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
RB14.3kb
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, .
F F
Fiiiggguuurrreee 222666...TTThhheee ppphhheeennnoootttyyypppeee ooofff mmmuuutttaaannnttt...Col-0, , ,and (lefttoright).
C o l0 ig i1 /IG I1 ig i1 /ig i1 ig i1 /ig i1 -R N A i
Relative expression levels
0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 1 0 0 0 1 5 0 0 2 0 0 0 2 5 0 0
F F
Fiiiggguuurrreee 222777...RRReeeaaalll---tttiiimmmeee PPPCCCRRR rrreeesssuuullltttfffooorrr mmmuuutttaaannnttt...Actin was usedfornormalizationanderrorbarsindicatedstandarddeviation.
D
D D. . .I I IG G GI I I1 1 1g g ge e en n ne e ei i is s ss s st t tr r ro o on n ng g gl l l y y ye e ex x xp p pr r re e es s ss s se e ed d di i in n na a an n nt t th h he e er r r
Tostudy theexpression pattern ofthe gene,wedeveloped aconstruct forhistochemicalGUS reporterassay.Inordertoinsertpredicted promoter region into pBI101.2 binary vector,PCR was carried outfor predicted promoter region.The PCR productand pBI101.2 vector were designed with
HIrestricting enzyme (Fig.28).These two fragments were ligated and namedas .Toconfirm the construct, HIand I restecitonenzymeswereused(Fig.29).Lane1andlane2werethefragments of HI- and I-digested Theconstructwasintroduced into wild type plant.In mature plants,GUS expression was detected strongly in antherpartand detected rarely in upperpartofstem,immature siliques,and root.However the GUS expression was notdetected in other partsuch as rossetteandcaulineleaves,maturesiliques,andmiddleandlowerstem.
Theexpression wasdetectedatlow levelin only hairzoneofprimary root and not detected in other part of 5-day-old seedlings (Fig.30A-D).The expressionwasdetectedmorestronglythan5-day-oldseedlingsinhairzoneof lateralrootbutwasnotdetectedin primary rootof10-day-oldseedlings(Fig.
30E-G). expression also observed only flower parts when the plants are about to start bolting (about 20 days after seedling). In the 33-day-old plants,the expression was strongly observed in antherofflower organ and weakly observed in upper stem and immature siliques (Fig.31).
expression showed tissue-specific gene expression which strongly expressed in theantherpart.To confirm theexpression patten of gene, theexpression levelwasexamined in differenttissuesby the realtimePCR.
Theexpression levelin flowerpartwasmuch higherthan in otherpartssuch astherosetteandcaulineleave,andstems(Fig.32).
NPTII
RB LB
β-glucuronidase NOS-Ter MCS
BamHI BamHI
Predicted IGI1 promoter
F F
Fiiiggguuurrreee 222888...TTThhheee cccooonnnssstttrrruuuccctttmmmaaappp fffooorrrGGGUUUSSS rrreeepppooorrrttteeerrraaassssssaaayyy iiinnn bbbiiinnnaaarrryyy vvveeeccctttooorrr p
p
pBBBIII111000111...222...The promoter region of gene was amplified with genomic specificprimerscontaining the H Irestriction siteand directly clonedinto binaryvectorpBI101.2.
M : maker 1: BamHI 2: XbaI
M M M
M 1 2 1 2 1 2 1 2
12.2kb, 12.6kb
0.9kb 0.5kb
F F
Fiiiggguuurrreee222999...DDDNNNAAA fffrrraaagggmmmeeennntttsssooofff HHH III--- aaannnddd III---dddiiigggeeesssttteeeddd c
c
cooonnnssstttrrruuuccctttiiinnn 111%%% aaagggaaarrrooossseee gggeeelll...The restriction enzyme sites of H Iand Iwere chosen to confirm the insert sizes and orientation.The lower fragments in lain 1 should be insert.M,1kb DNA size maker;1, H I restrictedfragment;2, Irestrictedfragment.
