全 文 ：第 38卷第 2期 上海师范大学学报(自然科学版) Vol.38, No.2
2 0 0 9年 4月 JournalofShanghaiNormalUniversity(NaturalSciences) 2 00 9 , Apr.
TheArabidopsisbHLHtranscriptionfactor
DYT1 isessentialforantherdevelopmentby
regulatingcalosedissolution
SONGYao＊ , LIHui＊ , SHIQi-long＊ , JIANGHua, CHENHui,
ZHONGXiao-li, GAOJu-fang, CUIYong-lan, YANGZhong-nan
(ColegeofLifeandEnvironmentSciences, ShanghaiNormalUniversity, Shanghai200234, China)
Abstract:Caloseformationanddissolutionduringandaftermeioticdivisionisauniquefeatureofmalemeiosisand
isimportantforpolendevelopmentinplants.Here, wereporttheidentificationandcharacterizationofamale-ster-
ilemutant, ms157, whichexhibitsiregulartapetumdifferentiationandcalosedissolutionduringantherdevelopment
inArabidopsis.MolecularmappingandgeneticanalysisindicatedthatMS157isanaleleofbHLHtranscriptionfactor
DYT1(At4g21330).Thus, themutantwasrenamedasdyt1-2.Ourtrans-activationactivityassayshowedtheacti-
vationdomainofDYT1 islocatedin250bpto504bp.YeasttwohybridassayalsosuggestedthattwoDYT1entirepro-
teinscouldformhomodimersinvivo.RT-PCRandReal-TimePCRanalysisindicatedthatcalasecomplexrelated
geneA6 wasdown-regulatedinthemutantbackground.Hence, DYT1playsanimportantroleinantherdevelopment
byregulatingcallosedissolution.
Keywords:Arabidopsis;callose;tapetum;polen;transcriptionfactor
CLCnumber:Q344+.4 Documentcode:A ArticleID:1000-5137(2009)02-0174-09
Receiveddate:2009-02-25
Foundationitem:NaturalScienceFoundationofChina(30530100).
Biography:SONGYao(1984-), male, graduatestudent, CollegeofLifeandEnvironmentSciences, ShanghaiNormal
University;YANGZhong-nan(1965-), male, professor, CollegeofLifeandEnvironmentSciences, ShanghaiNormalUniver-
sity.
＊ Equalcontributionstothiswork.
1 Introduction
Infloweringplants, theanther, whichusualyhasafour-lobedstructureconsistingofhighlyspecialized
reproductiveandnonreproductivetissues, yieldsmaturepolengrains[ 1, 2] .Thenonreproductivetissuesorso-
maticcellayers, includingtheepidermis, endothecium, middlelayer, andtapetum, arerequiredfornormal
polendevelopmentandthereleaseofpolengrains[ 2, 3] .Thetapetum, whichsurroundsthedevelopingrepro-
ductivecels, playsanimportantroleinpolendevelopmentbycontributingtomicrosporerelease, nutrition,
polen-walsynthesisandsporopolenindeposition[ 4] .Thedysfunctionoftapetumcelsindiferentiationand
secretingusualyresultsinabnormalformationandsterilityofthepolen.Sofar, severalgeneshavebeeni-
dentifiedtoberequiredfortapetumdevelopment.EXCESSMICROSPOROCYTES1/EXTRASPOROGENOUS
CELLS(EMS1/EXS)mediatessignalsthatcontrolthefateofreproductivecels[ 5, 6] .Excessmicrosporocytes
wereformedinems1 /exsmutantwithabsenttapetalcels, andthemiddlelayerpersistedbeyondthenormal
stageofitspresence.TheTAPETUMDETERMINANT1(TPD1)encodesasmalproteinwhichplaysanim-
portantroleinthediferentiationoftapetalcels, posiblyincoordinationwiththeEMS1/EXSgeneproduct, a
Leu-richrepeatreceptorproteinkinase.KnockoutofTPD1causedtheprecursorsoftapetalcelstodiferenti-
ateanddevelopintomicrosporocytesinsteadoftapetum[ 7] .Recently, abHLH transcriptionfactor
DYSFUNCTIONALTAPETUM1(DYT1)wasidentifiedbyDStransposon.Insituhybridizationsuggestedthat
theDYT1 expressionispreferentialydetectedintapetum andmeiocytesatstage5 and6 ofpolen
development[ 8] .
