Molecular characterisation of a MYB transcription factor from a succulent halophyte involved in ...

35 Pages·2015·0.64 MB·English

Research Article
Molecular characterization of an MYB transcription factor
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from a succulent halophyte involved in stress tolerance lo
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Pushp Sheel Shukla1,2, Parinita Agarwal1, Kapil Gupta1 and Pradeep K. Agarwal1,2* fro
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1 WastelandResearchDivision,CSIR-CentralSaltandMarineChemicalsResearchInstitute(CSIR-CSMCRI),CouncilofScientificand h
IndustrialResearch(CSIR),GijubhaiBadhekaMarg,Bhavnagar364002,Gujarat,India ttps
2 AcademyofScientificandInnovativeResearch,CSIR-CentralSaltandMarineChemicalsResearchInstitute(CSIR-CSMCRI), ://a
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CouncilofScientificandIndustrialResearch(CSIR),GijubhaiBadhekaMarg,Bhavnagar364002,Gujarat,India a
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Received:21February2015;Accepted:8May2015;Published:17May2015 m
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AssociateEditor:TimJ.Flowers .o
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Citation:ShuklaPS,AgarwalP,GuptaK,AgarwalPK.2015.MolecularcharacterizationofanMYBtranscriptionfactorfromasucculent .c
o
halophyteinvolvedinstresstolerance.AoBPLANTS7:plv054;doi:10.1093/aobpla/plv054 m
/a
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Abstract. Abiotic stresses like drought, salinityand extreme temperature significantlyaffect crop productivity. la
/a
Plantsrespond atmolecular, cellularandphysiological levels for management ofstresstolerance. Functional and rtic
regulatorygenesplayamajorroleincontrollingtheseabioticstressesthroughanintricatenetworkoftranscriptional le
-a
machinery.Transcriptionfactorsarepotentialtoolsformanipulatingstresstolerancesincetheycontrolalargenum- b
s
berof downstreamgenes. In the presentstudy, wehave isolated SbMYB44 from asucculent halophyte, Salicornia tra
c
brachiataRoxb.SbMYB44withanopen-readingframeof810bpencodesaproteinof269aminoacids,withanesti- t/d
o
matedmolecularmassof30.31kDaandanisoelectricpointof6.29.TheinsilicoanalysisrevealedthattheSbMYB44 i/1
0
proteincontainstheconservedR2R3imperfectrepeats,twoSANTdomainsandpost-translationalmodificationsites. .1
0
TheSbMYB44transcriptshowedup-regulationinresponsetosalinity,desiccation,hightemperature,andabscisicacid 9
3
and salicylic acid treatments. The SbMYB44 recombinant protein showed binding to dehydration-responsive cis- /ao
b
elements(RD22andMBS-1),suggesting its possibleroleinstresssignalling.OverexpressionofSbMYB44 enhanced p
la
thegrowthofyeastcellsunderbothionicandosmoticstresses. /p
lv
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Keywords: Abioticstress;halophyte;R2R3-MYB;Salicorniabrachiata;transcriptionfactor;yeast. 4/2
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Introduction y
throughanintricatenetworkoftranscriptionfactors(TFs). g
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Evolution has equipped plants with a wide spectrum Thetranscriptionalmachineryhasevolvedfromprimitive s
t o
of adaptationsto combat environmental perturbations tocomplexlifeformswithmoreregulatorgenesinplants n
2
(DinakarandBartels2013).Abioticstresses,likedrought, when compared with yeast (Riechmann et al. 2000). 8
M
salinityand extreme temperature, negatively influence Transcription factors can be classified into more than a
rc
plantgrowthandyield.Plantsrespondtothesestresses 50 different families on the basis of their DNA-binding h
2
bychangesinbothregulatorandfunctionalgenes.Tran- domains (Riechmann et al. 2000). These regions are 0
1
9
scriptionalcontrolisthemajormechanismthatregulates responsible for binding to the specific cis-elements in
