Soybean Trihelix Transcription Factors GmGT-2A and GmGT-2B Improve Plant Tolerance to Abiotic ...

14 Pages·2009·2.85 MB·English

Soybean Trihelix Transcription Factors GmGT-2A and
GmGT-2B Improve Plant Tolerance to Abiotic Stresses in
Transgenic Arabidopsis
Zong-Ming Xie1,2,3., Hong-Feng Zou1,2., Gang Lei1,2, Wei Wei1,2, Qi-Yun Zhou1,2, Can-Fang Niu1,2,
Yong Liao1,2, Ai-Guo Tian1, Biao Ma1, Wan-Ke Zhang1, Jin-Song Zhang1*, Shou-Yi Chen1*
1NationalKeyLaboratoryofPlantGenomics,PlantGeneResearchCenter,InstituteofGeneticsandDevelopmentalBiology,ChineseAcademyofSciences,Beijing,China,
2GraduateSchoolofChineseAcademyofSciences,Beijing,China,3XinjiangAcademyofAgriculturalandReclamationScience,Shihezi,China
Abstract
Background:Trihelix transcription factors play important roles in light-regulated responses and other developmental
processes. However, their functions in abiotic stress response are largely unclear. In this study, we identified two trihelix
transcription factor genes GmGT-2A and GmGT-2B from soybean and further characterized their roles in abiotic stress
tolerance.
Findings:Bothgenescanbeinducedbyvariousabioticstresses,andtheencodedproteinswerelocalizedinnuclearregion.
In yeast assay, GmGT-2B but not GmGT-2A exhibits ability of transcriptional activation and dimerization. The N-terminal
peptideof153residuesinGmGT-2Bwastheminimalactivationdomainandthemiddleregionbetweenthetwotrihelices
mediatedthedimerizationoftheGmGT-2B.TransactivationactivityoftheGmGT-2Bwasalsoconfirmedinplantcells.DNA
binding analysis using yeast one-hybrid assay revealed that GmGT-2A could bind to GT-1bx, GT-2bx, mGT-2bx-2 and D1
whereasGmGT-2Bcouldbindtothelatterthreeelements.OverexpressionoftheGmGT-2AandGmGT-2Bimprovedplant
tolerance to salt, freezing and drought stress in transgenic Arabidopsis plants. Moreover, GmGT-2B-transgenic plants had
moregreenseedlingscomparedtoCol-0underABAtreatment.Manystress-responsivegeneswerealteredinGmGT-2A-and
GmGT-2B-transgenic plants.
Conclusion:TheseresultsindicatethatGmGT-2AandGmGT-2Bconferstresstolerancethroughregulationofacommonset
ofgenes and specific setsof genes.GmGT-2B also affects ABAsensitivity.
Citation:XieZ-M,ZouH-F,LeiG,WeiW,ZhouQ-Y,etal.(2009)SoybeanTrihelixTranscriptionFactorsGmGT-2AandGmGT-2BImprovePlantToleranceto
AbioticStressesinTransgenicArabidopsis.PLoSONE4(9):e6898.doi:10.1371/journal.pone.0006898
Editor:HanyA.El-Shemy,CairoUniversity,Egypt
ReceivedMarch19,2009;AcceptedAugust13,2009;PublishedSeptember4,2009
Copyright:(cid:1)2009Xieetal.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsunrestricted
use,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.
Funding: This work was supported by the National Basic Research Project (2006CB100102, 2009CB118402), National High Tech program (2006AA10Z113,
2007AA021402),andtheprojectfromChineseAcademyofSciences(KSCXZ-YW-N-010).Thefundershadnoroleinstudydesign,datacollectionandanalysis,
decisiontopublish,orpreparationofthemanuscript.
CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.
*E-mail:[email protected](JSZ);[email protected](SYC)
.Theseauthorscontributedequallytothiswork.