Folowingtetradformationinantherdevelopment, exinesynthesisbeginsinthemicrosporesuroundedby
calose.Duringthisprocess, β -1, 3-glucanase(calase)issecretedbythetapetumcelsandreleasedintothe
locularspace.Finaly, thecalosewalisdegradedandmicrosporesarereleasedinthelocularspace[ 9, 10] .
Thetimingofcalaseactivityinanthersisimportantandtheprematureordelayedcalaseactivitywilcause
malesterileinplants[ 11 ～ 13] .Therefore, calasetakesavitalroleinmicrosporeseparationandantherdevelop-
ment.β-1, 3-glucanasesareadiversefamilyofhydrolyticenzymesandcanbeclassifiedasendoglucanasesor
exoglucanasesdependingonthenatureoftheirenzymaticactiononβ-1, 3-glucans.Themajorityofendoglu-
canaseactivityislocatedintapetum, theimmediatesuroundingofthemeiocytes.Nowlitleisknownabout
themajorityofexoglucanaseactivityinplantanther.Severalcandidategenesencodingtheendo-β-1, 3-glu-
canasecomponentofcalasehavebeenreported.Hird(1993)reportedthatA6 geneisspecialyexpressedin
Arabidopsisanther, whichishomologouswithβ -1, 3-glucanaseinnucleotidesequence[ 14] .Althoughthetem-
poralandspatialexpressiondataofA6 coupledwithtetraddissolution, itsfunctionformicrosporereleasefrom
tetradhasnotbeenconfirmeddirectly, andtheregulationofcalosedissolutionhasnotbeenreportedyet.
Thebasic/helix-loop-helix(bHLH)proteinsareasuperfamilyoftranscriptionfactorsthatbindasdimers
tospecificDNAtargetsitesandthathavebeenwelcharacterizedasimportantregulatorycomponentsindi-
versebiologicalprocesses[ 15] .Previousstudyhasreportedthatthems157 mutantwasobtainedusingEMSmu-
tationstrategyandthegenewasmappedtoaregionof74KBthroughamap-basedcloningmethod[ 16] .Inthis
work, weisolatedMS157(DYT1), andperformedfunctionalanalysis.Wecharacterizedthehomodimerof
DYT1 invivoandfounditscriticalroleincalosedissolutioninantherdevelopment.AlthoughtheMS157isan
aleleofDYT1, theinformationprovidedinthispaperaretheimportantadditionsofDYT1function.
2 Materialsandmethods
2.1 Plantmaterials
TheArabidopsismutantsms157 (Landsbergerecta)werescreenedusinganethylmethanesulfonate
(EMS)mutagenesisstrategy.Beforephenotypicanalysis, ms157 hadbeenbackcrossedtowildtypeLer3
times.Seedsweresownonvermiculiteandalowedtoimbibefor3dat4℃.Plantsweregrownat22℃ under
a16-hr-light/8-hr-darkphotoperiod.AtransposontaggedlineofAt4g21330 (pst18843)wasbought
fromthemutantcolectioninRIKEN(htp://rarge.gsc.riken.jp).
2.2 Methods
2.2.1 Lightmicroscopy
FlowerbudsatdiferentdevelopmentalstageswerefixedovernightinFAA(ethanol50% (v/v), acetic
acid5.0% (v/v), andformaldehyde3.7% (v/v)), dehydratedinagradedethanolseries(50% ×2,
60%, 70%, 80%, 90%, 95%, and100%×2)andtransferredtoxylene, ultimatelyembeddedinSpur s
175 第 2期 宋 垚 ,李 晖 ,石其龙 ,等:拟南芥 bHLH家族转录因子 DYT1在花药发育过程中调控胼胝质降解
epoxyresin.Semi-thinsectionsof1μmweremadeonPowertomeXL(RMCProductsbyBoeckeler)using
glassknivesandwereheatedtofixtoglassslides.Beforestainingwithtoluidineblue, thesectionswereincu-
batedinasaturatedsolutionofsodiummediummethoxidefor2min.Thestainedsectionswererinsedinpure
waterthreetimesandair-dried.Bright-fieldphotographsoftheanthercross-sectionsweretakenusingO-
lympusDX51digitalcamera.Photographspresentedherearerepresentativeofpolendevelopmentfromatleast
fiveindividualplantsandshowtypicalresults.Forexaminationofcalose, sectionswerestainedwith0.05%
(w/v)analinebluein0.067 Mphosphatebufer(pH8.5), andviewedunderUVilumination.
