IIIGGGIII111iiisssiiinnnvvvooolllvvveeedddiiinnnaaaxxxiiillllllaaarrryyy bbbrrraaannnccchhhiiinnngggiiinnn

I I IG G GI I I1 1 1i i i s s si i i n n nv v vo o ol l l v v ve e ed d di i i n n na a ax x xi i i l l l l l l a a ar r ry y y b b br r ra a an n nc c ch h hi i i n n ng g gi i i n n n

애 애

애기기기장장장대대대에에에서서서 액액액아아아발발발생생생을을을 조조조절절절하하하는는는 IIIGGGIII111의의의 분

분분자자자유유유전전전학학학적적적 연연연구구구

2 2 20 0 00 0 08 8 8년 년 년 8 8 8월 월 월 2 2 25 5 5일 일 일

조 조

조선 선 선대 대 대학 학 학교 교 교 대 대 대학 학 학원 원 원

생생생 명명명 공공공 학학학 과과과

황 황 황 인 인 인 덕 덕 덕

I I IG G GI I I1 1 1i i i s s si i i n n nv v vo o ol l l v v ve e ed d di i i n n na a ax x xi i i l l l l l l a a ar r ry y y b b br r ra a an n nc c ch h hi i i n n ng g gi i i n n n

지 지

지도도도교교교수수수 정정정 현현현 숙숙숙 이

이

이 논논논문문문을을을 이이이학학학박박박사사사학학학위위위 신신신청청청논논논문문문으으으로로로 제제제출출출함함함

2 2

20 0 00 0 08 8 8년 년 년 4 4 4월 월 월

조 조

조선 선 선대 대 대학 학 학교 교 교 대 대 대학 학 학원 원 원

생생생 명명명 공공공 학학학 과과과

황 황 황 인 인 인 덕 덕 덕

C C

COOONNNTTTEEENNNTTTSSS

L L LI I IS S ST T T O O OF F F F F FI I IG G GU U UR R RE E ES S S· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·ⅲ ⅲ ⅲ L L LI I IS S ST T T O O OF F F T T TA A AB B BL L LE E ES S S· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·ⅳ ⅳ ⅳ A A AB B BB B BR R RE E EV V VI I IA A AT T TI I IO O ON N N· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·ⅵ ⅵ ⅵ A A AB B BS S ST T TR R RA A AC C CT T T· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·ⅶ ⅶ ⅶ

Ⅰ.Introducti on· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·1

A.Pl antarchi tecture· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·1

B.Photoreceptorforl i ghtpercepti on· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·4

1.Phytochromes· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·4

2.Cryptochromes· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·5

3.Phototropi ns· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·5

C.Api caldomi nanceandbranchi ng· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·6

1.Api calDomi nance· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·6

2. genesi nvol vedi nbranchi ng· · · · · · · · · · · · · · · · · · · · ·8

3.Hormoneeffectsonbranchi ng· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·9

Ⅱ.Materi al sandMethods· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·15

A.Pl antmateri al sandgrowthcondi ti on· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·15

B.Mutantscreenandsel ecti on· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·15

1.Thescreeni ngformorphol ogi calmutant· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·15

C.TAIL-PCR· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·16

1.Pl antgenomi cDNA preparati on· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·16

ⅰ

2.TAIL-PCR condi ti on· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·16

D.Genotypi ng· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·17

E.Constructs· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·22

1.Recapi tul ati on· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·22

2.Constructforgenesi l enci ng· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·22

3.Constructforhi stochemi calassayofGUS acti vi ty· · · · · · · · · · · · ·23

F.Pl anttransformati on· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·28

G.RNA preparati onandcDNA synthesi s· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·29

H.Real -ti me(Quanti tati on)PCR· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·29

I.Hi stochemi calanal ysi s· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·30

J.Decapi tati onanal ysi s· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·31

Ⅲ.Resul ts· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·34

A.T-DNA taggi ngmutantscreeni ng· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·34

B.Cl oni ngofthe · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·39

C.Recapi tul ati on· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·43

D.IGI1genei sstrongl yexpressedi nanther· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·55

E.IGI1i nteractswi thLSH1andSUB1· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·61

F.Hormonesdoseresponse· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·61

Ⅳ.Di scussi on· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·73

Ⅴ.References· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·80

ⅱ

L

LLIIISSSTTT OOOFFF FFFIIIGGGUUURRREEESSS

Figure1.GeneraldevelopmentofArabidopsisthalianaColumbia···3

Figure2.Developmentalstagesofapicaldominance···7

Figure3.Theschemeofauxinandcytokininfunctioninthecontrolof budoutgrowth···12

Figure4.Modelforregulationofstagesofbudoutgrowth···13

Figure5.ThemapofpSKI015vector···14

Figure6.TAIL-PCR procedure···18

Figure7.A generalprincipleofGenotyping···21

Figure8.ThemapofpMN20vector···24

Figure9.ThemapofpHANNIBAL vectorfunctionalregion···25

Figure10.GeneralstructureofpCAMBIA vector···26

Figure11.Schematicdiagram ofgenetargetingtoknock-down···27

Figure12.pBI101.2T-DNA region···32

Figure13. mutantsphenotypes···37

Figure14.Axillarybranchingpatternin mutants···38

Figure15.T-DNA positioninthe mutants···40

Figure16.ThegenotypingresultforT-DNA locus···41

Figure17.Theexpressionlevelofneighboringgenes···42

Figure18.ThemapandconfirmedimageforIGI1-RC construct···45

Figure19.Recapitulationofplantphenotypes···46

Figure20.Recapitulationofplantphenotypes···47

Figure21.Relativeexpressionlevelforrecapitulationlines···48

Figure22.Theplantheightandnumberofinflorescence···49

Figure23.ThemapandconfirmedimageforIGI1-RNAiintermediated construct···50

Figure24.ThemapandconfirmedimageforIGI1-RNAiconstruct····51

Figure25. mutantgenotypingforigilocus···52

ⅲ

Figure26.Thephenotypeof mutant···53

Figure27.Real-timePCR resultfor mutant···54

Figure28.TheconstructmapforGUS reporterassayinbinaryvector pBI110.2 ···56

Figure29.DNA fragmentsofBamHIandXbaIdigested constructin1% agarosegel···57

Figure30.ExpressionpatternoftheIGI1gene···58

Figure31.ExpressionpatternoftheIGI1genein33dayoldplant····59

Figure32.TherelativeexpressionlevelsforIGI1geneindifferent tissues···60

Figure33.Diagram ofIGI1genomicDNA···63

Figure34.AminoacidsequenceoftheIGI1···64

Figure35.AP1expressionlevelsin mutnats···66

Figure36.Relativeexpressionlevelof and after decapitation···67

Figure37.Real-timePCR analysisofmultiplebranchingcontrolgenes ···68

Figure38.Cytokininresponse···69

Figure39.Real-timePCR analysisofcontrolgenesofhormones···70

Figure40.Cytokinindoseresponseunderlightanddarkcondition···71

Figure41.Auxindoseresponseunderlightanddarkcondition···72

Figure42.Diagram summarizingthepathwaythatcontrolbranchingin ···78

Figure43.Diagram summarizingthepathwaythatcontrolbranchingin ···79

ⅳ

L

LLIIISSSTTT OOOFFF TTTAAABBBLLLEEESSS

Table1.CyclingconditionsusedfortheTAIL-PCR···19

Table2.ArbitraryprimersandspecificprimersontheT-DNA···20

Table3.Primersfordetectionseveralgeneexpressionlevel.···33

Table4.Segregationratioofthe progeny···36

Table5.Theresultofyeast-twohybridscreening···65

ⅴ

A A

AB B BB B BR R RE E EV V VI I IA A AT T TI I IO O ON N N

2,4-D 2,4-DichlorophenoxyaceticAcid AD ArbitraryDegenerate

AP1 Apetala1

BLAST BasicLocalAlignmentSearchTool BRC1 Branched1

CRY Chyptochromes

DAD DecreasedApicalDominance EST ExpressedSequenceTag FMN FlavinMononucleotide IAA Indolyl-3-AceticAcid

IGI1 InflorescenceGrowthInhibitor1 LOV Light,Oxygen,Voltage

LSH1 Light-dependentShortHypocotyls1 MAX4 MoreAxillaryGrowth4

MS MurashigeandSkoog

MPSS MassivelyParallelSignatureSequencing PCR PolymeraseChainReaction

PERK Proline-richExtensinlikeReceptorKinase PHY Phytochrome

PHOT Phothotropins RNAi RNA Interference

SUB1 ShortUnderBluelight1 SMS ShootMultiplicationSignal SPS Supershoot

TAIL ThermalAsymmetricInterlaced TB1 TeosinteBranched1

TFL1 Terminalflower1

ⅵ

AAABBBSSSTTTRRRAAACCCTTT

I

I IG G GI I I1 1 1i i i s s si i i n n nv v vo o ol l l v v ve e ed d di i i n n na a ax x xi i i l l l l l l a a ar r ry y yb b br r ra a an n nc c ch h hi i i n n ng g gi i i n n n

IndeokHwang

Advisor:Prof.HyeonsookCheong,Ph.D.