Introduction sequenceis59-G-Pu-(T/A)-A-A-(T/A)-39[14],andareinvolvedin
awidearrayofplantbiologicalprocesses.GTelementswerefirst
Transcriptional regulation of gene expression plays a primary identifiedinthepearbcS-3Agenepromoterasalight-responsive
roleinplantdevelopmentandinenvironmentalstimuliresponses. elementnamedBoxII/GT1box(59-GTGTGGTTAATATG-39)
Expressions of the stress-responsive effector genes are largely [17,18]andlaterinmanypromotersofothergenes,someofwhich
controlled by several classes of transcription factors, such as were not responsive for light [14]. For instance, a GT element
members of the MYB, ERF/AP2, bZIP, WRKY and NAC namedSite1,foundintheribosomalproteingenerps1promoter,
families,throughbindingofthecorrespondingcis-actingelements has been shown to repress transcription in non-photosynthetic
[1–7].Thepotentialforimprovingplanttolerancebyengineering tissuesorcells[19,20].BoxII-related/GT-1likeelementsfoundin
ofstress-regulatedtranscriptionfactorsishighlightedrecently[8]. the promoter region of soybean chs gene and Pr-1A gene from
SeveralclassesoftranscriptionfactorssuchasAP2/ERF,DOF, tobacco are likely responsive to elicitor treatments and pathogen
YABBY and Trihelix families are unique to plant so far [9–14], infection [21,22]. The pathogen- and NaCl- induced soybean
suggesting that they may be implicated in plant-specific gene SCaM-4 gene contains GT-1 like element in the promoter region
tuning[15].MembersofTrihelixfamily,alsoknownasGTfactors [23].
(DNA binding proteins with specificity for GT-elements), are Rice GT-2 and tobacco GT1a/B2F were the first two nuclear
among the first transcription factors identified inplants [16]. GT proteins identified via affinity screening using GT2sequence and
elements arehighly degeneratedandthededuced consensuscore BoxIIsequence[24–26].Sincethen,moremembersofGTfactor
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GTFactorsinStressTolerance
(trihelix transcriptional factor) family have been isolated from wereidentifiedinGmGT-2A(Fig.1C).However,onlythreeNLSs
Arabidopsis, pea, soybean and rice [16,27–32]. The trihelix were found in GmGT-2B and the second NLS appeared not
transcriptional factor family has 28 members in Arabidopsis present in this protein (Fig. 1C). It should be noted that an
genome[33],22membersinricegenome[34]andputatively 13 asparagine-richregionwasfoundinthemiddleoftheC-terminal
members in soybean genome [11], and is defined based on the trihelix domains of the GmGT-2A and GmGT-2B but not in
highly conserved trihelix domain (helix-loop-helix-loop-helix). otherproteinscompared(Fig.1C).Thisfeaturepossiblysuggestsa
Members of trihelix family can be divided into three subgroups potentially specific function for this region in regulation of the
thatbindtofunctionallydistincttypesofGTelements.GT-1-type transcription factor activity insoybean.
factorscontainonlyasingletrihelixdomainthatisresponsiblefor The amino acid sequences of the GmGT-2A and GmGT-2B
specific binding to the Box II core sequence, whereas GT-2-type were compared with those from other homologous proteins
factors contain twin trihelix domains with the N-terminal one (Fig. 1C). At the whole amino acid level, the GmGT-2A showed
preferentially binding to GT3-bx (59-GAGGTAAATCCGCGA- 39%identitywiththeGmGT-2(O’Gradyetal.,2001),but,25%
39) and the C-terminal one to GT2-bx(59-GCGGTAATTAA-39) identity with GmGT-2B and the other two proteins compared.
[29,30].AlthoughAtGT-3aandAtGT-3baresimilartoGT-1in GmGT-2B showed 22% identity with GmGT-2 and similar
structure that containing one single trihelix motif, both of them identity with other proteins. For the N- and C-terminal trihelix
bindspecificallytoGT-3asite(coresequence59-GTTAC,i.e.Site domains, GmGT-2A had 74% and 67% identity with those of
1),nottoBoxII,GT2-bxorGT3-bxinvitro,suggestingthatthey GmGT-2, and 40% and 53% identity with those of GmGT-2B.
belong toathird subgroupof GT factors [27]. GmGT-2B had 35% and 56% identity with those of GmGT-2.
It is generally believed that trihelix factors are involved in the These results indicate that the GmGT-2A is more closely related
regulationoflight-responsivegenes[14,35].Theexpressionofall toGmGT-2butnottheGmGT-2B.Clusteranalysisalsosupports
ofthetrihelixfactorsclonedthusfarappearstobeubiquitousand this conclusion (Fig. 1B). In addition, the soybean GmGT-2A,
independent of light, except that AtGTL1 mRNA is more GmGT-2BandGmGT-2arecloselyrelatedtotheGT-2groupof
abundant in siliques, and soybean GmGT-2 and rice rml1 (rice Arabidopsis but not clustered with other group members from
generegulatedbyMagnaporthegriseaandlight)weredown-regulated Arabidopsis (Fig.1B)[36].
