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BRC1 BRC2 MAX1 REV SPS1
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Fiiiggguuurrreee 333888...CCCyyytttoookkkiiinnniiinnn rrreeessspppooonnnssseee...1,0 nM Kinetin + 50 nM 2,4-D;2,50 nM Kinetin+50nM 2,4-D;3,200nM Kinetin+50nM 2,4-D;4,1000nM Kinetin +50nM 2,4-D.
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ARR4 ARR5 CYP79B2 CYP79B3 Col0
igi1/IGI1 igi1/igi1
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Fiiiggguuurrreee 333999... RRReeeaaalll---tttiiimmmeee PPPCCCRRR aaannnaaalllyyysssiiisss ooofff cccooonnntttrrrooolllgggeeennneeesss ooofff hhhooorrrmmmooonnneeesss... Graphshowsrelativeexpressionlevelsofcontrolgenesofhormones.ThePCR reaction wasperformedusing cDNA from 10-day-old plantwild typeand mutants.The actin transcriptlevels were used for normalization.Error bars indicatestandarddeviation.
AAA
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Figure40.Cytokinindoseresponseunderlightanddarkcondition.
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Root length (mm)
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Col0 igi1/igi1
Figure41.Auxindoseresponseunderlightanddarkcondition.
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Ⅳ...DDDiiissscccuuussssssiiiooonnn
In plants,mutations can be caused by copying errors orchanging mRNA expression levelin thegeneticmaterialusing somemethodssuch asexposure toultraviolet,chemicalmutagenslikeEMS,oractivation-taggingusingT-DNA (agrobacterim mediated transformation). Despite the limitations such as functional redundancy and early embryonic or gametophytic lethality in organisms,thelossoffunction mutation techniquestillplaysan importantrole instudying geneticpathway.Activationtagging,amethodthatenablesgainof function mutation, can be used for genetic screening to overcome such limitationsoflossoffunction mutaion.An activation-tagging vectorcontaining fourmultimerized transcriptionalenhancersderived from thecauliflowermosaic virus 35S gene was used to generate the gain of function mutants.
Over-expressionofthegenesneartotherandomlyinsertedT-DNA mayresult in new phenotypes,and the relevantgenes can be identified.TheT-DNA of the activation tagging vector,pSKI015,can enhance the expression levelof neighboring genes in the insertion position (Weigeletal.,2000).Additionally, theT-DNA including enhancerscan induceknock-outofthegeneby T-DNA insertion (Hwang etal.,2007). mutants have T-DNA insertion in 200bp upstream of gene start codon (Fig. 15), that indicate gene overexpression which was confirmed by Real-time PCR (Fig. 17). The phenotypewasreverted by reducing themRNA levels(i.e.,RNA interference, RNAi) (Fig.26).The recapitulation was failed in lines.This is probably becausetheexpression levelwasnothigh enough torecapitulatethe phenotype(Fig.21).Thehigherlevelexpression may benecessary togetthe
mutantsphenotype.
Auxin and cytokinin areimportantphytohormonesforaxillary branching and apicaldominance (Cline,1997).The main inflorescence grows more strongly
than otherrosetteand caulineinflorescence.Apicalbud isstimulated to grow while the lateralbuds are inhibited.Auxin treatmentinhibits the lateralbud outgrowth ofdecapitated plantwhich isstimulated by cytokinin treatmentand overexpression of the cytokinin biosynthesis gene (Thimann et al., 1933;
Medford etal.,1989).The branching phenotypes were observed in auxin and cytokinin mutants (Booker et al., 2003; Tantikanjana et al., 2001). The phenotypeof mutantincludesincreased branching and reduced plant height(Fig.22).To testtheeffectofendogenousauxin and cytokinin in the mutants, the transcript levels of auxin biosynthesis genes and cytokinin response genes were examined.In mutants,the transcripts levelofthe auxin biosynthesis component, was increased and those of the cytokininresponsegenes and weresimilarwiththatofwildtype (Fig 37,Fig 39).Although thetranscriptlevelofauxin biosynthesiscomponent was increased, mutant showed opposite phenotype in the axillary branchingpattern.Also,exogenous2.4-D didn'tinfluencetherootelongationof the mutant(Fig.41).Cytokinin induced celldivision and greening of hypocotyl-derivedcalliwerepartiallyinhibitedincytokininreceptormutantsuch as and mutants(Higuchietal,2004).When thecytokinin sensitivity was tested with callus induction assay,the cytokinin response of the mutants were slightly reduced in the greening and induction of hypocotyl-derivedcalli(Fig.40).Thesephenotypeswerealsooppositeresponse to cytokinin similarto the auxin response in the axillary branching pattern.
Theseresultsindicatenotonly thehormonalregulationby auxin andcytokinin butalsootherpathwayinfluencedtheaxillarybranching.