DepartmentofBiotechnology

GraduateSchoolofChosunUniversity

Geneticapproach in mutantscreening isan importanttoolforstudying gene function in plant.We selected phenotypically distinctplants in the activation tagged lines and chose a mutant, (Inflorescence Growth Inhibitor1)for furtheranalysis.SegregationratiooftheF2generation,TAIL-PCR walkingand genotyping PCR showed that a single T-DNA was inserted at the 200bp upstream ofthe coding region.Realtime PCR indicated thatthe expression levelof gene was increased approximately by 1,000 to 3,000 foldin homozygousmutant.Thehomozygousmutantdisplayedabnormal phenotypes during reproductive stgage and was sterile. The heterozygous mutant plants produced primary inflorescence with short internode, and secondary inflorescences begin to emerge straightand axillary branching was dramatically increased.We attempted RNA interference (RNAi)to revertthe phenotypeby reducing themRNA levels. expression levelwasdecreased in RNAilinesof heterozygousmutants,and thephenotypesoftheRNAi linesweresimilartothoseofwildtypeplants.Theresultshowed mutants phenotypes were caused by overexpresssion of the gene.Auxin and cytokininareinvolvedinshootbranchingandapicaldominance.Inthecytokinin and auxin response, mutants showed opposite phenotypes in the axillary meristem development.The transcriptlevelofauxin biosynthesis component,

ⅶ

was increased in the mutants.In the celldivision and callus greening testforcytokinin response, mutantsshowed similarpattern with wildtype.Histochemicalanalysisby GUS staining showedtissue-specificgene expressionin theanther.Theexpressionlevelsofthe geneinapicalpart and flowerwere higherthan in otherparts.The IGI1 possesses proline rich domain in N-terminalregion and kinase domain in C-terminalregion.The proline rich domain in N-terminal region interacted with SUB1,which is negative regulatorofthe photomorphogenesis and regulates positive regulator HY5 in thephotomophogenesis.Taken togetherwith thefactthatHY5 binds the promoterregion of gene,my results suggestthatthere mightbe a noblepathway fortheaxillary meristem developmentthatinvolvesIGI1andis modulatedbylightregulatedpathway.

ⅷ

Ⅰ

Ⅰ

Ⅰ...IIInnntttrrroooddduuuccctttiiiooonnn

A

A A. . .P P Pl l la a an n nt t ta a ar r rc c ch h hi i it t te e ec c ct t tu u ur r re e e

Plant architecture is influenced by genetic control and environmental conditions such as light,temperature,humidity,nutrient conditions and etc.

During vegetative phase shootapicalmeristem produces leaf primordia,and inflorescence meristem is initiated from shoot apical meristems and then inflorescence meristem develop into flowers or branches during reproductive phase(Smithetal.,2004;McSteenetal.,2007).

Flowering plants have determinate and indeterminate type for inflorescence architecture.The inflorescences are indeterminate shoot,and the flowers are determinateshoots.Determinateinflorescensesarisewhentheterminalmeristem isconvertedtoaterminalflower.Theflowersinanindeterminatemeristem are of lateral origin (Singer et al., 1990). Wild type have an indeterminate growth patten.The plant generally produces 5 to 15 normal flowersinprimaryinflorescenceandthenterminateswithaflower.Schultzand Haughn (1993) divided the development of the shoot into four stagesbased on nodemorphology atmaturity:juvenilerosette,maturerosette, earlyinflorescence,andlateinflorescencestage.Intheearlyinflorescencestage, the apical meristem initiates several lateral branches. After the primary inflorescence came outfrom apicalmeristem,lateralinflorescence (rosette and caulineleaveinflorescence)isinitiatedintherosetteandcaulineleaveaxils.In the late inflorescence phase,nodes lack leaves and lateralmeristems develop into flowers (a number of lateraland terminal flowers) instead of lateral inflorescence (Fig. 1). The disorder of a gene involved in inflorescence architecturecausesthechangeofinflorescencestructuresbetweenindeterminate anddeterminate.ArabidopsisTFL1(terminalflower)playsan importantrolein

maintaining properfunctionoftheapicalmeristem oftheinflorescence(Schultz and Haughn,1993). mutantdevelopsa terminalflowerattheapex ofthe inflorescence(Ohshimaetal.1997). initiatesflowering early andterminates theinflorescencewithfloralstructuresandthephenotypesweresimilartothat ofthe mutant. doublemutantterminatestheinfloresccencewithout development of lateralflowers. gene product regulates the meristem responsetolightsignalsaffecting thedevelopmentoftheplant.TFL2function influences developmentalprocesses controlled by APETALA1 (AP1),which is flowermeristem identitygeneandpromotesthetransitionfrom inflorescenceto floralmeristem (Weigeletal.,1992;Larssonetal.,1998).

F F

Fiiiggguuurrreee111...GGGeeennneeerrraaallldddeeevvveeelllooopppmmmeeennntttooofff CCCooollluuummmbbbiiiaaa...

B

B B. . .P P Ph h ho o ot t to o or r re e ec c ce e ep p pt t to o or r rf f fo o or r rl l l i i ig g gh h ht t tp p pe e er r rc c ce e ep p pt t ti i io o on n n

The lightis one ofthe crucialenvironmentalsource notonly for energy during photosynthesis also for plant development and growth such as seed germination,de-etiolation,phototropism,andflowering.Planthaveatleastthree majorphotosensory receptors:red/far-red (600-750nm)absorbing phytochrome (PHY), blue/UV-A (320-500 nm) absorbing chyptochromes (CRY) and phothotropins(PHOT),and UV-B (282-320nm)absorbingunknownfactors.In ,there are five phytochrome members,atleastthree cryptochrome members,and two phototropin members (Neffetal,2000;Nagy and Schafer, 2002;LinandShalitin,2003;HuqandQuail,2005).

1

1 1. . .P P Ph h hy y yt t to o oc c ch h hr r ro o om m me e es s s

The phytochromes are dimeric chromoproteins consisting of polypeptide subunits thatcarry a tetrapyrrole chromophore in the amino-terminaldomain.

The carboxy-terminaldomain functions in dimerisation and contains a region with sequence similarity to prokaryotic two-componenthistidine kinases.The photosensory activity ofthe phytochrome molecule resides in its capacity to undergo reversible,light-induced interconversion between two conformers:the biologically inactive, R-absorbing Pr form and the biologically active R-absorbing Pfrform.Thephytochromesarecytosolically localised in theirPr form,butaretriggeredtotranslocateintothenucleusupon photoconversion to theirPfrform (Quail,2002).

2 2 2. . .C C Cr r ry y yp p pt t to o oc c ch h hr r ro o om m me e es s s

CRY were characterized in with mutantthatwas isolated from T-DNA taggedlines.WhengrownunderblueorUV-A light, mutant showedalongerhypocotylthanwildtype(AhmadandCashmore,1993).CRY1 showed sequence similarity to photolyases and the gene encodes a protein family thatmediaterepairofUV-damaged DNA (Sancar,2003).CRY1protein affects anthocyanin production,chalcone synthase gene expression,flowering timeandhypocotylelongation.CRY2alsoaffectedhypocotylelongation.(Ahmad etal.,1995).Analysesof doublemutantandover-expressionphenotype of CRY1 and CRY2 showed that these two CRY play redundant roles in photomorphogenicresponsetobluelightresponse(Ahmadetal.,1998;Linetal, 1998).In seedling stage,GUS fusion protein GUS-CRY1 was localized in the nucleus under dark condition.When the seedlings were grown under white light,theGUS-CRY1wasdepleted from thenucleusand primarily founded in the cytoplasm (Yang etal.,2000).In contrast,CRY2 (second memberofthe Arabidopsis cryptochrome family)was localized in the nucleus in both light- anddark-grownseedlings(Guoetal.,1999).