by light [24–26,29,30,32]. Arabidopsis PETAL LOSS (PTL) gene
encodingaGT-2-typefactoristhefirstmemberoftrihelixfamily GmGT-2A and GmGT-2B gene expressions in different
known to control morphogenesis [28]. A rice Shattering 1(SHA1) soybean organs and in response to abiotic stresses
gene encoding a GT-1-type factor plays an important role in TheexpressionsoftheGmGT-2AandGmGT-2Bwereexamined
activationofcellseparation,andamutationinthetrihelixdomain indifferentorgansofthesoybeanplants.TheresultsinFigure2A
resultedintheeliminationofseedshatteringincultivatedrice[31]. showedthattheGmGT-2Awasexpressedinahigherlevelinstems
Morerecently,ASIL1,belongingtoanewsubfamilyofthetrihelix thanthoseinothersorganstested,andnoexpressionofthisgene
transcription factors, has been found to function as a negative was detected in soybean seeds. For the GmGT-2B, its expression
regulator of a large subset of Arabidopsis embryonic and seed washigherinpodsbutlowerinotherorgansexamined.Similarly,
maturation genes inseedlings[36]. theGmGT-2B showedno expressionin soybeanseeds.
Althoughtherolesofthetrihelixfactorsaregraduallydisclosed, Thesoybeanseedlingsweretreatedwithvariousstressesandthe
the regulatory function of this kind of transcription factors in GmGT-2AandGmGT-2Bgeneexpressionswereinvestigated.Both
abiotic stress response remains largely unknown. In a previous geneexpressionscanbeenhancedbyABA,cold,droughtandsalt
work,wehaveidentified13putativetrihelixgenesinsoybean[11]. treatments (Fig. 2B). However, the expression patterns were
Fromthese,twoabioticstress-upregulatedgenesencodingputative different.TheGmGT-2Atranscriptsaccumulatedtoahigherlevel
GT-2-type proteins (named GmGT-2A and GmGT-2B, respec- at12hafterinitiationofthetreatmentswithABA,coldordrought
tively)wereclonedfromsoybeaninthisstudy.BothGmGT-2Aand stress, whereas the GmGT-2B expression had peak levels at 1 h
GmGT-2B overexpression in Arabidopsis plants increased plant and/or3 hafterthethreetreatments.TheupregulationofGmGT-
tolerance to abiotic stresses. The downstream genes regulated by 2Aexpressionafter12-hABAtreatmentmaybeanindirecteffect.
GmGT-2A and GmGT-2B were also investigated. GmGT-2A and At 12h after these treatments, the GmGT-2B expression was
GmGT-2Bmayrepresentthefirsttwomembersoftrihelixfamily apparentlydeclined(Fig.2B).Undersaltstress,theGmGT-2Aand
that are responsible for the stress tolerance in transgenic GmGT-2B inductions were similar in patterns. These results
Arabidopsis plants through regulation of downstream genes. suggest that the GmGT-2A and GmGT-2B may be involved in
regulationofplantresponsestoabioticstresses.BecausetheGT-2
Results geneshowedsimilaritytotheGT-2AandGT-2B,wefurthertested
if this gene is responsive to stresses. However, after the four
Genecloningandstructuralanalysisofthetrihelixfamily treatments, the GT-2 expression was not significantly altered,
genes GmGT-2A and GmGT-2B indicating that the gene may not be involved in abiotic stress
AmongthirteenESTsthatbelongtotrihelixfamilygenesfrom responses(Fig. 2B).
soybean, two were identified to be responsive to various abiotic
stresses. The full-length coding regions of the two corresponding Subcellular localization of the GmGT-2A and GmGT-2B
geneswerefurtherobtainedusingRACEmethod.Becausebothof Because the GT-2A and GT-2B contained putative NLSs, we
them encoded proteins similar to those from the GT-2 group of examinedthesubcellularlocalizationofthetwoproteins.Eachof
the trihelix family [36], we named the two genes as GmGT-2A the two genes was fused to the GFP and then transfected into
(EF221753) and GmGT-2B (EF221754) respectively and further Arabidopsis protoplasts to observe the localization of the GFP
analyzed. fusion proteins. The green fluorescence from GFP control was
SMART analysis revealed two trihelix domains in both the localizedinbothnuclearregionandcytoplasmwhereasthegreen
GmGT-2A and GmGT-2B proteins (Fig. 1A, C). Between these fluorescence of the GmGT-2A-GFP and GmGT-2B-GFP fusion
twotrihelixdomains,acoiled-coilregionof31residueswasnoted proteins was abundant in nuclear region of the protoplasts
in GmGT-2A. Four putative nuclear localization signals (NLS) (Fig. 2C). The red fluorescence indicated the position of
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GTFactorsinStressTolerance
Figure1.SchematicrepresentationandaminoacidsequencealignmentoftheGmGT-2AandGmGT-2B.(A)Schematicdiagramofthe
GmGT-2AandGmGT-2B.(B)ClusteranalysisoftheGmGT-2AandGmGT-2BwithotherGT-2groupfactors.Theanalysiswasperformedbyusingthe
MEGA4.0programwithneighborjoiningmethodandwith1000replicates.Numbersonthefigurearebootstrapvalues.Thesequencesarefrom
soybeanandArabidopsisplants[36].(C)MultiplealignmentsoftheaminoacidsequencesfromvariousGTfactors.NLSindicatesputativenuclear
localizationsignal.Accessionnumbersareasfollows:GmGT-2(AF372498),GmGT-2A(EF221753),GmGT-2B(EF221754).Arabidopsisgenecodesareas
in[36].