geneexpressionassociatedwithbothabioticandbiotic the promoters. The control and co-ordination among
stresses,growth,metabolismanddevelopmentofplants, specific set of genes is accomplished by the combined
*Correspondingauthor’se-mailaddress:[email protected]
PublishedbyOxfordUniversityPressonbehalfoftheAnnalsofBotanyCompany.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.org/
licenses/by/4.0/),whichpermitsunrestrictedreuse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
AoBPLANTS www.aobplants.oxfordjournals.org &TheAuthors2015 1
Shuklaetal.—AnR2R3SbMYB44showedstresstoleranceinyeast
interaction among TFs, between TFs and non-DNA- of nutrient-richsaltofplantorigin (US patentno.6929
bindingproteins,andbetweenTFsandcis-regulatoryele- 809). Salicornia brachiata adapts to high salinity and
ments(Agarwaletal.2013).TheTFscorrespondto5–7% drought by accumulation of compatible osmolytes
ofthetotalgenesandhaveincreasedinnumber,presum- and reducing stress-induced oxidative damage (Parida
ably due to the complexity of plant metabolism (Shiu and Jha 2010, 2013). Identification of stress-induced
etal.2005). ESTs in Salicornia showed that 4.8% ESTs belonged to
MYBproteinsconstituteoneofthelargestTFfamiliesin stress-tolerantgenecategory(Jhaetal.2009).SbDREB2a,
plants(Lindemoseetal.2013).Theseproteinspossessthe a DREB TF isolated from S. brachiata, induced tolerance D
o
w
highlyconservedMYBdomainscomprisingsingle,double to both salinity and desiccation in transgenic tobacco n
lo
or triple imperfect repeats. Each repeat contains 50–53 (Guptaetal.2010,2014).Inthepresentstudy,wereport a
d
amino acids (aa) with three a-helices. The second and an SbMYB44, an R2R3-type MYB TF from Salicornia and ed
thirdhelicesofeachrepeathavethreeregularlyspacedtryp- itstranscriptregulationinresponsetodifferentdevelop- fro
m
tophan(W)residuesformingahelix–turn–helix(HTH)with mental stages, abiotic stresses and by signalling mole- h
ahydrophobiccore(Ogataetal.1996).Thethirdhelixof cules. The SbMYB44 also showed binding with different ttps
eachrepeatservesas‘recognitionhelix’facilitatingintercal- cis-elementsofabioticstress-relatedpromoters.Overex- ://a
c
ationofDNAinthemajorgroove(Jiaetal.2004).MYBpro- pressionofSbMYB44enhancedthegrowthofyeastcells ad
e
teinsare classifiedintofoursubfamiliesnamely R1-MYB, inthepresenceofsalinityanddehydrationstress. m
ic
R2R3-MYB,3R-MYBand4R-MYBdependingonthenumber .o
u
of adjacent repeats in the MYB domain (Dubos et al. Methods p.c
o
2010).ThefirstplantMYB,c-MYB-likeTF,involvedinantho- m
cyaninbiosynthesis,wasisolatedfrommaize(Paz-Aresetal. Plantmaterialandstresstreatment /ao
b
1987);sincethenanumberofMYBTFshavebeenisolated Seeds of S. brachiatawere harvested from dried plants pla
aarnedhcighhalryacreteprriezseednftreodmindpiflfaenretsntwpitlhan1t5s7.TinhemRa2izRe3-(DMuYBetTaFls. cInodllieac(tGedPSfr2o1m83t5h.6e3c4o′aNsatanlda7re2a81n6e.7a8r6B′hEa).vTnhaegsaer,eGdusjwarearte, /article
2012)andmorethan100inArabidopsis(Yanhuietal.2006). germinated and grown in plastic pots with garden soil -ab
s
The R2R3-MYBs are considered to have evolved from an under natural conditions. One-month-old seedlings tra
c
R1R2R3-type ancestral gene, from which the first repeat were transferred tothe hydroponicmedium(1/2major t/d
o
was lost (Braun and Grotewold 1999). The three- and and minor salts of Murashige and Skoog’s medium, i/1
0
four-repeatMYBTFshavealsobeenidentifiedinArabidopsis Murashige andSkoog1962)inaplant growthchamber .1
0
(BraunandGrotewold1999).Theroleofdifferentmembers (CU-36L, Percival Scientific, Perry, IA, USA) with light/ 9
3
ofthe MYBfamilyhavebeenstudied duringcellular pro- dark (300–350mmolm22s21 spectral flux photon of /ao
b
cesses including growth and development (AtMYB123, photosynthetically active radiations) cycle of 16/8h at p