3

3 3. . .P P Ph h ho o ot t to o ot t tr r ro o op p pi i in n ns s s

The PHOT was most recently characterized blue/UV-A light absorbing photoreceptors in plant.The PHOT proteins have two distinct domains,a C-terminalserine/threonine kinase domain and and N-terminalregion which encode two LOV (Light,Oxygen,Voltage)sub-domain.The LOV domain of POHT,which bind thechromophoreflavin mononucleotide(FMN),isinvolved in a response for blue lightabsorption (Kagawa,2003).PHOTs have been

knowntoregulatephototropism andchloroplastrelocationmovementinreponse tobluelight(Sakaietal.,2001).Analysesofcombinatorialmultiplemutantsof blue lightreceptors ( , , , mutant)and microarray analysis suggestedthatCRYsplaymajorroleswhereasPHOTsplayminorrolesinthe transcriptionalregulationofblue-light-responsivegenes(Ohgishietal.,2004).

C

C C. . .A A Ap p pi i ic c ca a al l ld d do o om m mi i in n na a an n nc c ce e ea a an n nd d db b br r ra a an n nc c ch h hi i in n ng g g 1

1 1. . .A A Ap p pi i ic c ca a al l lD D Do o om m mi i in n na a an n nc c ce e e

Theshootapex affectvariousdevelopmentalprocessesincluding axillary bud growth,the orientation oflaterals,the growth ofrhizomes and stolons,leaf abscission,and others (Imre A.Tamas).In ,apicalbud generally initiate at shoot apex in shoot apicalmeristem.The apical bud controls (inhibits) lateral bud outgrowth. The phenomenon is the apical dominance. MG Cline divided apical dominance and its release into four developmentalstages:(I)lateralbud formation,(II)imposition ofinhibition on lateralbudgrowth,(III)releaseofapicaldominancefollowing decapitation,and (IV)branch shootdevelopment.Theinitialbud outgrowth occursin StageIII during thefirstfew hoursofrelease,which isinhibited by auxin.Thelateral bud outgrowth continuesin StageIV during daysorweeksafterdecapitation, whichmaybepromotedbyauxinandgibberellin(Fig.2)(Cline,1997).

Figure2.Developmentalstagesofapicaldominancebeforeandafterreleaseby decapitationoftheshootapex(Cline,1997).

2 2

2. . . g g ge e en n ne e es s si i in n nv v vo o ol l lv v ve e ed d di i in n nb b br r ra a an n nc c ch h hi i in n ng g g

The function of Petunia DAD (decreased apical dominance) regulating branching in plant architecture have been identified by plant grafting, morphology studies, double-mutant characterization, and gene expression analysis.The mutation in gene caused an increase in basalaxillary branchesandadecreaseinplantheight(Napoli,1996;NapoliandRueeau,1996, Napolietal,1999).Recentstudies showed that is an ortholog ofthe

(MoreAxillary Growth4)in and (Ramosus1)gene in pea (Sorefan etal.,2003;Snowden etal.,2005). MAX4 has homology to carotenoid cleavage dioxygenases required to produce a mobile branch-inhibiting signal,acting downstream of auxin (Sorefan et al.,2003).

encodesaplastidicdioxygenasethatcancleavemultiplecarotenoids,and is required forthe synthesis ofa novelcarotenoid-derived long-range signal that regulates shoot branching (Booker et al.,2004).MAX2 is an F-box, leucine-rich repeat-containing memberofthe SCF family ofubiquitin ligases (Stirnberg etal.,2002).MAX1 controls vegetative axillary bud outgrowth by the regulation of the flavonoid pathway, acts downstream of MAX3/4 to produceacarotenoid-derivedbranch-inhibiting hormone,andencodesamember of the CYP450 family, CYP711A1. Analysis of the mutants demonstrates thatbranching is regulated by atleastone carotenoid-derived hormoneandfour genesactinginasinglepathway,withMAX1,MAX3, and MAX4 acting in hormone synthesis,and MAX2 acting in perception (Bookeretal.,2005).

Anotherbranching signalcomponentBRC1 (branched 1)involved in MAX pathway encodes TCP transcription factor in closely related to

( ) of maize. expression was localized in developing buds and down-regulated in branch outgrowth. RNAi (RNA

interference)and double mutantexperimentindicated that gene prevents rosette branch outgrowth and control branching in downstream of MAX pathway. Also, it is required of auxin induced apical dominance (Aguilar-Martínezetal.,2007).

The (supershoot)mutanthas massive-overproliferation ofshoots.

gene encodes a cytochrome P450 and mutantshowed increased levels in Z-type cytokinins. The result indicates that SPS might modulate shoot branching byregulating cytokininlevelsatsitesofbudinitiation(Tantikanjana etal.,2001).

3

3 3. . .H H Ho o or r rm m mo o on n ne e ee e ef f ff f fe e ec c ct t ts s so o on n nb b br r ra a an n nc c ch h hi i in n ng g g

It has been well-established that auxin among the plant hormones is involved in shootbranching and apicaldominance (Fig.3).The outgrowth of lateralbuds was suppressed in decapitated plants with an auxin treatment (Thimannetal.,1933).Ithasbeenknownthatotherfactorssuchascytokinins alsoregulateshootbranchingandapicaldominanceassecondmessengers(Liet al.,1995,Tantikanjana et al.,2001).Cytokinin application to axillary buds induced bud outgrowth of (King and Van Staden,1988).When endogenous cytokinin levels was increased by overexpression of cytokinin biosynthesis genes,isopentenyltransferase,axillary bud is released in tobacco (Medfordetal.,1989).

Dun et al.(2006) explained the branching control into three hypothesis, classicalhypothesis,auxin transporthypothesis and bud transition hypothesis, involved a rolefortheplanthormoneauxin (Fig.4).Theclassicalhypothesis suggests auxin functions to regulate shoot branching with secondary messengerssuchascytokinin(SachsandThimann,1967;Bangerth,1994;Liet al.,1995).The auxin transporthypothesis suggests thatthe shootbranching

controlisaffectedbyauxinmovementintheauxintransportstream opposedto the actualauxin level.The auxin in the shoottip ofthe main inflorescence generally is loaded into the polar auxin transport stream and moved in a basipetalofplant.Theaxillarybudoutgrowthisinhibitedentirelybytheauxin transportedinthemaininflorescence(Morris,1977;LiandBangerth,1999).The bud transition hypothesis suggeststhatthebud entersdifferentdevelopmental stages thathave varying degrees ofsensitivity orresponses to long-distance signalsincluding auxin (Stafstrom andSussex,1992;Shimizu-SatoandMorris, 2001;Morrisetal.,2005).

Theshootbranching wasincreased and plantheightwasdecreased in mutants.Themutantseedlingsareresistantto 2,4-DichlorophenoxyaceticAcid (2,4-D)and Indolyl-3-acetic acid (IAA)than wild type (Lincoln etal.,1990).

Theoutgorwthoflateralinflorescencesfrom excisedcaulinenodesofwildtype plants is inhibited by apicalauxin,but nodes are resistant to the inhibition.Themeansthattheauxin may inhibitbud outgrowth (Stirnberg et al.,1999).