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GTFactorsinStressTolerance
Figure2.ExpressionsoftheGmGT-2AandGmGT-2B.(A)Geneexpressionsindifferentorgansofsoybeanplants.RT-PCRmethodwasusedand
tubulin gene was amplified as a control. (B) Expressions of the GmGT-2A and GmGT-2B in response to ABA and stress treatments revealed by
Northern analysis. GmGT-2expression was also compared. (C) Subcellular localization of the GmGT-2A and GmGT-2B as revealed by GFP fusion
proteins.GreenfluorescenceindicateslocationoftheGFPcontrolortheGFPfusionproteins.Redfluorescenceindicatespositionsofchloroplasts.
doi:10.1371/journal.pone.0006898.g002
chloroplasts. These results indicate that the GmGT-2A and 2B(CT)]didnothadthetranscriptionalactivationability.TheN-
GmGT-2B arenuclear proteins. terminal halfof theGmGT-2Bwas furthertruncated andtheN-
terminalpeptideof153residues[GT-2B(N1-153)]wereenoughto
havetheactivationability.FurthertruncationsincludingtheGT-
Transcriptionalactivation,dimerizationandDNAbinding
2B(N1-89),GT-2B-N (90-153)orthemiddlepartoftheGT-2B
GT
analysis of the GmGT-2A and GmGT-2B
[GT-2B-M(150-424)]didnothaveanyactivationability(Fig.3C).
Because the GmGT-2A and GmGT-2B belong to the The dimerization ability of the two proteins was also analyzed
transcriptionfactorsoftrihelixfamily,westudiedthetranscription andwefindthatthefull-lengthproteinoftheGmGT-2B,theN-
activation ability of the two proteins using the yeast assay system terminal half and the C-terminal half can form homodimers
(Fig.3A).Constructs,whichharboredvariousversionsofGmGT- respectively (Fig. 3D). Other combinations did not produce any
2A and GmGT-2B (Fig. 3B), were made in pBD vector and homo- or -herterodimers (Fig. 3D). Each version of proteins in
transfectedintoyeaststrainYRG-2.Theyeasttransformantswere pBDvectorpluspADvector,oreachversioninpADvectorplus
examined for their growth on selection medium (SD-His) due to pBD vectordid not generate positive response (datanot shown).
activation of the reporter HIS3 gene, or determined for their b- BecausetheGmGT-2B(NT)canformdimer,wethenexamined
galactosidase activity due to the activation of the reporter LacZ which part is responsible for the dimerization. Figure 3E showed
gene(Fig.3A).Full-lengthoftheGmGT-2A[GT-2A(FL)]didnot thatthemiddlepartGT-2B-M(150–424)canformdimerswhereas
hadanytranscriptionalactivationability.ItsN-terminalhalf[GT- theN-terminaltrihelixN orC-terminaltrihelixC themselves
GT GT
2A(NT)]orC-terminalhalf[GT-2A(CT)]didnothavetheactivity can’t form dimers. Other combinations did not generate
either (Fig. 3C). However, the full-length GmGT-2B had dimerizations.
