SEBthe plant journalSorietyThePlant Journal (2011)68,657-669doi:10.1111/j.1365-313X.2011.04718.xTheArabidopsisMERISTEMDISORGANIZATION1geneisrequired forthe maintenance of stem cells throughthereduction of DNA damageYuma Hashimura and Chiharu Ueguchi"Bioscienceand BiotechnologyCenter,NagoyaUniversity,Chikusa-ku,Nagoya 464-8601,JapanReceived 11 May 2011; revised 6 July 2011; accepted 19 July 2011; published online 31 August 2011.*For correspondence (fax +81 52 789 5214; e-mail cueguchi@agr.nagoya-u.ac.jp).SUMMARYInplants,stem cells reside in apical meristems,and providethe descendants required for post-embryonicgrowth anddevelopmentthroughoutthe lifeofaplant.Toidentifyanovelfactorrequiredforthemaintenanceofstemcells,weisolatedanArabidopsismutant,namedmeristemdisorganization1-1(mdo1-1),thatexhibitsseveral developmental defects,such asabnormal phyllotaxy and plastochron,stemfasciation and retardedroot growth.We found that themutant plants fail to maintain stem cells,resulting in the differentiation ordeath of stem cells.The mutant plants also showedseveral phenotypes related to DNA damage,suggestingthat themutant cells areexposed constitutively to DNA damage even without external genotoxic stress.Thegrowthdefectand thehypersensitivitytoDNA-damagingagents of mdo1-1were enhanced significantlywhencombinedwithalesionoftheATAXIA-TELANGIECTASIAMUTATED(ATM)gene,butnotoftheATM/RAD3-RELATED(ATR)gene,suggestingthatthefunction oftheMDO1gene iscloselyrelatedtothat ofATMkinaseTheMDO1geneencodes an unknown protein that is conserved ina widevariety of land plants.Theresultsthus suggestedthatthe MDO1geneproduct isrequiredforthe maintenance of stem cells throughareductionin DNA damage.Keywords: stem cell, apical meristem, DNA damage, cell death, cell differentiation.INTRODUCTIONin the SAM is dynamicallyregulated bytheWUS-CLAVATAIn plants, stem cells reside in apical meristems, and providethedescendantsrequiredforpost-embryonicgrowthand(CLVnegativefeedbackloop(Schoofetal.,2000).Asimilardevelopmentthroughout the lifeof a plant.Plantstemcellsbut distinct mechanism also operates in root tips.Thecan remain in an undifferentiated state by accepting signalsWUSCHEL-RELATEDHOMEOBOX5(WOX5)genefunctionsfrom organizing centers in specific microenvironmentsinQC cells to prevent the initialsfromundergoing differen-tiation,and overexpression of the CLV3-likegene CLE40calledstemcell niches.Intheshootapicalmeristem(SAM)reduces the meristematic activity of the RAM (Hobe et al.,stem cells are maintained in thecentral zone under thecontrol ofWUSCHEL(WUS)-expressing cellslocated just2003;Fiers etal.,2005;Sarkaretal.,2007).In additionbeneath the stem cell population (Mayer et al., 1998). In theseveral transcription factors specifically requiredfor themaintenance of apical meristems have been identified.Therootapicalmeristem(RAM),asetofspecializedstemcellsSHOOTMERISTEMLESS(STM)geneencodingaKnotted1calledinitialssurroundquiescentcenter(QC)cellsthatactasa root-organizing center (Dolan et al.,1993; van den Bergclass homeoboxprotein is necessaryfortheestablishmentetal.,1997).ThehighlyorganizedstructureofapicalmeroftheSAMduringembryogenesis,andforfurthermainte-istems is apparently indispensable for proper growth andnance in post-embryonic development (Barton and Poethig,development, e.g.the production of lateral organs in a1993;Endrizzi et al.,1996; Long etal.,1996).In the RAM,twospatially and temporally regulated manner (phyllotaxy andGRASfamilytranscriptionfactorgenes,SHORTROOT(SHR)plastochron,respectively).and SCARECROW(SCR),areinvolved inmaintainingtheExtensive genetic studies have revealed several factorsstem cell population (Di Laurenzio et al., 1996; Helariuttaet al.,2000; Sabatini et al.,2003).The PLETHORA1(PLT1)specifically involved in the maintenance of stem cells in eachtypeofapical meristem.The sizeofthe stemcellpopulationand PLT2 genes encoding AP2 transcription factors are2011TheAuthors657The Plant Journal 2011Blackwell Publishing Ltd