la
Nesietal.2000),embryogenesis(AtMYB118,Zhangetal. 258C. After acclimatization, plants were treated with /p
lv
2009), abiotic stresses (OsMYB3R-2, Dai et al. 2007; 250mM NaCl, desiccation (wrapped in tissue paper at 05
4
OsMYB55, El-Kereamy et al. 2012; CaMYB, Seong et al. room temperature), 100mM ABA, 2.5mM SA, and heat /2
0
2008;TaPIMP1,Zhangetal.2012;TaMYB73,Heetal.2012; (378C)for0,0.5,1,6,12,24and48h.Thetreatedplant 14
9
OsMYB,Yangetal.2012),defenceresponsestopathogens samples were frozen in liquid nitrogen and stored at 9
b
(BOS-1, Mengiste et al. 2003; TiMYB2R-1, Liu et al. 2013) 2808Cfortranscriptanalysis. y g
u
andasrepressorsandenhancersoflignification(EgMYB1, e
s
Legay et al. 2010; AtMYB61, Newman et al. 2004). MYBs IsolationofSbMYB44gene t o
n
areinvolvedintheabscisicacid(ABA)-,salicylicacid(SA)- TotalRNAwasisolatedfrom1-month-oldseedlingstreated 2
8
and jasmonic acid (JA)-dependent signalling pathways with 500mM NaCl for 15 days bythe guanidinium thio- M
a
(Abeetal.2003;Shanetal.2009). cyanate method (Chomczynski and Sacchi 1987). The rc
h
Although MYB TFs have been isolated from a large totalRNA(2.5mg)wasusedforfirst-strandcDNAsynthesis 20
1
numberofglycophytes,theircharacterizationfromhalo- usingRevertAidcDNAsynthesiskit(ThermoScientific).De- 9
phytesislimited.SalicorniabrachiataRoxb.(Amarantha- generate primers (forward 5′-AYGGAYCGGRTYAARGGYCC
ceae)isaleaf-less,annual,succulent,obligatehalophyte RTGGAG-3′ andreverse5′-TCTTTATCATYYCYTGCATCAC-3′)
growingabundantlyinthecoastalareaofGujarat,India. were designed from the conserved region of nucleotide
Salicorniabrachiataaccumulatessaltinitsstemsandcan alignment made from MYB sequences of Capsicum an-
surviveashighas2MNaClinthefield(Reddyetal.1992, nuum (EF222025), Malus domestica (DQ074461), Vitis
1993).SalicorniabrachiataaccumulatesNaClto30–40% vinifera(AY953543),Glycinemax(DQ822911)andSolanum
ofitsdryweight.Therefore,itsbiomasswasutilizedsuc- tuberosum(AF122051).Apolymerasechainreaction(PCR)
cessfullyatourinstitute(CSIR-CSMCRI)forthepreparation was carried out using cDNA as template with 150ng
2 AoBPLANTS www.aobplants.oxfordjournals.org &TheAuthors2015
Shuklaetal.—AnR2R3SbMYB44showedstresstoleranceinyeast
ofprimers,200mMdNTPsand2.5UTaqDNApolymerase ExpressionanalysisofSbMYB44transcript
ina50mLreactionvolumeunderthefollowingconditions: byreal-timePCR
948C,5minfor1cycle;948C,1min;558C,1minand728C, Total RNA was extracted from various stress-treated
1minfor35cycles;andlast728C,7minfor1cycle.The Salicorniashootsandfirst-strandcDNAwassynthesized
amplicon was gel-purified, cloned in pJET 1.2 vector using RevertAid cDNA synthesis kit (ThermoScientific).
(Thermo Scientific) and sequenced at Macrogen (Seoul, Real-timePCRwasperformedinaCFXdetectionsystem
Korea).AfterconfirmationofthesequencebyBLASTsearch, (Bio-Rad,USA)with1×SsoAdvancedSYBRgreensupermix
the 5′ and 3′ rapid amplification of cDNA ends (RACE) (Bio-Rad)using60ngofSbMYB44primers(forward5′-CTGA Do
wereconducted.The5′ RACEwasdoneusingInvitrogen CGTTGAGTTTCATCGCCCT-3′ andreverse5′-GAGGAGGTGAA wn
lo
kit (USA)withthehelpofthefollowinggene-specificpri- TCGGAAGAAA-3′)andb-tubulinprimers(forward5′-GGAG ad
mers: GSP R1 (5′-GATCTCCGAGAATTTCCTCTTC-3′), GSP R2 TCACCGAGGCAGAG-3′ and reverse 5′-ATCACATATCAGAAA ed
(5′-ACCGAACCTAGCGTGAGCTTTGA-3′)andGSPR3(5′-GGCA CCACAAAG-3′)underthefollowingPCRconditions:948C, from
CGATGTTCAACTTCCGGTG-3′).For3′RACE,cDNAwassynthe- 2min for 1 cycle; 948C, 30s, 558C, 30s and 728C, 30s h
sizedusingthePK1primer(5′-CCAGTGAGCAGAGTGACGAG for 40 cycles; 728C, 7min for 1 cycle. At the end of the ttps
GACTCGAGCTCAAGC(T)17-3′).Theprimary3′ RACEPCRwas PCRcycles,theproductsweresubjectedtomeltcurveana- ://ac
carried out using an adaptor primer PK2 (5′-CCAGTGA lysistoverifythespecificityofPCRamplification.Threeinde- ad