transcriptional activation activity, and its N-terminal half [GT- DNA-binding ability was investigated using yeast one-hybrid
2B(NT)]alsohadtheactivity(Fig.3C).TheC-terminalhalf[GT- system.TheeffectorplasmidsharboringtheGmGT-2A,GmGT-
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GTFactorsinStressTolerance
Figure3.Transcriptionalactivation,dimerizationandDNAbindinganalysis.(A)Schematicdiagramsfortranscriptionalactivation(left
panel)anddimerization(rightpanel)inyeastassay.(B)DifferentversionsofGmGT-2AandGmGT-2Busedfortheanalysis.FL:full-lengthprotein.NT:
N-terminalregion.CT:C-terminalregion.Numbersindicateaminoacidpositions.(C)Transcriptionalactivationactivityofdifferentversionsofthe
GmGT-2AandGmGT-2B.GrowthoftransformantsonSD-HisandbluecolorinthepresenceofX-Galindicatethatthecorrespondingproteinshave
transcriptionalactivationability.(D)DimerizationanalysisofGmGT-2AorGmGT-2B.GrowthofthetransformantsonSD/-Trp-Leu-Hisplus10mM3-
AT(SD-3)andpresenceofbluecolorindicatepositiveinteractions.EachversionofproteinsinpBDvectorpluspADvector,oreachversioninpAD
vectorpluspBDvectorservedasnegativecontrols.(E)IdentificationofdomainsinGmGT-2Bfordimerization.Othersareasin(D).(F)DNAbinding
analysis.GrowthofthetransformantsonSD/-Trp-Leu-Hisplus3-ATindicatesthatthecorrespondingproteinscanbindtothecis-DNAelements.D1:
59-catctacagttactagctagt-39; GT-1bx: 59-gtgtggttaatatg-39; GT-2bx: 59-tggcggtaattaactg-39; mGT-2bx-1: 59-tggctttaattaactg-39; mGT-2bx-2: 59-
tggcgggcattaactg-39;mGT-2bx-3:59-tggcggtacgtaactg-39;mGT-2bx-4:59-tggcggtaattgcctg-39;CaM-4:59-gatccgcgtag-39;GT-3b:59-taagaaaaataa-39.
(G)TranscriptionalactivationassayinArabidopsisprotoplasts.GALDBDisanegativecontrolandVP16isapositivecontrolfortransactivationability.
TheGmGT-2BhastransactiationactivitywhereastheGmGT-2Adoesnothavetheactivity.
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2B or their truncated forms were made in pAD vector, and the firefly luciferase (LUC) genewere co-transfected intoArabidopsis
reporter plasmid was made by inserting four tandem repeats of protoplasts and the relative LUC activity was determined.
various cis-DNA elements into the pHIS2, which contained a Figure 3G showed that the GmGT-2B protein activated higher
reportergeneHIS3.Aminimalpromoterwaspresentdownstream level of reporter LUC activity than the negative control
ofthetestedcis-DNAelementsbutupstreamfromtheHIS3gene. GALDBD.However,theGmGT-2Adidnotpromotethereporter
The effector plasmids and the reporter plasmids were transfected LUC activity. These results indicate that the GmGT-2B has
into yeast strain Y187 and growth of the transformants on SD/- transcriptional activation ability in the protoplast assay whereas
Trp-Leu-His plus 3-AT indicates binding of the transcription theGmGT-2Adoesnot have thisability.
factorstothecorrespondingcis-DNAelements.Figure3Fshowed
that the GmGT-2A, GmGT-2B, and their N-terminal or C- Seedling growth of the transgenic Arabidopsis plants
terminaltruncatedversionsallcanbindtotheGT-2bx,themGT- overexpressingtheGmGT-2AorGmGT-2BunderABAand
2bx-2, and the D1 element. However, the C-terminal region of osmotic stress
GmGT-2AorGmGT-2BappearedtohavelowaffinityfortheD1 To investigate the biological function of the GmGT-2A and
element. In addition, the GmGT-2A, its N-terminal and C- GmGT-2Bgeneinplant,wetransformedthetwogenesdrivenby
terminalregioncanbindtotheGT-1bx(Fig.3F).TheGmGT-2A, the 35S promoter into Arabidopsis plants and homozygous
GmGT-2B or their truncated versions could not bind to other transgeniclineswithhighergeneexpressionswereusedforfurther
elements tested. These results indicate that the GmGT-2A and analysis(Fig.4A).Throughouttheplantgrowthanddevelopmen-
GmGT-2B had common features in DNA binding and the tal stages, no significant difference was observed for these
GmGT-2A alsohadspecialty inthisability. transgenic lines in comparison with the wild type plants grown
The transcriptional activation ability was further examined in undernormal condition.
Arabidopsis protoplast assay system.Effector plasmids containing Because the GmGT-2A and GmGT-2B expressions were
the GmGT-2A or GmGT-2B, and a reporter plasmid containing a upregulated by ABA and various abiotic stresses (Fig. 2B), we
Figure 4. Morphogenesis of GmGT-2A- and GmGT-2B-transgenic seedlings under ABA and mannitol treatment. (A) Transgene
expressioninvarioustransgeniclinesasrevealedbyNorthernanalysis.(B)SeedlingmorphogenesisunderABAandmannitoltreatment.(C)Green
seedlingrateinresponsetoABA.(D)Greenseedlingrateundermannitoltreatment.For(C)and(D),eachdatapointisaverageofthreeexperiments
(n=150foreachexperiment)andbarsindicateSD.Asterisksindicatehighlysignificantdifference(P,0.01)fromCol-0.