Decapitation,shoottipremoval,hasbeenusedtostudybudoutgrowth.After removing the shoot tip, lateral shoot come out from axillary bud. The outgrowth of axillary buds after decapitation may be essential to provide replacementsites forreproductive development.The bud outgrowth could be quickly led after decapitation and might also divert resources away from
reproductive structures developing elsewhere on the plant. This implies communication between axillary buds and the shoot tip (Shimazo-Sato and Mori,2001;Christineetal.2003). transcriptwasdramatically increasedin 4hourafterdcapitation (Fig.36).TheresultindicatesthatIGI1isinvolved in the communication between axillary buds and the shoottip foraxillary bud development.
The increased branching patterns were observed in ,and mutants.MAX-dependentcarotenoidhormonemovesuptheplantfrom rootand preventsbud outgrowth (Bainbridgeetal.,2005;Stirnberg etal.,2002).BRC1 and BRC2 preventaxillary bud outgrowth in the downstream ofthe MAX pathway (Aguilar-Martinez et al.,2007).SPS1 is the cytokinin biosynthesis regulator(Reintanzetal.,2001;Tantikanjanaetal.,2008).Itisthoughtthatthe pathwaythroughIGI1isnotconnectedwiththesepathwaylikeMAX andSPS pathway.
Theaminoacidof containstheprolinerichdomaininN-terminalregion andkinasedomainsignatureintheC-terminalregion(Fig.34).Theprolinerich region ispresentin thereceptorprotein family such asPERK in . In the study using three differentapproaches,EST (expressed sequence tag), MPSS (massively parallelsignature sequencing),NASCArrays detabases,and RNA blot analyses, by Alina Nakhamchik et al., proline-rich extensin like receptor kinase ( ) gene family was classified with 15 predicted receptor kinases.They classified into .The basic structural features of conserved sequence regions is consisted of proline-rich domain, transmembrane domain and kinase domain. Some of members were identified as tissue-specific genes while others were more broadly expressed.The EST and MPSS data indicated that
membershavevariableexpression patterns.Someweremorehighly expressed based on the number ofESTs detected and the higher MPSS values.For example, and showed thehighestvalue, showed
no data, and showed low values in MPSS. Also, in NASCArrays,strong expression wasseen for and in flower buds.RNA gelblot analyses demonstrated that the majority of the famly are expressed in buds (Nakhamchik etal.2004).Ourresults alsoshowedsimilarresultswith PERK family expression patterns,which were highlydetectedinflowerbudsbyrealtimePCR andGUS staining.
Interestingly,light response components such as SUB1 and LSH1 were screened in yeasttwo-hybrid screening (Table 5).SUB1 is a Ca2+ binding protein involved in cryptochrome and phytochrome coaction.The mutant showed no sign ofphotomorphogenic developmentin the dark and had same phenotype with the wild type plantin red light.The , and doublemutantexperimentshowed SUB1actassignaltransducerof cry1 and cry2 but as a modulator of phyA signaltransduction.SUB1 is apparently enriched in the nuclear periphery region surrounding the nucleus.
Guo et al.proposed a hypothesis,which SUB1 is a negative regulator of photomotphogenesis,whereasthecryptochromessuppresstheactivity ofSUB1 toactivatethelightresponse.Theanalysisof doublemutantindicated that is epistatic to (Guo etal.,2001).SUB1 interacted with only N-terminalregion ofIGI1 including proline rich domain,which was expected cytoplasmicregion,suggesting thatthereispossibilitythatIGI1isthereceptor relatedwithasignaltransduction(Beckeretal.,2003).Thetransctiptionfactor, HY5isthebZIP proteinandpromotesphotomorphogenesis(Quail,2002;Chenet al.,2004;Oyamaetal.,2008).HY5binding targetstend tobeenriched in the earlylight-responsivegenesandtranscriptionfactorgenes(Leeetal.,2007).In the transcription factor-promoter interactions analysis by Gao et al., the promoterregion of geneexhibited high HY5binding affinity (Gao etal., 2004).AP1belong totheclassA floralorganidentitygenes,whichisrequired foractivation ofB classandC classgenes(Gomez-Menaetal.,2005;Sridhar et al., 2006; Weigel and Meyerowitz, 1993). In mutant, is
down-regulated by theoverexpression of gene(Fig.35),suggesting that thefunction ofIGI1 isassociated with AP1 expression in thefloralmeristem transition.These results indicate thatIGI1 mightbe the nuclearreceptorand regulatedbythelightresponseandflowering.Thedevelopmentalphenotypesof heterozygous mutant become noticeable after transition from vegetative to reproductive phase. The heterozygous mutant plants produce primary inflorescence with reduced internode elongation,and secondary inflorescences begin toemergestraight.Theheterozygousplantscontinuetoproduceaxillary inflorescence from the axils ofboth rosette and cauline leaves.Consequently, the heterozygous mutanthave dramatically increased axillary branches.These results suggestthatthere may be a noble pathway ofthe axillary meristem developmentthrouth IGI1,which was modulated by lightregulated pathway (Fig.43,Fig.44).
A B