e
TGACAACGAAAGATTGGAGC-G3-′3).′)ThaendseacGonSPd1a-rFypPrCimRwera(s5′c-aCrGriCeTdAoGuGtTuTCsiGnGg pexepnrdeesnsitoenxpwearsimdeentetsrmwienreedpeursfionrgmtehdeaLnivdatkhmereetlhaotidve(Lgievnaek mic.ou
1:50 dilution of primary PCR product with PK3 (5′-GAG andSchmittgen2001).TranscriptexpressionofSbMYB44 p.c
GACTCGAGCTCAAGC-3′)andGSP2-F(5′-GTCTGATGTCAGCG at0hforeachtreatmentservedascontrolandwasused om
TCTCTGG-3′)primers,andtheamplifiedproductwascloned for normalization of CT values of each treatment (Livak /aob
in pJET 1.2 vector (Thermo Scientific) and sequenced. andSchmittgen2001). pla
Tcfuahltel-iloesnen,qgu5the′nSRcbAeMCgYEBea4nn4erdsaet3qe′udReuAnscCienEg,inwdseailgsiceocn.oeFmurarbtthieneeprdmrimotoreer,oatbhmteapifnluifilla-- TransactivationassayofSbMYB44 /article-ab
length gene was amplified and cloned using SbMYB44 F Ayeastone-hybridassaywasperformedtostudythetran- stra
(5′-ATGGAAAGCCACCAAACTTTATCATCG-3′)andSbMYB44R scriptional activation of the SbMYB44 protein (He et al. ct/d
(5′-TTAATGGAAATGACCGTACCGAGAAAGGGAT-3′) primers. 2F01(25)′.-CSCBGMGYABA4T4TCcADTNGAGAwAaAsGCaCmApCliCfiAeAdAuCsTiTnTgA-S3b′)MYaBn4d4 oi/10
Forstudyingthegenomicorganization,thegenomicDNA SbMYB44 R (5′-CGCGGATCCTTAATGGAAATGACCGTACCG-3′) .10
was PCR amplified with gene-specific primers SbMYB44 F 9
3
andSbMYB44R. withtheEcoR1andBamH1flankingrestrictionsites,respect- /ao
ively.ThedigestedSbMYB44wasclonedinEcoRIandBamHI b
p
sites of pGBKT7 vector (Clontech). Plasmids of SbMYB44 la/p
Insilicoanalysis andvectoraloneweretransformedseparatelyintoyeast lv0
5
strainAH109(Clontech)andgrownonsyntheticdropout 4
A phylogenetic tree of SbMYB44 was constructed with /2
amino acid sequences of different MYB TFs using the mediumlackingtryptophan(SD/-Trp).HIS3activitywas 014
assessed by conducting a viability test on a histidine- 9
maximum-likelihoodmethodbytheMEGA6software. 9
ThesecondarystructureofSbMYB44waspredictedusing lackingmedium(SD/-His).LacZactivitywastestedbyper- by g
formingthegalactosidasefilterliftassay(Maetal.2009). u
thePSIPREDsoftware.Solventaccessibilityofaproteinisde- e
s
terminedusingPredictproteinserver(Jones1999;Rostetal. t o
n
2004).MEMSAT3andMEMSATSVMsoftwarewereusedto CloningofSbMYB44cDNAinpET28aexpression 2
8
predictthetransmembranehelixofSbMYB44(Nugentand vectorandpurificationofrecombinantprotein M
a
Jones2009).ConserveddomainsinSbMYB44wereidentified SbMYB44 open-reading frame (ORF) was PCR amplified rc
h
viatheSMARTsoftware(http://smart.embl-heidelberg.de/). using SbMYB44 PF (5′-CCGGAATTCATGGAAAGCCACC 20
A nuclear localization signal of SbMYB44 was predicted AAACTTTA-3′)andPR(5′-CCGCTCGAGTTAATGGAAATGACC 19
using cNLS mapper (http://nls-mapper.iab.keio.ac.jp/cgi- GTACCG-3′) with flanking restriction sites of EcoRI and
bin/NLS_Mapper_form.cgi). Primary SbMYB44 and other XhoI sites, respectively, and cloned in pJET1.2 vector.
MYBproteinsequenceswereanalysedforN-glycosylation The digested SbMYB44 was cloned in pET28a vector.
sites,proteinkinasessites,myristoylationsitesandphos- Plasmids of pET28a-SbMYB44 and vector alone were
phorylationsitesbyMotifScan(http://myhits.isb-sib.ch/cgi- transformed separately in Escherichia coli BL-21 Star
bin/motif_scan).SequencelogosforR2andR3MYBrepeats (DE3) cells (Invitrogen). Recombinant protein was in-
of SbMYB44 were obtained from http://weblogo.berkeley. ducedwith1mMisopropylb-D-1-thiogalactopyranoside
edu/logo.cgi(Crooksetal.2004). (IPTG) at 378C for 2, 4, 6h and purified under native
AoBPLANTS www.aobplants.oxfordjournals.org &TheAuthors2015 3
Shuklaetal.—AnR2R3SbMYB44showedstresstoleranceinyeast
conditions using Ni–NTA Fast Start Kit,usingmanufac- 7mL from each dilution were spotted on SD/-Ura
turer’sprotocol(Qiagen,Germany). mediumandincubatedat308Cfor3days.