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examined the effects of ABA and stress treatment on seed (Fig.6C).TheseresultsindicatethattheGmGT-2AandGmGT-2B
germination and seedling growth of the transgenic plants. Seed conferdroughttolerancetothetransgenicplants.
germination was not significantly affected by the ABA, mannitol Water loss represents another parameter for estimation of the
andsalttreatmentswhencomparedwiththewildtypeplants(data planttolerancetodroughtstress.Transgenicplantsoverexpressing
notshown).However,themorphogenesisofseedlingswasaltered. theGmGT-2AorGmGT-2Ballhadreducedwaterlossincomparison
Under treatments with both 0.5 mM and 0.75mM ABA, the withthe wild typeplants when detached leaves wereused forthe
GmGT-2B-transgenic lines (L11 and L69) exhibited significantly desiccationanalysis(Fig.6D).Thewholeplants(six-week-old)were
higher green seedling rates (,30% to ,40%) when compared alsowithheldfromwaterforvarioustimesandtheaerialpartswere
with the rates in wild type plants (,9% to ,16%) (Fig. 4B, C). measuredforwatercontent.Figure6Eshowedthat,atday8and
Under the same ABA treatments, the GmGT-2A-transgenic lines day 12 after the treatment, the water content was significantly
(L19 and L27) did not show significant change in the green higher in one GmGT-2A-transgenic line and two GmGT-2B-
seedlingrateincomparisonwiththatinthewildtypeplants.These transgenic lines compared to the control plants, suggesting that
results indicate that the GmGT-2B can promote seedling thesetransgenicplantsaretoleranttodroughtstress.
morphogenesis in the presence of ABA, possibly through Nonacclimated or cold-acclimated (5 d at 4uC) 12-day-old
suppression of ABA function. The transgenic seeds were also seedlings were exposed to freezing temperature for 6 h and then
germinatedonmediumwithmannitolandthegreenseedlingrates the plants were returned to normal conditions for evaluation of
were not significantly changed in the GmGT-2A- and GmGT-2B- performanceafter7-drecovery.Allthetransgeniclinesshoweda
transgenicplants(,70%to,77%)incomparisonwiththeratein bettergrowththantheWTcontrol,andtheacclimatedtransgenic
wild type plants (71%) (Fig. 4B, D). These results indicate that plantshadhighersurvivalrateafterfreezingtreatmentatdifferent
overexpression of the GmGT-2B conferred reduced sensitivity to temperatures when compared to the nonacclimated transgenic
ABAin thetransgenicplants. plants (Fig. 6F, G). Under normal condition, all the plants grew
very well (Fig. 6F). In addition, the GmGT-2B-overexpressing
Effects of salt stress on the transgenic plants plants had a higher survival rate than the GmGT-2A-overexpress-
ing plants under both acclimated and nonacclimated condition
overexpressing the GmGT-2A or GmGT-2B
(Fig. 6G). These results indicate that overexpression of both the
Performance of the GmGT-2A-, GmGT-2B-transgenic plants
GmGT-2Aand GmGT-2Bimproved plant tolerancetofreezing.
under NaCl treatment was examined. Under normal condition,
all the transgenic lines showed no significant difference when
GmGT-2A and GmGT-2B regulate expressions of stress-
comparedwiththewildtypeplantsintermsofthephenotypeand
responsive genes
thesurvivalrate(Fig.5A,B).Treatmentswith75mMor125mM
NaCldidnotaffectthesurvivalofallthetransgeniclinescompared The GmGT-2A and GmGT-2B may function in plant stress
either(Fig.5A;datanotshown).Under150mMNaCltreatment, tolerancethroughregulationofdownstreamgenes.Weselected33
,12%ofthewildtypeplantsweredeadwhereasalltheGmGT-2A- stress-responsive genes for further quantitative real-time PCR
analysis. Figure 7 showedthat 17genes including AZF1, MYB74,
andGmGT-2B-transgenicplantssurvived(Fig.5A,B).Thesurvival
MYB75,PAD3,LTP3,STZ,MYB73,LTP4,At4g30650,UGT71B6,
ratewasfurtherreducedatthe180mMNaCltreatment,withthe
RHL41, COR13, MYB77, CYP707A3, LTI30, RCI3, and DREB2A
wild type plants having a survival rate of around 70%. On the
wereenhancedinallthetransgeniclinesharboringtheGmGT-2A
contrary, the GmGT-2A- and GmGT-2B-transgenic plants had a
or GmGT-2B. The AZF1, STZ and RHL41/Zat12 encoded plant-
survivalrateofmorethan90%(Fig.5A,B).