A B C D
E F G
F F
Fiiiggguuurrreee333000...EEExxxppprrreeessssssiiiooonnnpppaaatttttteeerrrnnnooofffttthhheee gggeeennneee...Theexpressionpatternwas detectedwith GUS reportergeneundercontrolof genepromoter.A-D.5 dayoldplant.E-G.10dayoldplant.
A B C D E F
G H I J
F F
Fiiiggguuurrreee333111...EEExxxppprrreeessssssiiiooonnn pppaaatttttteeerrrnnn ooofffttthhheee gggeeennneeeiiinnn 333333dddaaayyy ooolllddd ppplllaaannnttt...The expression pattern showed tissue specific gene expression. A. Primary inflorescence.B.Secondary inflorescence.C.Primary inflorescencewith silique.
D.Matureseed.E.Middlestem.F.Root.G-H.Flower.I-J.Anther.
Rosettes leves Cauline leves flower part Lower stem Middle stem Upper stem
Relative expression levels
0 5 10 15 20 25 30 300 350 400 450
F F
Fiiiggguuurrreee 333222... TTThhheee rrreeelllaaatttiiivvveee eeexxxppprrreeessssssiiiooonnn llleeevvveeelllsss fffooorrr gggeeennneee iiinnn dddiiiffffffeeerrreeennnttt t
t
tiiissssssuuueeesss...The flowerparthashigherexpression levelthan otherparts.Actin wasusedfornormalizationanderrorbarsindicatestandarddeviation.
E
E E. . .I I IG G GI I I1 1 1i i in n nt t te e er r ra a ac c ct t tw w wi i it t th h hL L LS S SH H H1 1 1a a an n nd d dS S SU U UB B B1 1 1
The gene consists of8 exons with over 2,800 base pair (bp).The predicted IGI1 protein contains 720 amino acid residues possessed proline rich domain in N-terminalregion andkinasesignaturedomain in C-terminalregion (Fig.33andFig.34).
To investigate protein-protein interactions,yeasttwo-hybrid screening was performed with IGI1prolinerich domain in theN-terminaland kinasedomain in theC-terminalasbaits.Table5summarizestheproteinwhichidentifiedby theyeasttwohybrid screening.Interestingly,lightresponseprotein SUB1and LSH1 were identified.LSH1 do interactwith both proline rich domain and kinasedomain in theyeasttwo-hybrid screen.TheSUB1 interactswith only the proline-rich domain in the N-terminal,implying that IGI1 might be a nuclear receptor and the light source may influence the phenotype of mutants.
F
F F. . .H H Ho o or r rm m mo o on n ne e es s sa a an n nd d da a ax x xi i il l ll l la a ar r ry y yb b br r ra a an n nc c ch h hi i in n ng g g
To investigate the apicaldominance and bud outgrowth,decapitation was performed with wild type plant.Afterprimary bolts come out(<10 cm),the primary shoot was removed. After Decapitation, lateral bud outgrowth is stimulatedbytheapicalsourceasauxin(IAA)orvariouscomponentingenetic pathway in less than severalhours.The gene expression levels were markedlyincreasedin4hoursafterdecapitation. alsohighlyincreasedin 4hoursafterdecapitation(Fig.36).
Differentgeneexpression profileswereanalyzed in mutantsto testthe expression ofthe gene which is related axillary bud outgrowth orbranching.
Thereareno visibledetectable increasein the expression levelsofbranching components,such as , , , ,and and cytokinin response genes,such as and ,cytokinin signaling component.In contrast,the expression levelof auxin biosynthesis component is significantly increased, and flower meristem identity component is decreasedin mutants(Fig.35,Fig.37,Fig.39).
Cytokinin normally stimulatecelldivision and greening ofhypocotyl-derived calli(Higuchietal,2004).To testcytokinin sensitivity,callusinduction assay wasperformed.Forthecallusinduction assay,theplanthypocotyls grown in dim lightfor15dayswereexcised with scissorsand cultured for20daysin 1/2MS․1S supplemented with 50 nM 2,4-D and varying concentrations of kinetin.The , mutantsrespondednormallytocytokinininthe assay(Fig.38).