Statisticalanalysis
DNAprobesandgelmobilityshiftassay
Each experiment was performed three times and data
ComplementaryoligonucleotidesofRD22(F5′-CTTCTAA
wererecorded.One-wayanalysisof variancewithrepli-
CCACTACGTGCCTTCTGCTCCTTCTGC-3′, and R 5′-GCAGAA
cateswasperformedinMicrosoftExcel.Criticaldifference
GGAGCAGAAGGCACGTAGTGGTTAGAAG-3′ (Abeetal.1997)
valueswerecalculatedatP≤0.05todeterminethesig- D
and MBS-1 (F 5′-CGAGACACCCTAACTGACACACATTCT-3′ ow
andR5′-AGAATGTGTGTCAGTTAGGGTGTCTCG-3′)(Nakagoshi nificance of difference between the means, across the nlo
treatments,oftranscriptexpressionatvariousintervals. a
etal.1990)weresynthesizedhavinguniquebindingsites Themeanvaluesthatweresignificantlydifferentwithin ded
(furonmdeArlrianbeidd)o.pTshiseRdDe2h9yadrpartoiomno-rteesrp(oFn5s′i-vTeAeAlAeAmGenAtTA(DTARCE-) treatmentfromeachotherareindicatedbydifferentlet- from
TTATTCGCGGAACAACTTGCAAGTGTTTCCGGCTAAAGTAATAAATCGTCT-T3′TAa-3n′d)wRasu5s′-eGdCTaTs tvearrsia.tTihoensintatnhdearredpdliceavtiaetsi.onwascalculatedtoshowthe https://a
anegativecontrol(Liuetal.1998;Agarwaletal.2007). c
a
Results d
Complementary oligonucleotides (2mg) from different e
m
promoter elements were annealed separately using CloningandsequenceanalysisofSbMYB44 ic.o
annealing buffer (100mM Tris–HCl, 5mM NaCl, 10mM u
ethylenediaminetetraacetic acid (EDTA)) by incubating Aprnimaemrsp.liAcfotnerocfo6n3fi0rmbpinwgaitssasemqpuleifinecdeubsyinNgCBdIegBeLAneSrTa,t5e′ p.com
at608Cfor5minandthenat378Cfor15min.Theelec- and 3′ RACE were conducted to obtain the full-length /ao
trophoretic mobilityshift assay (EMSA) was carried out b
ruescinogmb8i0nanngtoSfbManYnBe4a4lepdropteroinbeinwbiinthdi4n0gmbugffoefrp[1u5rifimeMd acDndNA5.3T3hbep5,r′easnpdec3t′ivReAlyC.EThsheoSwbMedYBa4m4pclDifiNcaAticoonmopfri3s6ed4 pla/artic
anORFof810bpencodinganR2R3-typeMYBproteinof le
HEPES, 35mM KCl, 4mM EDTA (pH 8.0), 1mM DTT, 269aawithacalculatedmolecularweightof30.31kDa -ab
1mMMgCl and 6%glycerol]at roomtemperaturefor s
20min. The2 reaction was terminated by adding 10× and an isoelectric point of 6.29 (GenBank accession trac
DNA loading dye and fractionated on non-denaturing number: KJ027517). The amino (N)-terminal region of t/do
10% acrylamide gel in 0.5× tris-borate-EDTA buffer. SbMYB44containtheconservedR2R3imperfectrepeats, i/10
which are involved inbindingtotarget DNA sequences .1
ThegelwasstainedwithSYBRGreendye. 0
[seeSupportingInformation—Fig.S1].TheR2andR3re- 9
3
peatsshowedthepresenceofthreeandtwoTrp(W)resi- /ao
StresstoleranceassayofSaccharomycescerevisiae dues,respectively.SbMYB44containstwoSANTdomains, bp
la
underdifferentstresses onepresent in the R2repeat (18–63aa) and the other /p
lv
TheSbMYB44ORFwasclonedintheEcoRIandXhoIsites presentintheR3repeat (71–113aa).TheLSPEmotifis 05
4
of the yeast expression vector pYES2 (Invitrogen). The presentinthelinkerregionofR2andR3repeats(Fig.1). /2
0
plasmidsofpYES2-SbMYB44andvectoraloneweretrans- ThePSIPREDproteinstructurepredictionservershowed 14
9
formedseparatelyinyeaststrainW303usingYeastmaker thepresenceof33.46%helixand66.54%loop[seeSup- 9
b
yeast transformation system (Clontech). A stresstoler- portingInformation—Fig.S2AandB].Solventaccessibil- y g
u
ance assay of recombinant yeast cells was performed ity of the protein revealed that the 46.47% region is e
s
asdescribedbyLietal.(2014)withminormodifications. accessible tosolvents,the17%regiontointermediate t o
n
YeastcellshavingpYES2-SbMYB44andvectoralonewere exposurewhilerestof35.69%isburied[inaccessibleto 2
8
growninSD/-Urabrothfor24hat308C.Afteradjusting solvents,SupportingInformation—Fig.S2C].Theregion M
a
theOD to0.4,500mLofculturewasaddedto10mL between 86 and 101aa showed transmembrane helix rc
600 h
of induction medium (SD/-Ura broth supplemented havinganextracellularcarboxy(C)-terminalregionand 20
1
with2%galactose)andgrownfor36htopromotetheex- a cytoplasmic N-terminal region while the region be- 9
pressionofSbMYB44gene.Theculturesweredilutedtoan tween 191 and 200aa had an extracellular N-terminal
OD 0.6,and500mLofculturewasinoculatedin10mL regionandacytoplasmicC-terminalregion[seeSupport-
600
SD/-Ura containing 2.5M NaCl, 5.0M NaCl, 15% poly- ingInformation—Fig.S3].ThePROSITEanalysisrevealed
ethylene glycol (PEG 6000) equivalent to 20.295MPa three glycosylation motifs (41–44, 113–116, 180–
of osmotic potential and 30% PEG 6000 equivalent 183aa),twocAMP-andcGMP-dependentproteinkinase
to 21.027MPa of osmotic potential and incubated at phosphorylationsites(118–121,216–219aa),sixcasein
308C for 36h. After stress treatment, the cultures kinaseIIphosphorylationsites(23–26,129–132,150–
were serially diluted (100, 1021, 1022, 1023, 1024) and 153,164–167,206–209,260–263aa),fiveproteinkinase
4 AoBPLANTS www.aobplants.oxfordjournals.org &TheAuthors2015
Shuklaetal.—AnR2R3SbMYB44showedstresstoleranceinyeast
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Figure1. SchematicrepresentationoftheSbMYB44TFsequence.R2andR3repeatDNA-bindingdomainsareunderlinedwithdouble-headed
arrows.Conservedtryptophanresidues(W)aremarkedbyasterisksandgreenbarsrepresenta-helicesinR2R3repeats.SANTdomainsare
demarcatedbyredoutlinedbox.LSPEmotifismarkedwithred-dashedline.Serinerichregionisrepresentedwithredsolidline.