specific transcription factors with Cys-2/His-2 zinc finger motifs,
The salt-stressed seedlings on plates (Fig. 5A) were further
and can be induced by various stresses. Overexpression of the
transferredinpotscontainingvermiculiteandtheirrecoveryat8d
STZ, Zat10 and Zat12 increased stress tolerance [37–39]. The
and 21d was observed. Treatments with 75mM and 125mM
four MYB genes MYB73, MYB74, MYB75 and MYB77 can be
NaCl did not significantly affect recovery of all the compared
induced by salt stress [40,41]. The LTP3, LTP4, PAD3 and
plants(Fig.5C).However,at150mMNaCl,therecoveryratewas
UGT71B6 were involved in ABA responses [42–44]. The
reduced to ,50% for the wild type plants whereas this rate was
At4g30650,COR13andLTI30werealsoinvolvedinabioticstress
morethan80%forallthetransgeniclines.At180mMNaCl,the
response[45,46].CYP707A3encodesanABA89-hydroxylase.The
recoveryforthewildtypeplantswas,25%.ForboththeGmGT-
cyp707a3 mutant plants are hypersensitive to ABA and exhibited
2A-andGmGT-2B-transgenicplants,therecoveryratesweremore
enhanceddroughttolerance[47].RCI3encodesaperoxidaseand
than90%(Fig.5C,D).AlltheseresultsindicatethattheGmGT-
overexpression of the gene conferred dehydration and salt
2AandGmGT-2B conferred plant tolerancetosalt stress.
tolerance[48].DREB2Aexpressiondoesnotactivatedownstream
genesundernormalgrowthcondition.However,overexpressionof
TheGmGT-2AandGmGT-2Bconferdroughtandfreezing
its constitutive active form leads to drought stress tolerance and
tolerance in their transgenic Arabidopsis plants slight freezing tolerance[49].
BecauseexpressionoftheGmGT-2AandGmGT-2Bgenescanbe TwogenesMYB90andAt4g02200wereonlyhighlyexpressed
inducedunderdroughtandcoldtreatments,weinvestigatedifthe intheGmGT-2A-transgenicplantscomparedtothecontrolandthe
performance of their transgenic plants was altered under these GmGT-2B-transgenic plants. Eleven genes including MYB47,
stresses.Plants(12-day-old)inpotsweresubjectedtodroughtstress MYB15, PAD3, LTP3, LTP4, CBF2, PMZ, COR13, HVA22E,
bywithholdingwaterfor16d.Afterthisstress,only,40%ofthe CBF1,andNCED3werehighlyexpressedinGmGT-2B-transgenic
wildtypeplantssurvivedwhereasmorethan90%oftheGmGT-2A- plants compared to the control plants and GmGT-2A-transgenic
andGmGT-2B-transgenicplantscansurviveandgrowwell(Fig.6A, plants. These genes have been found to be responsive to various
B).Alltheplantsundernormalconditiongrewnormally(Fig.6A). abiotic stresses and/or ABA response [41,42,50,51]. The NCED
Plantsthatstarttohavesiliqueswerefurthertreatedunderdrought genes have been found to be responsible for the biosynthesis of
stressbywithholdingwaterfromplantsfor16d.Theaerialpartsof ABA precursor and are also involved in regulation of plant
these plants were harvested and the dry weights of the transgenic responses to abiotic stresses [52,53]. Six genes including LHY1,
plants were significantly higher than that of the wild type plants MYB2, At5g02840, VAMP711, At3g09600, and CCA1 were
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GTFactorsinStressTolerance
Figure5.PerformanceoftheGmGT-2A-andGmGT-2B-transgenicplantsundersaltstress.(A)GrowthofthetransgenicseedlingsinNaClmedium.
(B)Survivalrateofthesalt-treatedplantsin(A).(C)Comparisonofgrowthofthesalt-treatedplantsinpots.Plantsfromtreatmentsasin(A)weretransferred
inpotsandthepicturesweretakenat8dand21daftertransfer.(D)Survivalrateofthesalt-treatedplantsinpotsfrom(C).For(B)and(D),eachdatapoint
isaverageofthreeexperiments(n=30foreachexperiment)andbarsindicateSD.Asterisksindicatehighlysignificantdifference(P,0.01)fromCol-0.
doi:10.1371/journal.pone.0006898.g005
downregulated in all the transgenic lines. The LHY1, CCA1, in the GmGT-2B-transgenic lines. These results indicate that
At5g02840 and MYB2 have been found to be responsive to stress GmGT-2AandGmGT-2Bregulatedacommonsetofgenesaswell
and/orABA[41,54].TransgenicplantsoverexpressingMYB2had as specific setsof genes forstress tolerance.
higher sensitivity to ABA and showed stress tolerance [55].