An activation-tagging vector, pSKI015 contains four multimerized transcriptionalenhancersderivedfrom thecauliflowermosaicvirus(Fig5).The four multimerized transcriptionalenhancers can induce the overexpression of genesneartotherandomly insertedT-DNA,causing new phenotypes.(Weigel etal.,2000).A phenotypicallydistinctplant, (InflorescenceGrowthInhibitor 1),wasselected in theactivation tagged lines.Themutantwasheterozygous line,and thehomozygousmutantlinesweresterile,with no inflorescenceand no seeds.The mutant also displayed abnormalflower development.In heterozygous mutant the internode elongation was reduced in the primary inflorescence.Additionally,secondary inflorescences begin to emerge straight from theaxilsofboth rosetteand caulineleaves,and,asaresult,themutant interestingly showed increased axillary branching pattern.These phenotypes were induced by overexpression ofa gene encoding proline-rich extensin-like

receptorkinase(PERK)namedIGI1. geneexpressionlevelswasdecreased and gene was overexpressed in the mutants.SUB1 (shortunder bluelight)and LSH1 (Light-dependentShortHypocotyls 1)wasidentified by yeast-two-hybridscreening,and mutantsshowedsimilarpatternwithwild typein hypocotylgrowth underdark condition,suggesting thatIGI1 modulate axillary branching through notonly the planthormone auxin butalso light signal.

Auxin

Cytokinin

Auxin

Cytokinin

X X X X

F F

Fiiiggguuurrreee 333...TTThhheee sssccchhheeemmmeee ooofffaaauuuxxxiiinnn aaannnddd cccyyytttoookkkiiinnniiinnn fffuuunnnccctttiiiooonnn iiinnn ttthhheee cccooonnntttrrrooolllooofff b

b

buuuddd ooouuutttgggrrrooowwwttthhh... The axillary buds were suppressed by auxin signal and cytokinininducesbudoutgrowth.

F F

Fiiiggguuurrreee 444...MMMooodddeeelllfffooorrr rrreeeggguuulllaaatttiiiooonnn ooofff ssstttaaagggeeesss ooofff bbbuuuddd ooouuutttgggrrrooowwwttthhh...The classical hypothesis is illustrated in a,b,c,and d; the auxin transport hypothesis is illustratedineandf;analternativeinterpretationoftheauxintransporthypothesis isillustrateding;hypothesizedfeedbackinteractionsareillustratedinh,j,k,andl; and the bud transition hypothesis is illustrated in a,b,c,d,g,h,i,j,k,and l. Arrowheadlinesindicatepromotionandflat-endedlinesindicateinhibition.Location of cytokinin is not specified. Long-distance feedback regulation of cytokinin resulting from the perception ofSMS (shootmultiplication signal)by RMS4 (not depicted explicitly) refers to xylem-sap cytokinin,whereas auxin regulation of cytokinin occursin shootsand roots.Feedback regulation ofauxin,which may be from an independentfeedback process,is notshown because itmay affectauxin levelsortransport(Dunetal.,2006).

F F

Fiiiggguuurrreee 555...TTThhheee mmmaaappp ooofffpppSSSKKKIII000111555 vvveeeccctttooorrr...Activation-tagging vector,pSKI015 constructed by IgorKardailsky in the Weigellab (Weigeletal.,2000).This vectoris composed with the CaMV 35S enhancers,basta resistance gene for selectioninplants,andampicillinresistancegeneforplasmidselectionin

andin .

Ⅱ

Ⅱ

Ⅱ...MMMaaattteeerrriiiaaalllsssaaannndddMMMeeettthhhooodddsss

A

A A. . .P P Pl l la a an n nt t tm m ma a at t te e er r ri i ia a al l ls s sa a an n nd d dg g gr r ro o ow w wt t th h hc c co o on n nd d di i it t ti i io o on n ns s s

Columbia-0 (Col-0)was used as the wild type.Seeds weresurfacesterilizedwith70% Ethanolcontaining0.05% tritonX-100and95%

Ethanol.Afterplatingonthemedium,theplatewaswarpedwithaluminium foil andcoldtreatedat4℃ for3days.Allseedsweregerminatedonhalfstrength Murashigeand Skoog (MS)medium supplemented with 1% sucrose(1/2MS․

1S)andtheantransferredtosoil.Transferredplantsweregrowninthegrowth room at22℃ under16hrslightand8hrsdarkcycle.

B

B B. . .M M Mu u ut t ta a an n nt t ts s ss s sc c cr r re e ee e en n ni i in n ng g g a a an n nd d ds s se e el l le e ec c ct t ti i io o on n n 1

1 1. . .T T Th h he e es s sc c cr r re e ee e en n ni i in n ng g gf f fo o or r rm m mo o or r rp p ph h ho o ol l lo o og g gi i ic c ca a al l lm m mu u ut t ta a an n nt t t

Activation-tagged columbia seeds were provided by Dr.Soo Young Kim (Chonnam NationalUniversity),and the plants were screened by examining morphologicalphenotypes on soil.Selected mutants were back-crossed with Col-0 forsearching singlecopy lines.Themutantswerethen back-crossed again with Col-0 to confirm singlecopy integration and compared with the originalmutantphenotypes.

C

C C. . .T T TA A AI I IL L L- - -P P PC C CR R R 1

1 1. . .P P Pl l la a an n nt t tg g ge e en n no o om m mi i ic c cD D DN N NA A A p p pr r re e ep p pa a ar r ra a at t ti i io o on n n

The DNeasy plantminikit(QIAGEN)was used for the plantgenomic DNA preparation.Plantleaf(0.1g)wasground in liquid nitrogen to afine powderusing themortarandpestle.Groundplantsweretransferredintothe 1.7㎖ microtube,and400㎕ ofbufferAP1and4㎕ ofRNaseA stocksolution were added.The mixture was incubated for10min at65℃.130ulofbuffer AP2wasadded toatubecontaining thelysate,andthetubewasincubated for 5min on the ice.The lysate was transferred to the spin column and centrifuged for2min atthemaximum speed.Theflow-through fraction was transferred to a new tubeand added 1.5 volumes ofbufferAP3/Ethanolto theclearedlysate.Again theflow-through wastransferredtoanew column and centrifuged atthemaximum speed.To wash,bufferAW wasadded to thecolumnandcentrifuged.100㎕ ofpreheated(65℃)bufferAE wasdirectly added onto the membrane,and it was incubated for 5min at the room temperatureandcentrifugedfor1minforelution.

2

2 2. . .T T TA A AI I IL L L- - -P P PC C CR R R c c co o on n nd d di i it t ti i io o on n n

TAIL(Thermal Asymmetric Interlaced)-PCR (polymerase chain reaction) (Liu etal,1995)was performed with long specific primers on the T-DNA and short arbitrary primers on the genomic DNA (Table 2).Three PCR reactions are carried outby using the LB150,LB100,and LB50 (Table 1).

Figure6 showsthatT-DNA specificprimers-LB150,LB100,LB50,and AD primerpairs make the specific productofthe genomic sequence flanking a T-DNA insertion.The last amplification band is eluted and carried out

sequencing.T-DNA insertion sitesofthetransgeniclineswereidentified by basiclocalalignmentsearchtool(BLAST)search.

D

D D. . .G G Ge e en n no o ot t ty y yp p pi i in n ng g g

T-DNA insertion sites and inserted T-DNA direction were determined by PCR procedure using T-DNA primers and genomic DNA primers.Also,the TAIL-PCR resultsgiveustheinformationwhetherthelinesarehomozygousor heterozygous.Figure 7 shows the principle of Genotypting.If the T-DNA present,thePCR productisn'tpresentwhen thePCR reaction wascarriedout with a genomic F primer and R primers,and when the PCR reaciton was carried outwith theT-DNA primer(RB and LB primer)and genomicprimer (R andF primer)thePCR productispresent.Ifitwasaheterozygous-line,the PCR productisalsopresentwhen thePCR reaction wascarried outwith the genomicF primerandgenomicR primer.

F F

Fiiiggguuurrreee 666...TTTAAAIIILLL---PPPCCCRRR ppprrroooccceeeddduuurrreee fffooorrrttthhheee ssspppeeeccciiifffiiiccc aaammmpppllliiifffiiicccaaatttiiiooonnn ooofffgggeeennnooommmiiiccc s

s

seeeqqquuueeennnccceeeffflllaaannnkkkiiinnnggg aaaTTT---DDDNNNAAA iiinnnssseeerrrtttiiiooonnn...T-DNA specificprimersandgenomic random primerwereusedforTAIL-PCR.TAIL-PCR tertiary reaction products are sequenced and T-DNA insertion sites of the transgenic line can be analyzedbyBLAST search.

T T

Taaabbbllleee111...CCCyyycccllliiinnngggcccooonnndddiiitttiiiooonnnsssuuussseeedddfffooorrrttthhheeeTTTAAAIIILLL---PPPCCCRRR (Liu ,1995).