C phoshphorylation sites (9–11, 53–55, 56–58, 115– phylogenetic tree of SbMYB44 was constructed on the
117,192–194aa) and three myristoylation sites(160– basisofalignmentofthecompletededucedpolypeptide
165, 197–202, 249–254aa). The maximum-likelihood sequences of different MYB TFs. Bootstrap values from
AoBPLANTS www.aobplants.oxfordjournals.org &TheAuthors2015 5
Shuklaetal.—AnR2R3SbMYB44showedstresstoleranceinyeast
1000replicateswereusedtoassesstherobustnessofthe and MBS-1 (dehydration responsive) (Fig. 5). The RD29
tree.Inthephylogenetictree,bootstrapvalues .50are cis-elementwasusedasanegativecontrolanditdidnot
shown.ThephylogeneticanalysisrevealedthatSbMYB44 showbindingwitharecombinantprotein.TheSbMYB44
isclusteredinsubgroup22oftheMYBfamilywithclose proteinshowedincreasedbindingwithincreasingconcen-
homology to FvMYB44 and MdMYB from Fragariavesca trationoftheprobe,themaximumbindingwasobserved
andMalusdomestica(Fig.2). using80ngprobewithallthecis-elements(Fig.5AandB).
Different amounts of recombinant protein were also
TranscriptexpressionofSbMYB44 analysedforbindingreactionwith80ngprobes(Fig.5C D
o
w
Salicorniabrachiataisadaptedtodifferentstresscondi- and D). The maximum binding was found with 40 and n
lo
tions such as salinity and drought, and to examine 80ngproteins. a
d
e
whether SbMYB44 contributes to its adaptability, tran- d
scriptexpressionofSbMYB44wasstudiedinthepresence SbMYB44confersenhancedstresstolerance fro
m
ofdifferentstressesandstress-relatedphyto-hormones. inyeast h
TheresultsshowedthatSbMYB44expressionwasinduced Inthepresenceofdifferentstresses,SbMYB44ORF,driven ttps
significantlybysalt,desiccation,cold,hightemperature, byagalactose-induciblepromoter,showedbettergrowth ://a
c
ABA and SA. In the presence of salinity, the maximum in recombinant S. cerevisiae (W303) compared with ad
e
transcript accumulation of SbMYB44 was observed at wild-type cells. The growth rate of pYES2-SbMYB44- m
ic
1h (8-fold) and then reduced with increasing time transformed yeast cells was similar to the growth rate .o
u
(Fig.3A).Underdesiccationstress,SbMYB44showedan ofpYES2-transformedyeastcellsundernon-stresscondi- p.c
o
early induction at 0.5h (64-fold) and reaches its max- tions (Fig. 6A). In the presence of 2.5 and 5.0M NaCl, m
/a
imum level (120-fold) at 12h (Fig. 3B). In response to pYES2-SbMYB44-transformedcellsshowedhighergrowth o
b
hightemperature,transcriptlevelsshowedtheirhighest thanpYES2alone(Fig.6BandC).Similarly,pYES2-SbMYB44- pla
expressionat24hofexposure(118-fold)(Fig.3C).During transformed yeast cells produce more cells than pYES2 /artic
coldtreatment,theexpressionwasfoundmaximumat alone, in thepresence of thedehydration stress with 15 le
1h(120-fold)andthenreduced(Fig.3D).Inthepresence and30%PEG(Fig.6DandE).Theseresultsindicatethat -ab
s
of ABA, SbMYB44 was maximally expressed at 30min SbMYB44wasfunctionalinyeastandimproveditstolerance tra
c
(6-fold), decreased till 6h and again increased at 12 tosalinityanddehydrationstress. t/d
o
and24h(Fig.3E).InthepresenceofSA,theexpression i/1
0
wasup-regulatedat0.5h(6-fold)andthengetsdown- Discussion .10
regulated(Fig.3F). 9
3
Plant TFs play important roles in regulating the gene /ao
TranscriptionalactivationofSbMYB44 expressionduringdevelopmentandabioticstresstoler- bp
la
Ayeast GAL4system was used to investigate the tran- ance. MYB TFs form a large family of proteins that are /p
lv
scriptionalactivationofSbMYB44.TheGAL4DNA-binding involvedinanarrayoffunctionssuchasprimaryandsec- 05
4
domain-SbMYB44recombinantplasmidwastransformed ondary metabolism, regulation of plant development, /2
0
into yeast strain AH109 and assayed for its ability to regulation of cell fate and identity and in response to 14
9
