
DREB DREB
CK CK
(c) (i)
0.9 0.9
0.8 0.8
0.7 0.7
Y(II) 00..56 Y(II) 00..56
0.4 0.4
0.3 0.3
0.2 0.2
0 50 100 150 200 250 0 5 10 15 20
NaCl content (mM) PEG content (%)
DREB DREB
CK CK
(d) (j)
Figure6:Continued.
10 InternationalJournalofGenomics
5.0 5.0
g) 4.5 g) 4.5
g/ g/
m m
4.0 4.0
nt ( nt (
e 3.5 e 3.5
nt nt
co 3.0 co 3.0
gars 2.5 gars 2.5
u u
e s 2.0 e s 2.0
bl bl
u u
ol 1.5 ol 1.5
S S
1.0 1.0
0 50 100 150 200 250 0 5 10 15 20
NaCl content (mM) PEG content (%)
DREB DREB
CK CK
(e) (k)
8.0 8.0
7.0 7.0
g/g) 6.0 g/g) 6.0
𝜇 𝜇
nt ( 5.0 nt ( 5.0
nte 4.0 nte 4.0
o o
c 3.0 c 3.0
n n
oli 2.0 oli 2.0
Pr Pr
1.0 1.0
0.0 0.0
0 50 100 150 200 250 0 5 10 15 20
NaCl content (mM) PEG content (%)
DREB DREB
CK CK
(f) (l)
Figure 6: Salinity and dehydration tolerance of transgenic tobacco. (a)–(f) Salinity tolerance of transgenic plants. (g)–(l) Dehydration
toleranceoftransgenicplants.(a),(g)Netphotosynthesisrate(𝑃𝑛).(b),(h)Stomatalconductance (𝐺𝑠).(c),(i)MaximalPSIIquantum
(Fv/Fm).(d),(j)EffectivePSIIquantumyield(YII).(e),(k)Solublesugarscontent.(f),(l)Prolinecontent.For(a)–(l),eachdatapointis
meansfromthreereplicates±SE.BarsindicateSE.
WT Transgenic WT Transgenic
𝛼-tubulin 𝛼-tubulin
ltp1 TOBPXD
Lea5 Cu/ZnSOD
+
H -ATPase B subunit Peroxidase
+
H -ATPase GST
Figure7:SemiquantitativeRT-PCRofstress-responsivegenes.
study,theDSAWandLWSYmotif,theconservedsequences in the AP2/ERF domain of SsDREB protein [7]. However,
inA1-subgroup(DREB1)[29],wasnotfoundinSsDREB. the19thglutamicacidresidueisreplacedbyleucineresidue
AnumberofreportshavesuggestedthatVal14andGlu19 in the SsDREB (Figure1(a)). Similar amino acid changes
intheAP2/ERFdomainareessentialforspecificbindingto have also been observed in other plant species. In rice,
DRE[7,25].Theabsolutelyconserved14thvalineresidue,an wheat, and barley, the DREB1-type factors harbor a valine
importantsitethatactsinDNAbinding,hasalsobeenfound residue at position 19 in the AP2/ERF domain [30, 31]. The