Reaction File no.

Cycle

no. Thermalcondion

Primary 1 2 3

4

5 1 4 1

14^a

1

93^OC(1min)→95^OC(1min)

94^OC(45sec)→62^OC(1min)→72^OC(2.5min)

94^OC(45sec)→25^OC(3min)→ramp to 72^OC in 3min (0.3^OC/sec)→72^OC(2.5min)

94^OC(20sec)→68^OC(1min)→72^OC(2.5min)→94^OC(20sec)

→ 68^OC(1min)→ 72^OC(2.5min)→ 94^OC(20sec)→

44^OC(1min)→72^OC(2.5min) 72^OC(5min)

Secondary 6

5

11^a

1

94^OC(20sec)→64^OC(1min)→72^OC(2.5min)→94^OC(20sec)

→ 64^OC(1min)→ 72^OC(2.5min)→ 94^OC(20sec)→

44^OC(1min)→72^OC(2.5min) 72^OC(2.5min)

Tertiary 7 5

20 1

94^OC(38sec)→44^OC(1min)→72^OC(2.5min) 72^OC(5min)

Note.Theprogram filesin each reaction werelinkedautomatically.^aTheseare nine-segmentsupercylces.

T T

Taaabbbllleee 222.Arbitrary degenerate(AD) primers on the genomic DNA and long specificprimers(LB150,LB100andLB50)ontheT-DNA (Liuetal,1995).

arbitrary degenerate primers on the genomic DNA

AD2 5'-NGTCGASWGANAWGAA AD5 5'-SSTGGSTANATWATWCT

long specific primers on theT-DNA

LB150 5'-CACGTCGAAATAAAGATTTCCG LB100 5'-CCTATAAATACGACGGATGCT

LB50 5'-ATAATAACGCTGCGGACATCA N=A,T,C orG S=C orG W=A orT

F F

Fiiiggguuurrreee 777...AAA gggeeennneeerrraaalllppprrriiinnnccciiipppllleee ooofffGGGeeennnoootttyyypppiiinnnggg...In case ofhomozygous line, the PCR product is not present for the T-DNA insertion when the PCR reactionwascarriedoutwithagenomicF primerandR primer.But,whenthe PCR reactionwascarriedoutwiththeT-DNA primer(RB andLB primer)and genomic primer(R and F primer),the PCR productis present.Ifitwas a heterozygousline,thePCR productpresentwhenthePCR reactionwascarried outwith agenomicF primerandR primer.Alsowhen thePCR reaction was carriedoutwiththeT-DNA primer(RB andLB primer)andgenomicprimer(R andF primer),thePCR productispresent.

E

E E. . .C C Co o on n ns s st t tr r ru u uc c ct t ts s s 1

1 1. . .R R Re e ec c ca a ap p pi i it t tu u ul l la a at t ti i io o on n n

pMN20vectorincluding thefourenhancerswereused forrecapitulation of phenotypes(Fig.8)(Weigeletal.,2000).The genewereproduced bythePCR amplification.PCR reactionsarecarriedoutbyusingthe50KC1F (5'-AAc tgc agA GGT ATC TGT TAC TTT CAC CTA-3')and 50KC1R (5'-TTctgcagG CCC ATC TAG ATT TCA CAT CAT-3').PstIrestriction siteswasusedforcloningthe gene.ThecloneswereconfirmedbyPCR reaction with genomic partprimerand T-DNA partprimer,digestion with severalrestrictionenzymesandsequencing.

2

2 2. . .C C Co o on n ns s st t tr r ru u uc c ct t ts s sf f fo o or r rg g ge e en n ne e es s si i il l le e en n nc c ci i in n ng g g

The pHANNIVAL vector was used for gene cloning (Fig. 9). Intron containing hairpin RNA constructshows silencing oftargeting gene.KpnI restrctionenzymeisusedforsenseorientationinsertionandBamHIrestrction enzymeisforanti-senseorientation.PCR amplificationswerecarried outon cDNA. The sequence of the PCR primers were: for the sense strand amplification,aforwardprimer5'-CGg gtaccA GAA TGT CTA ACA CAT GCA GC-3'named RIKpnI2F and a reverse primer5'-CGg gta ccT AGC GCC GGA ATA TGC ATC A-3'named RIKpnI2R,and forthe anti-sense strand,aforwardprimer5'-CGg gatccA GAA TGT CTA ACA CAT GCA GC-3'named RIBamHI2F and areverseprimer5'-CGg gatccT AGC GCC GGA ATA TGC ATC A-3'namedRIBamHI2R.Theseamplificationproducts weredigested with KpnIand BamHIrestrction enzymesand directly cloned into the pHANNIVAL.The construct were digested with SacI and PstI

restrcitonenzymes.ThefragmentsubclonedintothepUC18multicloningsite ofabinary vectorpCAMBIA1302(Fig.10and Fig.11).Allconstructswere confirmedbyPCR reaction,enzymedigestionandsequencing.

3

3 3. . .C C Co o on n ns s st t tr r ru u uc c ct t ts s sf f fo o or r rh h hi i is s st t to o oc c ch h he e em m mi i ic c ca a al l la a as s ss s sa a ay y yo o of f fG G GU U US S S a a ac c ct t ti i iv v vi i it t ty y y

The predicted promoterregion ofthe genewas amplified from wild type genomic DNA by the PCR amplification with the primers 5'-TCg gat ccG GCG ACT CGC CTA AGT CTG ACA T-3' named KCPCF6 and 5'-AGg gat ccT ATA CTA AGA TCA CGT TAC TTG CC-3' named KCPCR5.ThePCR productwasdigestedwithBamHIrestrictionemzymeand thendiretlyclonedintopBI101.2binaryvector(Fig.12).

F F

Fiiiggguuurrreee888...TTThhheeemmmaaapppooofffpppMMMNNN222000vvveeeccctttooorrr...Thevectorincludes4copiesofCaMV 35S enhancerandareusedtoconfirm activation-taggedgenes.Alsothevector containsnptIIgeneforplantselectiononMS mediaandspectinomycingenefor plasmidselectionin and .

F F

Fiiiggguuurrreee 999...TTThhheee mmmaaappp ooofffpppHHHAAANNNNNNIIIBBBAAALLL vvveeeccctttooorrr fffuuunnnccctttiiiooonnnaaalllrrreeegggiiiooonnn...The PCR fragmentcould be inserted in the sense orientation into the XhoI.EcoRI.KpnI polylinker and in the anti-sense orientation into ClaI.HindIII.BamHI.XbaI polylinker.The construct will produce an hairpin RNA for silencing with targetinggene.

F F

Fiiiggguuurrreee 111000...GGGeeennneeerrraaalllssstttrrruuuccctttuuurrreee ooofffpppCCCAAAMMMBBBIIIAAA vvveeeccctttooorrr...The pCAMBIA vector backboneisderivedfrom thepPZP vectors.pCAMBIA vectorsofferhigh copy number in E.colifor high DNA yields,pVS1 replicon for high stability in ,bacterialselectionwithchloramphenicolorkanamycin,andplant selectionwithhygromycinB.

F F

Fiiiggguuurrreee111111...SSSccchhheeemmmaaatttiiicccdddiiiaaagggrrraaammm fffooorrrRRRNNNAAAiiicccooonnnssstttrrruuuccctttsss...TheKpnIand BamHI fragmentforgenesilencing isclonedintothepHANNIVAL andsubclonedinto thebinaryvectorpCAMBIA1302.

F

F F. . .P P Pl l la a an n nt t tt t tr r ra a an n ns s sf f fo o or r rm m ma a at t ti i io o on n n

Allcloned vectors were introduced into the A

) GV3101 stain by freeze and thaw method.Competentcellof wasmixedwithabout1㎍ DNA in micro-tube.Themixturewas frozenwithliquidnitrogen(LN2)for2minutes(min)andmovedin37℃ water.