activatetranscriptionofthedualreportergeneHis3and biotic and abiotic stress tolerance (Dubos et al. 2010; 9
b
LacZ. Yeast cells with recombinant plasmid harbouring Agarwaletal.2013).Inthisstudy,anR2R3-typeSbMYB44 y g
u
SbMYB44grewonSD/-Hismedium,andshowedbluecol- wasisolatedfromS.brachiata.Theinsilicoanalysisrevealed e
s
ourinX-galsolution(Fig.4AandB)indicatingitsactivity thatSbMYB44hassimilarHTHrepeatsaspresentinother t o
n
asaTF. R2R3-typeMYBs(Romeroetal.1998).SbMYB44possesses 2
8
a bipartite type of nuclear localization signal similar to M
a
BindingofSbMYB44recombinantproteinto MdMYB,FvMYB44andGmMYB44.Themotifscancompari- rc
h
cis-elements sonofSbMYB44showedthepresenceofmyristoylation,gly- 20
The6×His-TagSbMYB44recombinantproteinof30.31kDa cosylationandotherpost-translationalmodification.These 19
was induced with 1mM IPTG for different time periods. post-translationalmodificationscouldhelptomediatethe
Theproteinshowedamaximuminductionat6h;therefore, finetuningofSbMYB44activity,althoughnofunctionhas
theproteinwasinducedfor6handpurifiedtonearhomo- yetbeenknownforthesepost-translationalmodifications.
geneity(Fig.4C). Myristoylationplaysavitalroleinmembranetargetingand
TheEMSAwasperformedtostudytheinvolvementof signaltransductioninresponsestoenvironmentalstress
SbMYB44indifferentsignallingpathwaysduringplantde- (PodellandGribskov2004);therefore,thepresenceofamyr-
velopmentandstressbyinteractionwithcis-elementsof istoylationsitesuggeststhatitmayplayaroleinregulating
different promoters like RD22 (dehydration responsive) theactivityofSbMYB44inresponsetodifferentstresses.The
6 AoBPLANTS www.aobplants.oxfordjournals.org &TheAuthors2015
Shuklaetal.—AnR2R3SbMYB44showedstresstoleranceinyeast
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Figure2. ThephylogeneticrelationshipofSbMYB44wasconstructedusingthemaximum-likelihoodmethod.Theproteinsequencesusedforthe
constructionofphylogenetictreeareasfollows:Salicorniabrachiata,SbMYB44(AHN50422),Citrussinensis,CsMYB(ABQ10816),Cucumissativus,
CsMYB44(XP004141899),Fragariavesca,FvMYB44(XP004305954),Glycinemax,GmMYB50(NP001238087),GmMYB68(NP001235715),GmMYB70
(NP001237693),GmMYB80(NP001237709),GmMYB112(NP001235142),GmMYB44(XP003524661),Malusdomesticus,MdMYB(ADL36760),
MdMYB6 (AAZ20429), Medicago truncatula, MtMYB8 (ABR28330), Solanum lycopersicum, SlMYB (XP004238123), Theobroma cacao, TcMYB
(XP007047395), Vitis vinifera VvMYB44 (XP002285015), Arabidopsis thaliana AtMYB44 (AED98326), AtMYB3R-2 (AF151647), AtMYB56
(AF062891),AtMYB45(AF062883),AtMYB61(AF62896),AtMYB97(AF176004),AtMYB84(Y14209),AtMYB30(AF062873),AtMYB47(AF062885),
AtMYB13(Z50869),AtMYB58(AF062893),AtMYB24(AF175987),AtMYB116(AF334815),AtMYB11(AF062863),AtMYB66(AF062900),AtMYB5
(X90380),AtMYB123(AF371981),AtMYB76(AF175992),AtMYB41(AF062882),AtMYB107(AF249310),AtMYB93(AF062917),AtMYB16(X99809),
Hordeumvulgare,MYBHv1(CAA50224).
AoBPLANTS www.aobplants.oxfordjournals.org &TheAuthors2015 7
Shuklaetal.—AnR2R3SbMYB44showedstresstoleranceinyeast
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Figure3. TranscriptprofilingofSbMYB44withdifferentstressconditions:(A)NaCl,(B)desiccation,(C)heat,(D)cold,(E)ABAand(F)SA. 1
9
MYB DNA-binding domain of SbMYB44 possesses highly replacesthefirsttryptophanintheR3repeat.R2R3-MYB
conservedandevenlydistributedWresidues,whichareim- belonging to subgroup 22 have consensus sequence
portantforformingthehydrophobiccoreandmaintaining (GxFMxVVQEMIxxEVRSYM)butSbMYB44hadGPELMAVMQE-
three dimensional structure of the MYB repeats (Stracke MIKKEVKSYMatC-terminalend.Phylogeneticrelationships
etal.2001).SimilartotheR2R3-MYBsofmaizeandArabi- revealedthatSbMYB44clusteredwiththememberofsub-
dopsis (Dubos et al. 2010; Du et al. 2012), SbMYB44 also group 22, such as AtMYB44 and AtMYB70 (Dubos et al.