2.2.2 RNAextraction, RT-PCRandreal-Time-PCR
TotalRNAwasisolatedfromfloraltissueofmaturesoilgrownArabidopsisplantsusingtheTrizolkit(In-
vitrogen).FirststrandcDNAwassynthesizedfrom2ugofRNAusingpoly(dT12-18)primer, AMVreverse
transcriptaseandaccompanyingreagentsfor60minat42℃andusedasRT-PCRtemplates.PCRwasper-
formedfor30 and35 cycles(94℃ for30 s, 55℃ for30 s, 72℃ for30 s).RT-PCRprimersforA6
(At4g14080)5-GGTGATGTTACCGTTGCTGAA-3and5-GGTGTACCGATTGGAGGACTT-3, β -tu-
bulin(At5g23860)5-GGACACTACACTGAAGGTGCTGAG-3and5-GGCTCTGTATTGCTGTGATC-
CACG-3, Real-TimePCRprimersforA6 5-TACCTAAACCGACGAACA-3and5-ATGCCAATAAATG-
GAGAC-3, β -tubulin5-GATTTCAAAGATTAGGGAAGAGTA-3and5-GTTCTGAAGCAAATGT-
CATAGAG-3, theβ -tubulinwasusedasconstitutiveexpressioncontrol.
2.2.3 Complementationanalysis
Togenerateacomplementationconstruct, a2.3 kbwild-typegenomicfragmentcontainingthepredicted
promotor, transcriptionregion, andthe3untranslatedregionofAt4g21330wasamplifiedbyPCRusingprim-
ersF:(5-CGTTAATTTAATCTTACCAGAGTTTCTTCTC-3)andR:(5-TACGATTTGAGTTCAAT-
AGAATTTTAAGACC-3), andthenclonedintoamodifiedpCAMBIA1300 Ti-derivedbinaryvector.The
plasmidwastransformedintoAgrobacteriumLBA4404, andthenwasintroducedintodyt1-2/+plants.The
transformedseedswerescreenedusing40mg/Lhygromycin, andtransferredintothesoil.TheT1 plantswere
genotypedtoidentifyhomozygousdyt1-2 backgroundwiththemappingprimers.
2.2.4 Yeasttransformation
Ful-lengthanddiferentregionsofDYT1 cDNAwereamplifiedbyPCRandfusedthemwiththepG-
BKT7 vector.Thentransformedthemintotheyeaststrain(AH109)andscreenedontheSDminimalmedia
(SD/-trp/-his/-ade+x-gal).Theprimersusedwerelistedasfolows:DYT1ful-lengthF:5-
GAATTCGGTGGAGGAAGCAGAT-3;DYT1ful-lengthR:5-GTCGACTGGATTGCTTCTCATAA-3;
DYT1N1F:(1 ～ 249)5-GAATTCGGTGGAGGAAGCAGAT-3;DYT1N1R:5-GTCGACTCATCAATCT-
CAGGAGGAGCTTCTT-3;DYT1C2F:(250 ～ 380)5GTCGACTGGATTGCTTCTCATAA-3;DYT1C2R5
-GTCGACCAAAACTTCCTCTCCCCAATCTTAC-3;DYT1C3F:(370 ～ 504)5GAATTCGGAATCGAG-
GAGAATGTGCAATT3;DYT1C3R5-GTCGACAGATTTCTCGGATTCGAGATTATCG-3;DYT1C3F:(494
～ 624) 5 - GAATTCAGATTTCTCGGATTCGAGATTATCG - 3 ; DYT1C3R: 5 - GTCGAC
TGGATTGCTTCTCATAA-3.