Themixture tube was incubated for5 min in waterbath to melt.The step, freezeand melt,wasrepeated onemoretime.Thetubewasincubated in ice for30min.Afterincubationinice,Thecellmixturewasspreadedwithabent glass rod on YEP (Bacto-peptone 10 g/ℓ,Bacto-yeastextract10 g/ℓ,and NaCl5g/ℓ)platescontainingantibiotics.Colonieswereconfirmedbydigestion andPCR reaction. -mediatedtransformationwascarriedoutbya dipping transformation protocolby Kim Hanson 5/95 forVacuum Infiltration, andadaptedfrom protocolbyAndrew Bent( Network1/11/94based on work ofNicoleBechtold,JeffEllisand GeorgesPelletier)with suggestions from PeterDoernerand TakashiAraki.Agrobacterium wasgrown to mid-log phasein YEP medium,pelletedand resuspendedwith infiltration medium (Half strength MS salt,Gamborg'svitamin 112 ㎎/ℓ(Duchefa,G0415),and 0.44uM benzylamino purine).Plants was inverted in infiltration media for15 minute.

Immediately,the plants were loosely covered with plastic wrap to maintain humidity.Theplasticwrapwasremovedafteraday.Thesoilwassufficiently dried abouta week and than watered.T1 seedswereselected on the1/2MS medium withantibioticstogettransformedplant,T2toselectsinglecopylines, andT3todistinguishhomozygousandheterozygouslines.

G

G G. . .R R RN N NA A A p p pr r re e ep p pa a ar r ra a at t ti i io o on n na a an n nd d dc c cD D DN N NA A A s s sy y yn n nt t th h he e es s si i is s s

Total RNA was isolated from plant tissues using Trizol reagent (Life Technologies).100㎎ oftheplanttissuewashomogenizedbyfreezingwiththe liquidnitrogenandgroundtotheveryfinepowder.1㎖ ofTRIzolreagentwas added to homogenized tissuesamples,mixed,incubated for10min attheroom temperature. 0.2 ㎖ chloroform was added to those samples, mixed and incubated for3min attheroom temperature.Thesampleswerecentrifuged at 13,000 rpm for 15 min at 4℃ and a colorless upper aqueous phase was transferred to the new tube.0.5 ㎖ ofisopropylalcoholwas added to the samplesandthesampleswerecentrifugedat13,000rpm for10minat4℃.The supernatantwasremoved,thepelletwaswashed with 70% ethanoland dried.

cDNA was synthesized by using the superscript Ⅱ reverse-transcriptase (Invitrogen).4㎕ (about2㎍)oftotalRNA and1uloftheoligodT (500㎍/

㎖)weremixed in thereaction tubeand itwas heated at65 ℃ for10 min.

Theenzymewasadded into thetubeand incubated at42℃ for50 min.The reactiontubewasincubatedat70℃ for15minforinactivation.

H

H H. . .R R Re e ea a al l l- - -t t ti i im m me e e( ( (Q Q Qu u ua a an n nt t ti i it t ta a at t ti i io o on n n) ) )P P PC C CR R R

Real-timePCR amplificationswerecarriedoutusingcDNA from mutantsand wild type.Forthe gene amplification,the forward primer5'-AGG CGA TTG AAG ATC TTG AGA CGG AGG A-3' named EPKF and a reverse primer5'-ATG TCA GAC TTA GGC GAG TCG CCG AGT TCT-3'named EPKR.Allthe primers were selected by computeranalysis with the PRIME program.Weused QuantiTectSYBR Green PCR Kit(QIAGEN)containing 2x SYBR Green PCR Master Mix with ROX as a passive reference dye,

HotStarTaqDNA PolymeraseanddNTP Mix.5pmolofbothprimers(Forward and Reverse),1 ㎕ cDNA and RNase-free waterwere added.For gene, PCR amplifiedwithadenaturationof15minat95℃ 40cyclesat95℃ 20s;5 5℃ 20s72℃ 40s;andafinalextensionof5minat72℃.Itwasmeasuredon a real-time DNA detection system (Corbettco,RG-3000,Australia).Forthe multiple branching control genes, cytokinin response genes, and auxin biosynthesisgenesprimerpairsweredescribedinTable3.

I

I I. . .H H Hi i is s st t to o oc c ch h he e em m mi i ic c ca a al l la a an n na a al l ly y ys s si i is s s

For GUS activity assay,MUG assay was performed.Sample was ground with150㎕ extractionbuffer(50mM sodium phosphatebuffer(pH 7.0),10mM EDTA,0.1% triton X-100,0.1% sarcosyl,10 mM β-mercaptoetahnol).The sampleswerecentrifuged at13,000rpm for10min at4℃ and upperaqueous phasewastransferredtothenew tube.Protein concentration ismeasuredwith Bradford reagentusing 25㎍ sample.8㎕ MUG stock solution wasadded to thesamplesandthemixturewasincubatedfor1hourat37℃.After1hour, 40㎕ samplemixtureand 160㎕ 0.2M Na2CO3weremixed to stop reaction, andtheactivitywasmeasuredat420nm wavelength.

Histochemical analysis using GUS staining was performed by incubation tissue in GUS staining buffer containing 2 mM cyclohexylammonium salt (Duchefa),100mM sodium phosphatebuffer(pH 7.0),10mM EDTA,0.5mM potassium ferrocyanide,0.5 mM potassium ferricyanide,and 0.1% triton X-100 (volumein volume,v/v).Sampleswereincubated for16hoursin thereaction bufferanddestainedwith70% (v/v)ethanol.

J

J J. . .D D De e ec c ca a ap p pi i it t ta a at t ti i io o on n ne e ex x xp p pe e er r ri i im m me e en n nt t t( ( (A A Ap p pi i ic c ca a al l ld d do o om m mi i in n na a an n nc c ce e et t te e es s st t t) ) )

Arabidopsis wild type plants,Col-0,were grown underlong day condition.

Afterprimary boltscomeout(<10cm),theprimary shootwasremoved.RNA wasextractedfrom decapitatedplantsin0hour(hr),1hr,2hr,4hr,8hr,12 hr,24hr,36hr,and48hrafterdecapitation.Theexperimentwasrepeatedtwo times.

F F

Fiiiggguuurrreee111222...pppBBBIII111000111...222TTT---DDDNNNAAA rrreeegggiiiooonnn...Thevectorcontainspromoterless,1.87kb GUS cassette,andbacterialandplantselectionwithkanamycin.

T T

Taaabbbllleee333...PPPrrriiimmmeeerrrsssfffooorrrdddeeettteeeccctttiiiooonnnssseeevvveeerrraaalllgggeeennneeeeeexxxppprrreeessssssiiiooonnnllleeevvveeelll.

G G

Geeennneeennnaaammmeee PPPrrriiimmmeeerrrnnnaaammmeee PPPrrriiimmmeeerrrssseeeqqquuueeennnccceee(((555'''→→→333''')))

MAX1 (At2g26170)

MAX1F MAX1R

ACATCAATGGTGGGAAGTTCTTGATCCA ACCATAAGAGATGGCCATATATACCA

BRC1 BRC1F

BRC1R

TTCCCAGTGATTAACCACCAT TCCGTAAACTGATGCTGCTC

BRC2 BRC2F

BRC2R

TCAAAGAGAAGAACAAAGACTATGGA CCGAGGTCTCAAATAATCTCATC

CYP79F1 (At1g16410)

SPS1F SPS1R

ACATCATGATGAGCTTTACCACATCAT GGAGTTTATGGTGATGGCACGGATGCCGGC

CYP79F2 (At1g16400)

CYP79F2F CYP79F2R

TGGCCGACCAGGATGGCCCATCCTCGG TGTCTCCAATGGACTCTACGATGGAAAGT

REV (At5g60690)

REVF REVR

AGTGTTGTTCGTTCAGAGTCTTCAA AGACCAACGACATGTGGATACGAGA

ARR4 (At1g10470)

ARR4F ARR4R

AGCTCGTCTATGGCCAGAGACGGTGG AGGCATACAGTAATCAGTGATGATC

ARR5 (At3g48100)

ARR5F ARR5R

CCCGAGATGTTAGATATCTCTAACGAC ATCCAGTCATCCCAGGCATAGAGTA

CYP79B2 (At4g39950)

CYP79B2F CYP79B2R

TGTGGCTATAACCTTAGTGATGCTACT GCCGTTAGAGAGGATCTTCTGAGCGTAAG

CYP79B3 (At2g22330)