The Arabidopsis MERISTEM DISORGANIZATION 1 gene is required for the maintenance of stem cells through the reduction of DNA damage Yuma Hashimura and Chiharu Ueguchi* Bioscience and Biotechnology Center, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan Received 11 May 2011; revised 6 July 2011; accepted 19 July 2011; published online 31 August 2011. *For correspondence (fax +81 52 789 5214; e-mail cueguchi@agr.nagoya-u.ac.jp). SUMMARY In plants, stem cells reside in apical meristems, and provide the descendants required for post-embryonic growth and development throughout the life of a plant. To identify a novel factor required for the maintenance of stem cells, we isolated an Arabidopsis mutant, named meristem disorganization 1-1 (mdo1-1), that exhibits several developmental defects, such as abnormal phyllotaxy and plastochron, stem fasciation and retarded root growth. We found that the mutant plants fail to maintain stem cells, resulting in the differentiation or death of stem cells. The mutant plants also showed several phenotypes related to DNA damage, suggesting that the mutant cells are exposed constitutively to DNA damage even without external genotoxic stress. The growth defect and the hypersensitivity to DNA-damaging agents of mdo1-1 were enhanced significantly when combined with a lesion of the ATAXIA-TELANGIECTASIA MUTATED (ATM) gene, but not of the ATM/RAD3- RELATED (ATR) gene, suggesting that the function of the MDO1 gene is closely related to that of ATM kinase. The MDO1 gene encodes an unknown protein that is conserved in a wide variety of land plants. The results thus suggested that the MDO1 gene product is required for the maintenance of stem cells through a reduction in DNA damage. Keywords: stem cell, apical meristem, DNA damage, cell death, cell differentiation. INTRODUCTION In plants, stem cells reside in apical meristems, and provide the descendants required for post-embryonic growth and development throughout the life of a plant. Plant stem cells can remain in an undifferentiated state by accepting signals from organizing centers in specific microenvironments called stem cell niches. In the shoot apical meristem (SAM), stem cells are maintained in the central zone under the control of WUSCHEL (WUS)-expressing cells located just beneath the stem cell population (Mayer et al., 1998). In the root apical meristem (RAM), a set of specialized stem cells called initials surround quiescent center (QC) cells that act as a root-organizing center (Dolan et al., 1993; van den Berg et al., 1997). The highly organized structure of apical meristems is apparently indispensable for proper growth and development, e.g. the production of lateral organs in a spatially and temporally regulated manner (phyllotaxy and plastochron, respectively). Extensive genetic studies have revealed several factors specifically involved in the maintenance of stem cells in each type of apical meristem. The size of the stem cell population in the SAM is dynamically regulated by the WUS-CLAVATA (CLV) negative feedback loop (Schoof et al., 2000). A similar but distinct mechanism also operates in root tips. The WUSCHEL-RELATED HOMEOBOX 5 (WOX5) gene functions in QC cells to prevent the initials from undergoing differentiation, and overexpression of the CLV3-like gene CLE40 reduces the meristematic activity of the RAM (Hobe et al., 2003; Fiers et al., 2005; Sarkar et al., 2007). In addition, several transcription factors specifically required for the maintenance of apical meristems have been identified. The SHOOT MERISTEMLESS (STM) gene encoding a Knotted 1 class homeobox protein is necessary for the establishment of the SAM during embryogenesis, and for further maintenance in post-embryonic development (Barton and Poethig, 1993; Endrizzi et al., 1996; Long et al., 1996). In the RAM, two GRAS family transcription factor genes, SHORT ROOT (SHR) and SCARECROW (SCR), are involved in maintaining the stem cell population (Di Laurenzio et al., 1996; Helariutta et al., 2000; Sabatini et al., 2003). The PLETHORA 1 (PLT1) and PLT2 genes encoding AP2 transcription factors are ª 2011 The Authors 657 The Plant Journal ª 2011 Blackwell Publishing Ltd The Plant Journal (2011) 68, 657–669 doi: 10.1111/j.1365-313X.2011.04718.x