3 Results
3.1 MolecularcloningoftheMS157 locus
Inthepreviouswork, anArabidopsisthalianamalesterilemutantEC2-157(ms157)wasisolatedusing
anethylmethanesulfonate(EMS)mutagenesisstrategy.MS157hasbeenmappedtoaregionof74kblocated
inBACcloneT6K22 onchromosomeIVusingamap-basedcloningstrategy[ 16] .Thisregioncontains11
176 上海师范大学学报(自然科学版) 2009年
genes, ofwhichAt4g21330 isaputativebHLHtranscriptionalfactor(Figure1a)whichidentifiedpreviously
asDYSFUNCTIONALTAPETUM1(DYT1)gene[ 8] .Thus, themutantwasrenameddyt1-2.Weclonedthe
genomicregionofthisgenefrombothmutantandwildtype.Sequenceanalysisindicatedthattherearethree
basepairchangesfromGAG-to-AAG, GAG-to-CAG, andGGA-to-CGAinthecodingregionthatre-
sultinGlu-to-Lys, Glu-to-Gln, Gly-to-Arginthemutantprotein(Figure1b).Ofthemutations,
theaminoacidtransitionfromGlutoArgwaslocatedinthebindingdomainofbHLHprotein.
(a)FinemappingofMS157toaregionof74kbbetweenIn/DelmarkersT6K22(HindII)andT6K22(EcoRV)onchromosome4
(b)TheDYT1genestructureandpositionsofthenucleotidechangesindyt1-2andDsinsertionmutantpst18843.Theblackboxindicatesthe
exonsofDYT1, andthelinesbetweentheexonsindicatethepositionsoftheintrons.ThearrowlineindicatestheDYT1genomicfragmentused
forthecomplementationofdyt1-2.
Figure1 MolecularcharacterizationoftheMS157(DYT1)Gene
TodeterminewhetherthemutationsofAt4g21330 wereresponsibleforthemalesterilityindyt1-2 mu-
tant, a2.3kbgenomicDNAfragmentincludingthepredictedpromoter, codingregion, and3nontranslated
region(Figure1b)wasclonedandintroducedintodyt1-2 heterozygousplantsbyinfiltration.Ofthe74
screenedtransformants(Figure2a), 16 lineswerefoundtobethedyt1-2homozygousplants.Thirteenofthe
16 linesshowednormalfertility(Figure2d).TheseresultsindicatedthatthemutationsofAt4g21330werere-
sponsibleforthemalesterilityindyt1-2 mutants, andthe2.3-kbgenomicfragmentfromchromosomeIV
containsalofthegeneticinformationrequiredforthenormalfunctionofDYT1.
InorderfurthertoconfirmthatthemutationsofAt4g21330 leadtomalesterilephenotypeindyt1-2
plants, aleleanalysiswasperformed.Atransposontagginglineofpst18843withaDselementinsertedinthe
promoterregionofAt4g21330 wasrequestedfromRIKEN(htp://rarge.gsc.riken.jp).Thislinealso
showedmalesterilephenotype.Whenitwasusedasafemaleparentincrossingwithdyt1-2 heterozygous
plants, theF1 plantsfromthecrossessegregatedas1∶1 (fertile∶sterile).Theseresultsindicatethatthe
knockoutofAt4g21330showedmalesterilephenotypeandMS157 isanaleleofDYT1.
3.2 CharacterizationofDYT1 asapositivetranscriptionregulator
BasedonTAIRdatabase(www.arabidopsis.org), DYT1 isamemberofArabidopsisbHLHtranscription
factorfamily.TodeterminewhetherDYT1 isinvolvedintranscriptionregulation, wefusedfourregionsof
177 第 2期 宋 垚 ,李 晖 ,石其龙 ,等:拟南芥 bHLH家族转录因子 DYT1在花药发育过程中调控胼胝质降解
(a)Screeningfortransgenicplant(T1)ontheHygRselectionmedium;
(b)Phenotypeofthewildtype(Col);
(c)Phenotypeofthedyt1-2mutant;
(d)Adyt1-2plantcontainingtheDYT1transgene
Figure2 Phenotypesofthewild-type, dyt1-2mutantandtransgenicplants(complementedforDYT1)
DYT1toyeastvectorpGBKT7respectivelyasdescribedbyFigure3a, andtransformedthemintoyeaststrain
AH109.TheresultsofscreeningonselectivemediumshowedthatthemiddleregionDYT1(250 ～ 380)and
DYT1(370 ～ 504)ratherthantheentireDYT1 proteinsignificantlyactivatedthereportergeneexpresionin
AH109(Figure3a), indicatingthatDYT1 mayserveasapositiveregulatoroftranscriptioninantherdevelop-
ment.ThisresultalsoimpliesthattheN-terminalorC-terminalregionmaybeinhibitsthefunctionofacti-
vationdomainandthecorectconfigurationoftheentireproteinmaybecriticalforthetranscriptionregulation.