showedhighconservationofphenylalanineresidue,which 2010), and show close homology with MdMYB (53%),
8 AoBPLANTS www.aobplants.oxfordjournals.org &TheAuthors2015
Shuklaetal.—AnR2R3SbMYB44showedstresstoleranceinyeast
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Figure4. TransactivationassayofSbMYB44.(A)Full-lengthSbMYB44ORFclonedinpGBKT7vector.(B)(i)Transformedyeastcell(AH109)contain- e
s
ingpGBKT7+SbMYB44andpGBKT7alonegrownonSD/-Trp/-His/-Uramedium.(ii)Schematicrepresentationoftheplating.(iii)Yeastcellstrans- t o
n
ferred on filter paper showed b-galactosidase (encoded by LacZ) activity using X-gal staining. (C) SDS–PAGEanalysis of expression of 2
8
recombinantSbMYB44proteininE.coliBL-21Star(DE3)cells.M,marker;UI,un-inducedprotein;I,inducedprotein. M
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FvMYB44 (58%), GmMYB68 (57%) and other members transcriptaccumulationofAtMYB2inresponsetodehy- 2
0
1
of the MYB family belonging to the subgroup 22. The dration, salt, ABA and binding to the cis-element of 9
C-terminalcharacteristicsofSbMYB44areconsistentwith RD22 and MBS-1 promoter (Urao et al. 1993; Abe et al.
itsclassificationinR2R3-MYBsubgroup22andmayalso 2003).TheAtMYB44 showedstronginductionwithsalt,
suggest functional conservation between SbMYB44 and drought,coldandABA(Kranzetal.1998),andstresstol-
othersubgroupmembers. erancebyABA-dependentstomatalclosureintransgenic
SbMYB44wasinducedbyNaCl,desiccation,heat-and Arabidopsis (Jung et al. 2008). AtMYB20 is also highly
stress-relatedphyto-hormones(ABAandSA).Theprotein expressed in the presence of NaCl, drought and ABA
alsoshowedbindingwiththedrought-induciblepromoter (Cuietal.2013).Similarly,theexpressionofPtsrMYB,an
RD22 and MBS-1. Similarly, Urao et al. (1993) showed R2R3-MYBfromPoncirustrifoliata,isinducedbydifferent
AoBPLANTS www.aobplants.oxfordjournals.org &TheAuthors2015 9
Shuklaetal.—AnR2R3SbMYB44showedstresstoleranceinyeast
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Figure5. TheEMSAstudyshowingbindingofSbMYB44proteinwithdifferentcis-elements.(AandB)Bindingof40ngofproteinwithdifferent /p
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amountsofprobe.(CandD)Bindingof80ngofprobewithdifferentamountsofprotein. 0
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stresses(Sunetal.2014).TheexpressionofSbMYB44was cross-talk between abiotic and biotic stress signalling 9
b
alsoinducedbythesalinity,buttherelative-foldinduc- (Zhangetal.2012). y g
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tionwaslower whencomparedwithotherstresseslike MYBproteinshaveahighlyconservedMYBDNA-binding e
s
heat, desiccation and cold. In the coastal region, the domainacrosstheeukaryotesincludingyeast,plantsand t o
n
seawaterisinundatedtwiceinamonthandremaining animals (Lipsick 1996). The R2R3-type MYB TFs have 2
8
times the soil is almost dry and temperature is high, evolved from MYB3R protein,and areclosely related to M
a
therefore, in addition to the salinity, S. brachiata also vertebrate c-MYB and MYB TFs from other eukaryotic rc
h
showstolerancetowardsdehydration and temperature group such as slime moulds and ciliates (Feller et al. 2
0
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alteration. SbMYB44 might be a TFwhich is involved in 2011);therefore, theseproteinspossiblyexisted before 9
toleranceoftheseplantstodesiccationandtemperature the divergence of plants and animals (Kranz et al.
stressratherthansaltandhelpsS.brachiatainrelieving 2000). The most distant relative of c-MYB is the BAS1
physiological drought condition. SbMYB44 was induced gene of S. cerevisiae, involved in the regulation of HIS4
both with ABA and SA, suggesting the involvement of gene. Sequence analysis revealed that BAS1 binds to
ABA and SA in MYB regulation. A wheat MYB TaPIMP1 the PyAACG/TG (closely related MYB response element)
showed resistance against Bipolaris sorokiniana and (LuscherandEisenman1990).Inourstudy,theSbMYB44
to drought stress through activation of SA- and ABA- proteinshowedbindingwithdifferentpromotershaving
responsive downstream genes suggesting its role in similar core sequence (TAACTG) motifs. Therefore, it
10 AoBPLANTS www.aobplants.oxfordjournals.org &TheAuthors2015