658Yuma Hashimura and Chiharu Ueguchiredundantly required to both establish and maintain stemtoxictreatments(FulcherandSablowski,2009).Unlikethecell niches in root tips (Aida et al., 2004).The OBERON 1caseinanimals,becauseplantstemcellsproducegerm-line(OBE1)and OBE2genes encoding homeodomain fingercells in the final phase of the life cycle,there is a risk that theproteins are required for the maintenanceof boththe SAMmutations fixed in stem cells during the vegetativegrowthand the RAM (Saiga et al.,2008).phaseareinheritedbytheoffspring.Thus,theprotectionofIt has been reported in Arabidopsis that the structuralstem cells against DNA damage,including exclusion ofdamaged stem cells, is crucial not only for developmentalorganizationandproperfunctioningofapicalmeristemsarealso affected by lesions of the genes with general cellularnormalitybutalsoforreproductivefitness.functions,suchas chromatin organization,DNA replicationHere, we isolated a novel mutant, named meristemand cell cycle regulation.Mutations of the FASCIATA(FAS)disorganization 1-1 (mdo1-1), that exhibits abnormal shootgenes encoding subunits of chromatin assembly factor-1,morphogenesis as well as retarded growth of roots.Wewhich supports nucleosome assembly on replicating DNA,found that the developmental defects observed in mdo1-1result in thedisorganizationofapicalmeristems,leadingtoresult from the failure of the maintenance of stem cells inapical meristems,whichleadstothedisorganizationofthepleiotropicdevelopmentaldefects(LeyserandFurner,1992;Kayaetal.,2001).TheMGOUN1(MGO1)geneencodingameristem structures. In mdo1-1 cells, an elevated level oftype-lBtopoisomerase,which is requiredforthe regulationDSBs and increased expression of DNA damage-inducedof DNA coiling during replication and transcription, wasgenes,the expression of which is regulated by ATM,revealed to be important for the proper functioning of thesuggested that the mutant cells are exposed constitutivelySAMandRAM(Laufsetal.,1998;Takahashiet al.,2002;GraftoDNAdamage,leadingtoDSBs.Themdo1-1phenotypeset al., 2010).Altered expression levels of a key cell-cyclewere enhanced when combined with an atm mutation.Theregulatorgene,RETINOBLASTOMA-RELATED(RBR),affectMDO1gene encodes an unknown proteinthatis conservedthe maintenance of stem cells in apical meristems (Wild-inawidevarietyoflandplants.Theseresultsthus suggestedwater et al.,2005; Borghi et al.,2010).Lesions ofthe TEBICHIthat the MDO1gene product is requiredforthe maintenance(TEB)gene,theproductofwhich isproposedtobe involvedof stem cells through the protection of cells from DNAincell cycleregulation,result inthedisorganizationof apicaldamage.meristems(lnagakietal.,2006,2009).TheMGO3/TONSOKURESULTS(TSK)/BRUSHY1(BRU1)geneproduct,anuclearprotein,isneeded for meristem function as well as other cellularIsolationofthemdo1-1mutantfunctions,such as DNAdamageresponses and epigeneticWe obtained a novel mutant exhibiting aberrant shootgene silencing(Guyomarc'h et al.,2004; Suzuki et al.,2004;morphogenesisfromapool of EMS-mutagenizedM,seed-Takeda et al., 2004).Alithough the underlying mechanismlings ofArabidopsisthaliana,anddesignateditasmdo1-1remains unclear, the results suggest the requirement ofbased on themeristem phenotype(seebelow).AboutproperDNAmetabolism for themaintenance of stem cellthree-quarters and one-quarter of the self-progeny of theniches.heterozygousmdo1-1/+plants,which showed noapparentLivingorganisms are atriskfor exposureto endogenousdefective phenotype, exhibited the wild-type and mdo1-1and environmental hazards causing DNA damage and,phenotypes, respectively (576 wild type and 189 mutantconsequently,mutations.Fixedmutations,ifcausedinstemplants; =0.04, P>0.05), suggesting that mdo1-1 is acells, would be inherited after cell division, and