ThebHLHtranscriptionfactorsareproposedtoformhomodimersormorecommonlyheterodimerswithan-
otherproteininvivo.TotestwhetherDYT1formshomodimerintransactivationactivity, weclonedtheentire
codingsequenceintopGBKT7 andpGADT7 vectorsandco-transformingthemintotheyeaststrainAH109,
Yeasttwohybridassayshowedthatthesesameproteinscouldinteractseachotherinvivo(Figure3b).These
resultssuggestedthatDYT1isatypicalbHLHtranscriptionfactorwhichformshomodimerinvivotoperformas
apositiveregulator.
3.3 Tapetumdifferentiationanddevelopmentisdefectiveindyt1-2
Tounderstandtheantherdevelopmentdefectsofdyt1-2, semi-thinsectionsofbothwildtypeandmutant
antherweregenerated.Atstage6 ofwild-typeanther, microsporemothercelsentermeiosis, middlelayer
degeneratesandtherearesmalvacuolesinsidethetapetalcels(Figure4a).However, largevacuolescanbe
observedinsidethetapetalcelsofdyt1-2 (Figure4e).Atstage7, middlelayerofdyt1-2 persistedandto-
getherwiththetapetum, enlargedsignificantlyand“crushed” themeioticproductswithinthelocules(Figure
4f).Atstage8ofwildtype, individualmicrosporesarereleasedfromtetrads(Figure4c).Bycontrast, tape-
talcelsarefurthervacuolatedandenlargedindyt1-2, andmeiocytesinitiatedegeneratedwithcrowdedtogeth-
erinsidetheloculeofmutantanther(Figure4g).Atstage12, tapetalcelsaredegradedandmaturedpolen
grainsareformedinwildtype(Figure4d).Whereas, therearenopolengrainsinthemutantanther, andthe
loculebecomescompressed(Figure4h).ThecytologicalobservationshowsthatDYT1 afectstapetum
developmentinArabidopsis.
3.4 DYT1regulatescalosedissolution
Calosesynthesisanddisolutionisauniquefeatureofmalemeiosisinplants.Ascalosedisolutionis
controledbycalasereleasedfromthetapetalcel, weutilizedanilinebluestainingtotestwhethercalosedeg-
178 上海师范大学学报(自然科学版) 2009年
(a)LocalizationofDYT1activationdomain.PartialsequencesofDYT1cDNAwererespectivelyclonedintopGBKT7 andfusedinframewith
theGAL4DNAbindingdomainandtransformedintoyeaststrainAH109.Thenumbersinthebracketsindicatethecorrespondingnucleotideacid
sitesofDYT1cDNA.Filterassayswerecariedoutbyusingx-a-galasasubstrate.pGBKT7, whichharborsthefulllengthDYT1cDNA,
wasusedasapositivecontrol; (b)Confirmationofself-activation.ThefullengthDYT1 cDNAwasclonedintopGBKT7 vectorand
pGADT7vectorandco-transformedintoyeastcels
Figure3 LocalizationofDYT1activationdomainandConfirmationofself-activation
(a, e)Wildtypeantheratstage6showingthetapetumbecomesvacuolated;thedyt1-2mutanttapetumandmiddlelayerbecomemoreexten-
sivevacuolated; (b, f)Atstage7, wildtypemicrosporemothercellscompletemeiosisandformtetrads;thetapetumandmiddlelayercels
ofdyt1-2wereexcessvacuolization; (c, g)Atstage8, microsporeswerereleasedfromthetetradinthewildtypeanther;meiocytesinitiate
degeneratedindyt1-2mutant; (d, h)Atstage12, wildtypeanthershowedthepolengainsandcolapsingtapetalcelswhilethemutantan-
thercrimpleswithoutpolengainsinside.E, epidermis;En, endothecium;Mc, meiccyte;ML, middlelayer;MSp, microspores;PG, pollen
gain;T, tapetum;Tds, tetrads.Bars=20μm
Figure4 Cytologicalobservationofantherandpolendevelopmentofwild-type(a, b, c, d)anddyt1-2(e, f, g, h)
radationwasalteredindyt1-2 mutants.Inbothdyt1-2 andwildtype, caloseandtetradcanbeclearlyob-
servedatstage7 (Figure5a, b).Atstage8, calosewasdissolvedandtetradcannotbeobservedinwild
type(Figure5c).However, indyt1-2anthers, calosewasdetectedfromstage7 tostage12withhighfluo-
rescencecomparedwithwildtype(Figure5d, f).ThisindicatedthatDYT1 afectedthecalosedissolutionin
Arabidopsisantherdevelopment.