CYP79B3F CYP79B3R

ACGACCAAGTCAAGTCTCGGAATGTCGT GGGATCACGTGAGTGTTTCCTAGACGCA

Ⅲ

Ⅲ

Ⅲ...RRReeesssuuulllttt

A

A A. . .M M Mu u ut t ta a an n nt t ts s sc c cr r re e ee e en n ni i in n ng g g

Geneticapproachinmutantsscreeningisanimportanttoolforstudyinggene function in plant.Wegotmorphologicalmutantplantsin theactivation tagged lines and chose a mutant.The mutant exhibited a number of phenotypes:

smaller silique,semisterility,bunchy stem and shortened inflorescence.When selfed, three phenotypic classes of progeny were observed with an approximately1:2:1ratio.Thefirstclasswassterileandseverely defective,the secondclasshadmany branching likeoriginalmutant,andthethirdclasswas normal,similar with wild type.To conform single copy,back-crossing with wild type to originalmutantwas performed.In F1 generation,the progeny showedasegregation ratioofapproximately 1:1(survivalplants:deadplants) in bastaplate.Allsurvivalplantshowedphenotypessimilartooriginalmutant insoil.InF2generation,thesevenplants,whichsurvivedinF1generation,the progenyshowedasegregationratioofapproximately3:1ratio(survivalplants:

deadplants)inbastaplate(Table4).Theviableplantsalsosegregatedseverely defective phenotypes and resemble phenotypes with original mutant with approximately 1:2ratioin soil.Theseresultsindicatethatoriginalmutantwas single copy line and heterozygous, and three phenotypic classes in next generationoforiginalmutantcorrespondedtoplantscontainingahomozygous,a hetetozygous,or no mutation.Upon selfpollination,plants looking wild type produced only wild type progeny,whereas alloriginalmutantsegregated out three phenotypic classes.In heterozygous mutant,the phenotype of young seedlings is similar to wild type,and homozygous mutant showed curled and smallerleaf(Fig.13A).The homozygous mutantshowed sterilityandhadnoinflorescence,noseedsandabnormalflowerorganafterthe plants start to flower (Fig.13B).The heterozygous mutant plants produce

primary inflorescence with reduced internode elongation, and secondary inflorescencesbegintoemergestraight.Thenumberofinflorescencedeveloping from the rosette leaves in wild type is usually 1 to 5 and fewer than 10 includinginflorescenceofcaulineleaves.Incontrast,heterozygousmutantplants continue to produce axillary inflorescence from the axils ofboth rosette and cauline leaves. Consequently, the heterozygous mutant have dramatically increasednumberofaxillarybranches(Fig.14).

T T

Taaabbbllleee444...SSSeeegggrrreeegggaaatttiiiooonnnrrraaatttiiioooooofffttthhheee ppprrrooogggeeennnyyy...Screenedmutantdidn'thave single T-DNA.After the mutantwas back-crossed with wild type and the genotyping PCR was performed in F2 generation for searching single copy lines.After fixing the originalmutantwhich have single T-DNA insertion, againthemutantwasback-crossedwithwildtypetotestthesinglecopy line or not. After seedling heterozygous mutant (expected) in 1/2MS medium containingbasta20mg/l,theplantswerecounted.

Plant(s)/cross

Numberofplants

Similarwith wildtype

Defective

phenotype deadplants Theoriginalmutant( )/self 226 119 150

× wildtype F1

F2(7plants)

38 1638

- 910

31 941

A B

F F

Fiiiggguuurrreee111333... mmmuuutttaaannntttsssppphhheeennnoootttyyypppeee...A.10dayoldplants.B.25dayoldplant.

From leftto right, heterozygous,and homozygous mutantplant.

A B C

D E F G

F F

Fiiiggguuurrreee 111444... AAAxxxiiillllllaaarrryyy bbbrrraaannnccchhhiiinnnggg pppaaatttttteeerrrnnn iiinnn mmmuuutttaaannntttsss... A. Wild-type axillary inflorescencefrom rosetteleaves.20day-old plant(B)and 40day-old plant(C)of homozygousmutantareshown.Thehomozygousmutant have no inflorescence and abnormal flowers. D-F. heterozygous mutant axillary inflorescence from rosette leaves. G. Multiple axillary inflorescencesemergefrom eachcaulineleafof mutant.

B

B B. . .C C Cl l lo o on n ni i in n ng g g o o of f ft t th h he e e

To determine T-DNA locus ofthe mutants,we extracted genomic DNA from heterozygous mutant,and performed thermalasymmetric interlaced-PCR (TAIL-PCR) using arbitrary genomic primers and T-DNA specific primers.

Segregation ratio analysis,TAIL-PCR walking and genotyping PCR results indicated a single T-DNA insertion atthe 200bp upstream ofthe

coding region in theBAC F5O8 (Fig.15).Wereferred to geneas (inflorescence growth inhibitor 1) and the mutation as for heterozygousand forhomozygousmutant.Afterdetermining T-DNA insertion site by sequencing of TAIL-PCR products containing the T-DNA/plant genomic DNA junction,Genotyping PCR was carried out to identify whetherthe plantassayed is wild type,homozygous orheterozygous mutant.Figure16isresultsofthegenotyping PCR in genomicDNA samples ofthe wild type and igi1 mutants.Because wild-type plants don'thave the T-DNA,therearenoband in thelane3and lane5.When only thegenomic DNA primer pairs were used,the PCR productin mutantdid not amplifybecauseofT-DNA insertion(lane2),butitwasamplifiedin

mutant.Thehomozygousandheterozygousplantswereselectedby segregation analysis and genotyping PCR reaction, and T-DNA direction also was determined.

AftertheT-DNA position and direction weredetermined,expression levels ofnearbygenesflankingT-DNA wereinvestigatedbyRealtimePCR reaction.

Theexpressionlevelfor , genewasincreasedapproximatelyby 1,000 to 3,000 fold in homozygous mutantand 500 to 1,000 fold in heterozygousmutant(Fig.17).Expressionofotherneighboring genes alsoincreased in ,butnotin mutant.Theseresultsindicate thattheover-expression ofthe genecausethe mutantsphenotypes suchassteriility,lackofinflorescenceandseeds,andabnormalflowerorganin

mutant and dramaticially increased axillary branches in mutant.

1kb

AT1G23540 AT1G23540AT1G23540 AT1G23540 AT1G23530

AT1G23530 AT1G23530

AT1G23530 AT1G23550AT1G23550AT1G23550AT1G23550 AT1G23560AT1G23560AT1G23560AT1G23560

F F

Fiiiggguuurrreee 111555...TTT---DDDNNNAAA pppooosssiiitttiiiooonnn iiinnn ttthhheee mmmuuutttaaannntttsss...BAC F5O8 clone are shown.There is T-DNA insertion in the 200 bp upstream ofthe

genecodingregion.

M 1 2 3 4 5 6 M 1 2 3 4 5 6 M 1 2 3 4 5 6 M 1 2 3 4 5 6

M: maker 1: Col0 2: igi1/igi1 3: igi1/IGI1 4: Col0 5: igi1/igi1 6: igi1/IGI1

F primer + R primer R primer + LB primer

F F

Fiiiggguuurrreee111666...TTThhheeegggeeennnoootttyyypppiiinnnggg rrreeesssuuullltttfffooorrrTTT---DDDNNNAAA lllooocccuuusssiiinnn ttthhheee mmmuuutttaaannntttsss... GenomicDNA wasamplifiedbyforwardandreversegenomicprimersinlain1 to3,andbyreversegenomicprimerandT-DNA specificprimerinlain4to6.

F F

Fiiiggguuurrreee 111777...TTThhheee eeexxxppprrreeessssssiiiooonnn llleeevvveeelllooofffnnneeeiiiggghhhbbbooorrriiinnnggg gggeeennneeesss nnneeeaaarrrttthhheee TTT---DDDNNNAAA... Realtime PCR resultshow that gene transcriptwas dramatically increased in mutants.Actin was used for normalization and error bars indicatedstandarddeviation.