When grown in dark,Col-0and mutantshowedsimilarpatternsin hypocotylgrowthandin2,4-D andkinetindoseresponse.Interestingly,theroot growthshowedinhibitionpatternin mutantunderlightcondition(0nM hormoneconcentration)andtherelativedeferencein rootgrowth between Col0 and mutantdiminishedinBAP doseresponse(Fig.40).Inthemedium supplementedwith2,4-D,thegrowthshowednoresponse(Fig.41).Theeffect oftheauxin andcytokinin onrootselongation wasexaminedin 7daysgrown seedlings.
500bp
intron exon
ATG TGA
F F
Fiiiggguuurrreee 333333...DDDiiiaaagggrrraaammm ooofffIIIGGGIII111 gggeeennnooommmiiiccc DDDNNNAAA...The gene consists of8 exons(blackrectangles)and7introns.
Protein kinase region
*
ATP binding region signature ★ active site signature proline**** ***** *
★
F F
Fiiiggguuurrreee 333444...AAAmmmiiinnnooo aaaccciiiddd ssseeeqqquuueeennnccceee ooofffttthhheee IIIGGGIII111...The predicted IGI1 protein contains 720 amino acid residues composed prolinerich domain in N-terminal regionandkinasedomainsignatureintheC-terminalregion.TheATP binding region signature is marked (★),the protein kinase region is underlined,and acivesitesignatureismaked(*).
T T
Taaabbbllleee 555...TTThhheee rrreeesssuuullltttooofffyyyeeeaaasssttt---tttwwwooo hhhyyybbbrrriiiddd ssscccrrreeeeeennniiinnnggg...The protein identified from thecDNA libraryinArabidopsis.
Bait;IGI1N-terminal
Locus Description A
A
Attt555ggg222888444999000 LLLSSSHHH111(((LLLiiiggghhhttt---dddeeepppeeennndddeeennntttssshhhooorrrttthhhyyypppooocccoootttyyylll111))) FFFuuullllll---llleeennngggttthhh A
A
Attt444ggg000888888111000 SSSUUUBBB111(((SSShhhooorrrtttuuunnndddeeerrrbbbllluuueeellliiiggghhhttt111))) FFFuuullllll---llleeennngggttthhh
At5g08720 Partial
At3g25070 RIN4(RPM1-interactingprotein4) Full-length
Bait;IGI1C-terminal
Locus Description A
A
Attt222ggg444222666111000 UUUnnnkkknnnooowwwnnn,,,hhhooommmooolllooogggooouuussstttoooLLLSSSHHH111 FFFuuullllll---llleeennngggttthhh A
A
Attt555ggg222888444999000 LLLSSSHHH111(((LLLiiiggghhhttt---dddeeepppeeennndddeeennntttssshhhooorrrttthhhyyypppooocccoootttyyylll111))) FFFuuullllll---llleeennngggttthhh At5g11740 AGP15(Arabinogalactanprotein1) Full-length At2g21230 bZIP proteinsimilartoCREB protein Partial At3g02780 IPP2(Isopentenylpyrophosphate2) Full-length
At4g18610 Unknown Partial
At3g11100 Transcriptionfactor,6b-interactingprotein1 Full-length
0 20 40 60 80 100 120
Col0 igi1/IG I1 igi1/igi1
F F
Fiiiggguuurrreee 333555...AAAPPP111 eeexxxppprrreeessssssiiiooonnn llleeevvveeelllsss iiinnn mmmuuutttnnnaaatttsss...Actin was used for normalizationanderrorbarsindicatestandarddeviation.
0 200 400 600 800 1000 1200 1400 1600 1800
0 hrs 1 hrs 2 hrs 4 hrs 8 hrs 12 hrs 24 hrs 36 hrs 48 hrs Hours after decapitation
Relative expression levels
IGI1 BRC2
F F
Fiiiggguuurrreee333666...RRReeelllaaatttiiivvveeeeeexxxppprrreeessssssiiiooonnn llleeevvveeelllooofff aaannnddd aaafffttteeerrrdddeeecccaaapppiiitttaaatttiiiooonnn... wasincluded aspositivecontrol.Actin wasused fornormalization,and errorbarsindicatestandarddeviation.
0 100 200 300 400 500 600 700 800 900 1000