179 第 2期 宋 垚 ,李 晖 ,石其龙 ,等:拟南芥 bHLH家族转录因子 DYT1在花药发育过程中调控胼胝质降解
(a, b)Atstage7, strongfluorescenceofcalosewalwasobservedinwildtypeandmutant(whitearow); (c, d)Atstage9, microsporewas
releasedfromtetradandcalosecouldnotbeobservedinwildtype.Calosewasstildetectableindyt1-2 anther(whitearow); (e, f)At
stage11, polengrainswereformedinwildtypewhilecalosestilremainedindyt1-2mutant(whitearrow); Tds:tetrads;Msp:microspore;
PG:pollengrain.Bars=20μm
Figure5 Calosefluorescencestainingofwildtype(a, c, e)andmutant(b, d, f)atdiferentstagesofantherdevelopment
PreviousinvestigationsuggestedthatA6 genewasrelatedwithcalosedissolution[ 14] .Thus, wefurther
analyzedwhetherA6 expressionwasalteredindyt1-2usingRT-PCRmethod.WhenwildtypetotalRNAwas
used, RT-PCRproductscouldbeobservedafteramplificationfor30 and35 cycles.However, thereisno
PCRproductafteramplificationfor30cycles, andaveryweakbandwasobservedafter35cycleswhenreverse
transcriptionproductofdyt1-2 totalRNAastemplates(Figure6a).ThisresultshowedA6 expressionwasse-
verelydown-regulatedindyt1-2.Real-TimePCRanalysiswasusedtofurtherconfirmtheresult.Theexpres-
sionlevelofA6indyt1-2 isonly1.5% ofthewildtype(Figure6b).Therefore, bothRT-PCRandReal-
(a)RT-PCRanalysisofA6expressionwith30and35cycles
inwildtype(WT)anddyt1-2background;
(b)Real-TimePCRanalysisofA6expresioninwildtypeand
dyt1-2background.TheexpressionlevelofA6indyt1-2
inflorescenceswasabout1.5% ofthewildtype
Figure6 A6geneexpressioninwildtype(Ler)anddyt1-2
TimePCRdemonstratedthatDYT1actsupstream
ofA6 geneinArabidopsisantherdevelopment.
4 Discussion
Inthispaper, weidentifiedthelocusof
MS157(DYT1)bygenomesequenceanalysis, ge-
neticcomplementationandaleleanalysis.DYT1
(At4g21330)encodesaputativebHLHtranscrip-
tionfactor.bHLHtranscriptionfactorsarepro-
posedtoformhomodimersormorecommonlyhet-
erodimerswithanotherprotein.Interestingly, they
havebeenreportedaseitheractivatingorrepress-
ingspecificsetsoftargetgenes[ 15] .Inthisstudy,
wefoundthattwoentireDYT1proteinscouldform
ahomodimerinyeastcel, andmiddleregionof
codingsequenceDYT1(250 ～ 380)andDYT1
(370 ～ 504)hadtranscriptionactivityinyeast, theseresultsindicatedthatDYT1 mayformhomodimersasa
positivetranscriptionregulatorinArabidopsis.However, theentireDYT1 proteincouldnotactivatethereport
geneinouryeastexperiment.Similarresultshavebeenpreviouslyreportedinothertranscriptionfactors, suchas
AtMYB21 andAtMYB59, whichshareaW/Y-MDDIWmotif.Thismotifaloneshowsnotrans-activationac-
tivityinyeast, however, itenhancesthetrans-activationactivityoftheneighborregionsgreatly[ 17] .
180 上海师范大学学报(自然科学版) 2009年
WhileweinvestigatedthefunctionofMS157, Zhangetal., (2006)reportedthemolecularcloningand
functionalanalysisofDYT1[ 8] .dyt1isamalesterilemutantwithtapetalandmiddlelayercelssweledwith
expandedvacuolesandfiledthecenterofthelocules.Itsmeiocytescolapsedanddegradedatstage7 and
stage8.Thesecytologicalobservationsisconsistentwithdyt1-2phenotype.Additionaly, DYT1alsoplaysan
importantrolefortapetum-specificgeneexpression[ 8] .BothMS157 andDYT1 belongtothesamelocus
(At4g21330)inArabidopsis.Althoughbothdyt1 anddyt1-2 aremalesterile, dyt1 isaleakymutantwith
DYT1expressedatareducedlevelcomparedwithwildtype.dyt1-2 isEMS-mutagenizedwiththreeamino
acidchangescomparedwithwildtype.Theinformationofdyt1-2mutantandDYT1functionalanalysisprovid-
edinthisworkisanimportantadditionofDYT1functioninantherdevelopment.