not onlysingleand recessive Mendeliantrait.prevent cells from functioning normally but also causeserious problems,suchas cancer.ToavoidsuchdeleteriousShoot phenotype of mdo1-1situations,inanimals,stemcellsshowalowtolerancetoThe mdo1-1 mutation affected shoot development. In theDNA damage, and have a tendency for differentiation orseedling stage,the emergence ofthefirstpairofleaves (firstprogrammed cell death (apoptosis) in response to DNAand second leaves)orthe second leaf was occasionallydamage (Rich et al.,2000; Sherman et al.,2011).Cellulardelayed (Figure 1b,c). In some cases,three leaves (second,responses to DNAdamage are mediated bytwoproteinthird and fourth leaves) were generated nearly simulta-kinases:ATAXIA-TELANGIECTASIA MUTATED(ATM)andneously (Figure 1e,arrowheads). In the subsequent vege-ATM/RAD3-RELATED (ATR).ATM and ATR are activated bytative growth phase,leaves continued to emergein aDNA double-strand breaks (DSBs) and single-stranded DNA,respectively,and in turn positively regulate downstreamspatiallyand temporally unregulated manner (Figure1g).Themutant leaves showeda slightlyabnormal shapeandareactions,suchasthetranscriptionalupregulation ofseveralDNA repair genes (Shiloh, 2006; Flynn and Zou, 2010).reduced size, resulting in a compact rosette structure. In thereproductivegrowthphase,themutant inflorescences wereLikewise,itwasdemonstrated recentlythatplantstem cellsand their early descendants selectively undergo ATM/ATR-frequentlyfasciated,andsomeofthemceasedtogrow,withnew inflorescences developing from the arrested shootmediated programmed cell death upon certain mild geno-2011TheAuthorsThePlantJournal2011BlackwellPublishingLtd,ThePlantJournal,(2011),68,657-669
redundantly required to both establish and maintain stem cell niches in root tips (Aida et al., 2004). The OBERON 1 (OBE1) and OBE2 genes encoding homeodomain finger proteins are required for the maintenance of both the SAM and the RAM (Saiga et al., 2008). It has been reported in Arabidopsis that the structural organization and proper functioning of apical meristems are also affected by lesions of the genes with general cellular functions, such as chromatin organization, DNA replication and cell cycle regulation. Mutations of the FASCIATA (FAS) genes encoding subunits of chromatin assembly factor-1, which supports nucleosome assembly on replicating DNA, result in the disorganization of apical meristems, leading to pleiotropic developmental defects (Leyser and Furner, 1992; Kaya et al., 2001). The MGOUN1 (MGO1) gene encoding a type-IB topoisomerase, which is required for the regulation of DNA coiling during replication and transcription, was revealed to be important for the proper functioning of the SAM and RAM (Laufs et al., 1998; Takahashi et al., 2002; Graf et al., 2010). Altered expression levels of a key cell-cycle regulator gene, RETINOBLASTOMA-RELATED (RBR), affect the maintenance of stem cells in apical meristems (Wildwater et al., 2005; Borghi et al., 2010). Lesions of the TEBICHI (TEB) gene, the product of which is proposed to be involved in cell cycle regulation, result in the disorganization of apical meristems (Inagaki et al., 2006, 2009). The MGO3/TONSOKU (TSK)/BRUSHY 1 (BRU1) gene product, a nuclear protein, is needed for meristem function as well as other cellular functions, such as DNA damage responses and epigenetic gene silencing (Guyomarc’h et al., 2004; Suzuki et al., 2004; Takeda et al., 2004). Although the underlying mechanism remains unclear, the results suggest the requirement of proper DNA metabolism for the maintenance of stem cell niches. Living organisms are at risk for exposure to endogenous and environmental hazards causing DNA damage and, consequently, mutations. Fixed mutations, if caused in stem cells, would be inherited after cell division, and not only prevent cells from functioning normally but also cause serious problems, such as cancer. To avoid such deleterious situations, in animals, stem cells show a low tolerance to DNA