SuppressionoftheVAMP711,ageneencodingaproteinforvesicle
Discussion
trafficking, in antisense transgenic plants improved salt tolerance
[56].The gene PK1/PK6, which was induced by different stresses Althoughrolesoftrihelixfamilytranscriptionfactorshavebeen
[57],wasinhibitedintheGmGT-2A-transgeniclinesbutpromoted discovered in light-relevant and other developmental processes,
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GTFactorsinStressTolerance
Figure 6. Performance of theGmGT-2A- andGmGT-2B-transgenic plants under drought and freezing stress. (A) Phenotype of the
transgenic plants under drought stress. (B) Survival rate of the transgenic plants under drought stress. Each data point is average of three
experiments(n=30foreachexperiment)andbarsindicateSD.(C)Comparisonofplantdryweightafterdroughtstress.BarsindicateSD(n=30).
(D)Waterlossindetachedleavesfromthetransgenicplants.BarsindicateSD(n=3).(E)Watercontentsinaerialpartofthepot-grownplantsafter
withholding water. Bars indicate SD (n=8). (F) Survival of the transgenic plants under freezing treatment after aclimation or non-acclimation.
(G)Survivalrateofthetransgenicplantsaftertreatmentin(F).Eachdatapointisaverageofthreeexperiments(n=30foreachexperiment)andbars
indicateSD.For(B),(C),(E),(G),asterisksindicateasignificantdifference(*P,0.05and**P,0.01)fromCol-0.
doi:10.1371/journal.pone.0006898.g006
their functions in abiotic stress response are largely unknown. In The GmGT-2B exhibited transcriptional activation activity in
the present study, two trihelix family transcription factor genes boththeyeastassayandtheprotoplastassay,whereastheGmGT-
GmGT-2AandGmGT-2Bfromsoybeanwereidentifiedtobestress- 2A did not have this activity. This difference in transcriptional
responsiveandconferredstress-toleranceintransgenicArabidopsis activationabilitymayresultinthedifferentialgeneexpressionsin
plants through regulation of downstream genes. This study adds thetransgenicplants,withmoregenesbeinghighlyorspecifically
thetrihelixfamilymemberstothosetranscriptionfactorsthatcan expressed in the GmGT-2B-transgenic plants (Fig. 7). The
improve plant stress-tolerance. activation domain of the GmGT-2B was also analyzed in detail
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GTFactorsinStressTolerance
Figure7.AlteredgeneexpressionsinGmGT-2A-andGmGT-2B-transgenicplantsincomparisonwiththeWT(Col-0).Foreachgene,two
transgeniclineswereused.ThequantitativeRT-PCRwasusedfortheanalysis.Totally33geneswereexaminedfortheirexpressions.BarsindicateSD
(n=4).
doi:10.1371/journal.pone.0006898.g007
and it appears that the N-terminal peptide of 153 residues is the be formed between GmGT-2A and GmGT-2B. The biological
minimal domain for transcriptional activation. Neither the N- significanceofsuchinteractionisnotknown.Itispossiblethatthe
terminaltrihelixdomain(90–153)noritsN-terminalsequence(1– interactionwouldmodifythetranscriptionalactivationabilityand
89)themselveshastheabilitytoactivatetranscription.Theroleof then affect the downstream gene expressions. Arabidopsis GT-3a
theminimalactivationdomainneedstobefurthertestedinplant and GT-3b could form homo or heterodimers, and the
system.AriceGT-2proteinhasalsobeenfoundtofunctionasa dimerization domain seemed to be located at the C-terminus.
transcriptionalactivator.However,theactivationdomainwasnot However, no interaction was observed between each of the two
identified[58].ArabidopsisGT-1alsohastransactivationfunction proteinswith theGT-1[27].
inbothyeastandplantcells[59].However,ASIL1functionsasa The trihelix domain is generally believed to be the DNA-
repressorforembryonicandseedmaturationgenesinArabidopis binding domain [29,30]. In the yeast one-hybrid assay, both the
seedlings [36]. The present GmGT-2A does not have transcrip- N-terminal and C-terminal trihelix-domain-containing region of
tional activation ability (Fig. 3C, G). Whether it has repression the GmGT-2A and GmGT-2B can bind to the three elements
activity needsfurther study. (Fig. 3F). In addition, the GmGT-2A can bind to the GT-1 bx
The GmGT-2B, unlike the GmGT-2A, has the ability to whereas theGmGT-2B can’t. Thesedifferent features imply that
dimerize, and the dimerization seems to happen through inter- the two genes may play some different roles in plant. However,
action of the middle part of the protein. No heterodimers could sincethetwogenesalsohavecommonfeaturesingeneexpression
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