C

C C. . .R R Re e ec c ca a ap p pi i it t tu u ul l l a a at t ti i io o on n n

To rescue the mutants phenotypes, recapitulation construct was generated.Recapitulationvector,pMN20,including thefourenhancerswasused (Weigel ,2000) for this experiment.3.3kb gene containing own promoterwas amplified by PCR reaction from wild type genomic DNA and clonedintothe IrestrictionsiteofpMN20vector(Fig.18A).Toconfirm the construct,itwasdigestedbyseveralrestrictionenzymes, I, I,and I (Fig.18B).Aftercloned,theconstructwastransformedtothewildtypeplant.

T3 homozygous lines were generated from T2 individuals carrying single insertion,which was identified in 3:1 segregation ratio on antibiotics medium.

The mRNA levelofthe recapitulation mutants was analyzed by quantitative real-timePCR. recapitulationlines#1( )and #5,which showed higherand middle expression levels among otherrecapitulation lines, wereselected.Theexpressionlevelsof genewerelowerin and

than thatof mutants(Fig.21).Figure19and Figure20show thephenotypesof and .Unexpectedly,wedidn'tfindsame phenotypes with mutants in population of recapitulation mutants. The

mutantshowedsamephenotypewithwildtype.However,

showed phenotypes somewhat similar to those of mutant.

Significantly,the inflorescence number was increased and plant height was reduced in mutant (Fig.22).It couldn't completely rescue mutantsphenotypespossiblybecauseoflowerexpressionlevelsinrecapitulation mutants.

Weattemptedtorevertthephenotypeof plantsby reducing themRNA levels with RNA interference (RNAi).The 300 base pairs (bp)of gene cording region were amplified from wild type plantusing primers thatwas added Isiteon theendsofoneproduct(sensestrand)and HIsiteon the ends of one the other product (anti-sense strand).These amplification

productsweredigested with Iand HIrestrction enzymesand directly cloned into thepHANNIVAL (Fig.23).Theconstructwassubcloned into the binary vectorpCAMBIA1302 (Fig.24)and transformed into mutant plants to revert the phenotype of mutants. The cloning region of pHANNIVAL vector contains a intron which induced self complementarity betweenthesenseandanti-sensetargetingRNA strand.

To select the mutant, segregation test in the medium supplement with hygromycin for RNAisingle locus (data not shown) and genotyping PCR for locusinF2generation(Fig.25)wereperformed.

expression level was efficiently decreased in RNAi transformed mutant, (Fig.27),andthephenotypeshowedsimilartowildtype(Fig.

26).ThephenotypewasrevertedbyreducingthemRNA levels,suggestingthat mutantsphenotypeswerecausedbyoverexpressionofthe .

A A A

nptII IGI1

(genomic DNA including promoter)

PstI PstI

enhancer

LB

3.3kb

14.3kb

B B B

M M M

M 1 2 3 1 2 3 1 2 3 1 2 3

M: maker 1: PstI 2: EcoRI 3: KpnI

11kb, 10.5kb, 11.7kb 3.8kb

3.3kb 2.6kb

F F

Fiiiggguuurrreee111888...TTThhheeemmmaaappp aaannnddd cccooonnnfffiiirrrmmmeeeddd iiimmmaaagggeeefffooorrr cccooonnnssstttrrruuucccttt...A.The constructforrecapitulationcontaininggenomicpartincludingthegenepromoter.

B.DNA fragmentsof I-, RI-,and I-digested constructin 1% agarosegel.Therestrictionenzymesitesof Iwaschosentoconfirm the insertsizes.The lowerfragments in lain 1 indicate the insert.M,1kb DNA sizemaker;1, Irestricted fragment;2, RIrestricted fragment;3, I restrictedfragment.

Igi1/igi1

Col0 Igi1/IGI1 IGI1RC#1 IGI1RC#5

F F

Fiiiggguuurrreee111999...RRReeecccaaapppiiitttuuulllaaatttiiiooonnn ppplllaaannntttsssppphhheeennnoootttyyypppeeesss...Theplantsweregrown for15 days (upper panel) or 20 days (below panel).From left to right,Col-0,

25 days plants

F F

Fiiiggguuurrreee222000...RRReeecccaaapppiiitttuuulllaaatttiiiooonnn ppplllaaannntttsssppphhheeennnoootttyyypppeeesss...Theplantsweregrown for25 days. From left to right, Col-0,

.

Col0 igi1/IGI1 igi1/igi1 IGI1-RC#1 IGI1-RC#5

Relative expression level

0 10 20 30 40 50 200 400 600 800 1000 1200 1400 1600

F F

Fiiiggguuurrreee 222111...RRReeelllaaatttiiivvveee eeexxxppprrreeessssssiiiooonnn llleeevvveeelllfffooorrr rrreeecccaaapppiiitttuuulllaaatttiiiooonnn llliiinnneeesss...Actin was usedfornormalizationanderrorbarsindicatedstandarddeviation.

A B

0 50 100 150 200 250 300 350 400

Col0 igi1/IGI1 IGI1-RC#1

primary inflorescence lenth(mm)

0 10 20 30 40 50 60

Col0 igi1/IGI1 IGI1-RC#1

number of inflrescence

F F

Fiiiggguuurrreee 222222...TTThhheee ppplllaaannnttt hhheeeiiiggghhhttt (((AAA))) aaannnddd nnnuuummmbbbeeerrr ooofff iiinnnffflllooorrreeesssccceeennnccceee (((BBB))) in 40-day-old mutantand .Errorbarsrepresentthestandard errorsofthemeans.

A A A

6.4kb

IGI1 kinase part

pHANNIVAL

KpnII BamHI

SacI SacISacI

SacI PstIPstIPstIPstI

CaMV35S promoter Intron OCS terminator IGI1 kinase part

pHANNIVAL

KpnII BamHI

SacI SacISacI

SacI PstIPstIPstIPstI

CaMV35S promoter Intron OCS terminator

B B B

M : maker

1: KpnI (pHIGI1KB) 2: BamHI (pHIGI1KB) 3: KpnI(pHANNIVAL only)

M M M

M 1 2 3 1 2 3 1 2 3 1 2 3

6.1kb, 6.4kb

0.3kb

F F

Fiiiggguuurrreee 222333...TTThhheee mmmaaappp aaannnddd cccooonnnfffiiirrrmmmeeeddd iiimmmaaagggeee fffooorrr iiinnnttteeerrrmmmeeedddiiiaaattteee c

c

cooonnnssstttrrruuucccttt...A.TheconstructmapforRNA interferenceincluding targeting site.

B.DNA fragments of I and HI digested intermediated constructin 1% agarosegel.Therestriction enzymesitesof Iand HI werechosentoconfirm theinsertsizesofsenseandantisensefragment.There arenolowerfragment(0.3kb)inlain3(vectoronly).M,1kbDNA sizemaker;

1, Irestricted fragment;2, HIrestricted fragment;3, Irestricted fragmentwithvetoronly.

A A A

Hygromycin®

LB RB

promoter ^MCS promoter mGFP5

promoter Intron terminator

SacI PstI

B B B

M: maker 1: SacI and PstI 2: SpeI

3: EcoRI

M M M

M 1 2 3 1 2 3 1 2 3 1 2 3

11.5kb, 15kb, 13.6kb

3.5kb 1.4kb

F F

Fiiiggguuurrreee 222444...TTThhheee mmmaaappp aaannnddd cccooonnnfffiiirrrmmmeeeddd iiimmmaaagggeee fffooorrr IIIGGGIII111---RRRNNNAAAiiicccooonnnssstttrrruuucccttt...A.

The constructmap forRNA interference in binary vectorpCAMBIA1302.B.

DNA fragmentsof I, I, I,and RIdigested construct in 1% agarosegel.Therestriction enzymesitesof Iand Iwerechosen toconfirm theinsertsizes.Thelowerfragmentsinlain1shouldbeinsert.M, 1kb DNA size maker;1, Iand Idouble restricted fragment;2,SpeI restrictedfragment;3, RIrestrictedfragment.

M 1 2 3 4 5 6 7 8

F F

Fiiiggguuurrreee 222555... mmmuuutttaaannntttgggeeennnoootttyyypppiiinnnggg fffooorrr lllooocccuuusss...The PCR reaction was performed with genomic primerpairs (lain 1,3,5,and 7),and genomic primerand T-DNA primer(lain 2,4,6 and 8).M,1kb DNA size maker;lain1and2,Col0;lain3and4, ;lain5and6, ;lain7

and8, .