damage, and have a tendency for differentiation or programmed cell death (apoptosis) in response to DNA damage (Rich et al., 2000; Sherman et al., 2011). Cellular responses to DNA damage are mediated by two protein kinases: ATAXIA-TELANGIECTASIA MUTATED (ATM) and ATM/RAD3-RELATED (ATR). ATM and ATR are activated by DNA double-strand breaks (DSBs) and single-stranded DNA, respectively, and in turn positively regulate downstream reactions, such as the transcriptional upregulation of several DNA repair genes (Shiloh, 2006; Flynn and Zou, 2010). Likewise, it was demonstrated recently that plant stem cells and their early descendants selectively undergo ATM/ATRmediated programmed cell death upon certain mild genotoxic treatments (Fulcher and Sablowski, 2009). Unlike the case in animals, because plant stem cells produce germ-line cells in the final phase of the life cycle, there is a risk that the mutations fixed in stem cells during the vegetative growth phase are inherited by the offspring. Thus, the protection of stem cells against DNA damage, including exclusion of damaged stem cells, is crucial not only for developmental normality but also for reproductive fitness. Here, we isolated a novel mutant, named meristem disorganization 1-1 (mdo1-1), that exhibits abnormal shoot morphogenesis as well as retarded growth of roots. We found that the developmental defects observed in mdo1-1 result from the failure of the maintenance of stem cells in apical meristems, which leads to the disorganization of the meristem structures. In mdo1-1 cells, an elevated level of DSBs and increased expression of DNA damage-induced genes, the expression of which is regulated by ATM, suggested that the mutant cells are exposed constitutively to DNA damage, leading to DSBs. The mdo1-1 phenotypes were enhanced when combined with an atm mutation. The MDO1 gene encodes an unknown protein that is conserved in a wide variety of land plants. These results thus suggested that the MDO1 gene product is required for the maintenance of stem cells through the protection of cells from DNA damage. RESULTS Isolation of the mdo1-1 mutant We obtained a novel mutant exhibiting aberrant shoot morphogenesis from a pool of EMS-mutagenized M2 seedlings of Arabidopsis thaliana, and designated it as mdo1-1 based on the meristem phenotype (see below). About three-quarters and one-quarter of the self-progeny of the heterozygous mdo1-1/+ plants, which showed no apparent defective phenotype, exhibited the wild-type and mdo1-1 phenotypes, respectively (576 wild type and 189 mutant plants; v2 = 0.04, P > 0.05), suggesting that mdo1-1 is a single and recessive Mendelian trait. Shoot phenotype of mdo1-1 The mdo1-1 mutation affected shoot development. In the seedling stage, the emergence of the first pair of leaves (first and second leaves) or the second leaf was occasionally delayed (Figure 1b,c). In some cases, three leaves (second, third and fourth leaves) were generated nearly simultaneously (Figure 1e, arrowheads). In the subsequent vegetative growth phase, leaves continued to emerge in a spatially and temporally unregulated manner (Figure 1g). The mutant leaves showed a slightly abnormal shape and a reduced size, resulting in a compact rosette structure. In the reproductive growth phase, the mutant inflorescences were frequently fasciated, and some of them ceased to grow, with new inflorescences developing from the arrested shoot 658 Yuma Hashimura and Chiharu Ueguchi ª 2011 The Authors The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 68, 657–669