Duringantherdevelopment, themicrosporocytesundergomeiosistoformtetradsofhaploidmicrospores.
Inalmostalhigherplants, thetetradsofmicrosporesaresuroundedbyacalosewal, whichissecretedbe-
tweenthecelmembraneandtheprimarycelwal[ 18] .Atacriticaldevelopmentpoint, thecaloseisdegraded
bycalasewhichissecretedbythetapetalcels.Folowingthedegradationoftheprimaryandcalosewalsof
thetetrad, themicrosporesarereleasedintotheantherloculeandcontinuetheirdevelopmentintomaturepol-
lengrains.Thetimingofcalaseactivityandtheresultingcalosebreakdownis, therefore, ofcriticalimpor-
tancetothedevelopingmicrospores.WorkbyWorraletal., (1992)showedthatprematureexpressionofan
engineeredβ-1, 3-glucanasetransgeneinthetapetumresultedinpartialortotalmalesterilityintobacco[ 19] .
Inthiswork, wefoundthatDYT1 regulatecalosedissolution.ThissuggestedthatDYT1 isacomponentof
complexgeneticnetworktoregulatemicrosporereleasefromtetradinArabidopsisantherdevelopment.Asthe
maincompositionofcaloseisβ-1, 3-glucan, calaseshouldbeaβ-1, 3-glucanaseoracomplexwiththemain
compositionofβ-1, 3-glucanase.Althoughseveralcandidategenesencodingtheβ-1, 3-glucanasehavebeen
reported[ 14, 20] , noneofthemhasbeenconfirmedtobecalase.Inourwork, thedefectofcalosedisolutionis
inquiteagreementwithdownregulationofA6 geneexpresionindyt1-2.ThissupportsthesuggestionbyHird
DL(1993)thatA6 maybecalaseorpartofthecalaseenzymecomplex.AsDYT1regulatescalosedissolu-
tion, itsmutantprovidesusanexperimentalapproachtofurtheridentify.Thefurtherfunctionalanalysisshould
helpustoidentifywhichgeneisforcalaseandtodisclosetheregulatorynetworkofcalosedissolutionwhich
isauniqueandimportantprocessofantherdevelopment.
Acknowledgments WethankProfessorHuangHai(InstituteofPlantPhysiology＆ EcologyShanghai
InstitutesforBiologicalSciences, CAS)forprovidingthemutantandtheRIKENRBCforprovisionofthe
transposon-taggedlinesinthisstudy.
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拟南芥 bHLH家族转录因子 DYT1在花药发育过程中调控胼胝质降解
宋 垚 ,李 晖 ,石其龙 ,江 华 ,陈 辉 ,钟晓丽 ,高菊芳 ,崔永兰 ,杨仲南
(上海师范大学 生命与环境科学学院 , 上海 200234)
摘要:胼胝质的合成和降解是雄配子体减数分裂过程中的一个重要特征 , 对后期花粉成熟有重要作用.在此研究中 ,分
离到了一个雄性不育突变体 ms157, 该突变体的绒毡层分化及胼胝质降解过程出现异常 ,导致花粉败育.图位克隆和遗
传分析表明:MS157基因与 bHLH家族转录因子 DYT1 (At4g21330)是同一基因.因此 ,将 ms157突变体改名为 dyt1-2.反
式激活作用实验揭示了 DYT1的激活功能域位于基因的 250 ～ 504bp之间.通过酵母双杂交实验发现 DYT1蛋白在体内
可以形成同源二聚体来执行其功能.RT-PCR及定量 PCR分析表明胼胝质酶相关基因 A6的表达在突变体背景下严重
下调.因此 , DYT1通过调控胼胝质的降解来影响花药发育过程.
关键词:拟南芥;胼胝质;绒毡层;花粉;转录因子
(责任编辑:郁 慧)
182 上海师范大学学报(自然科学版) 2009年