DNA damage and stem cell maintenance 659inflorescence meristems in the early reproductive growthphase were used for the following analysis. Scanning electron microscopic analysis indicated thatthe wild-type SAMshowed adome-shapedstructurewithasmoothsurface(Figure 2a).We did not observe sucha regular structure in themdo1-1 shoot apex: the apical region of mdo1-1, a region米米米surrounded byfloral buds,was expandedand coveredwithalot of small convex structures (Figure2b).Longitudinal sections revealedthatthe mdo1-1shoot apex wasflat,enlargedlaterally and was not covered by a canonical surface layerstructure,i.e.theL1and L2 layers,as is usually observed inthe wild-type SAM (Figure 2c,d). In the mutant apical region,especiallyinthesurfacearea,weobservedalotofenlargedcells,suggesting thatthecellshad startedto differentiate.Toclarify this further, we carried out in situ hybridization anal-ysis ofthe STMand AINTEGUMENTA(ANT)genes,thetranscripts of whichcan mark undifferentiated and differen-tiating cells, respectively (Elliott et al., 1996; Long et al.,1996).STMexpression wasdetectedinthecentral regionofthe wild-type SAM and downregulated in the incipient floralbuds (Figure2e),ashasbeen reportedpreviously (Long etal.,1996).Inthemdo1-1shootapex,STMexpressionwasreducedinthesurfacecells(Figure2f),or,insomesamplesFigure1.Shoot phenotypes of the mdo1-1mutant.(a-c)Morphology ofmultipleSTM expression domainscould be observed(Fig7-day-old seedlingsPlants were grown on MS gellan gum plates with 16-h light/8-h darkure 2g).Although ANTexpression was downregulated in thefluorescent illumination at 22C.Col (a) and mdo1-1 mutants showingwild-type central region (Figure 2h),it was detected in a widedelayed emergence of the first pair of leaves (b) and the second leaf (c) arerange of cells in the mdo1-1 shoot apex (Figure 2i). Theseshown-Scalehars:5mn(d, e)Col (d) and mdo1-1(e)seedlings (10 days old) are shown.Thearrowandresults thus suggest that the structural organization of thearrowheads indicate the first leaf, and second,third and fourth leavesSAM in mdo1-1isdisrupted bythe failure of maintaining therespectively.Scalebars:5mm(f-h) Morphology of 26-day-old rosettes. Plants were grown on MS gellanundifferentiated stateof stem cells.gum plates for 10 days and then on rockfiber with 16-h light/8-h darkRAMphenotypeofmdo1-1fluorescent illumination at22°C.Inflorescencestems wereremoved.Col (f)mdo1-1 (g) and the mdo1-1 carrying genomic MDO1 allele (h) are shown.Themdo1-1 mutation also affected root growth.The rootScalebars: 1 cm.(i-l) Inflorescence phenotypes of 35-day-old plants. Plants were grown on MSelongation rate was substantially decreased in the mdo1-1gellan gum plates for 10 days and then on rock fiber with 16-h light/8-h darkmutant (Figure 3a), suggesting a functional defect of thefluorescent illuminationat22°C.Col (i),mdo1-1(j,.k)and mdo1-1carrying thegenomic MDO1allele (l) are shown.ThearrowmutantRAM.To examinethisfurther,weinvestigatedthein panel (j) indicates an inflorescence showing the stop-and-go phenotypeexpressionofmarkergenesspecifyingseveraltypesofstemScale bars: 1 cm.cellnichecells.QC25expression wasobservedinQCcells inthewildtype(Figure3b;Sabatinietal.,2003),whereasQC25expressionwasreducedinsomeQCcellsorwastotallyapices (the stop-and-go phenotype; Figure 1j,arrow).Theabsent in mdo1-1 (Figure 3c,d). This suggested that thephyllotaxy of the floral buds was also distorted (Figure 1k)maintenanceofQCfunction is,atleastpartially,impaired byTheabnormaldevelopmentalpatternofthelateral organsasthe mdo1-1mutation.Inthe wild type, columella initialswell as the frequently observed stem fasciation in the mdo1-1locateddirectlybelowtheQCweremaintainedinanundifplants strongly suggested that the mutant SAM is impairedferentiatedstate sothattheydidnotaccumulate starchstructurallyand/orfunctionally.Thus,wefocused uponthegranules that were visualized by Lugol staining (Willemsenmutational effect on apical meristemfunctioning.et al.,1998;Figure3b).In contrast,cells justbeneaththeQCcells accumulated starch granules in mdo1-1 (Figure 3c,d),SAMphenotypeofmdo1-1indicating a failure in themaintenanceof the undifferentiTo determine whether or not the structure of the SAM is af-ated state of columella initials,presumably because ofafected by the mdo1-1mutation, we inspected the mutantmalfunction of the QC cells and/orcolumella initialsperse.shoot apiceshistologically.Because severe phenotypes,WeobtainedessentiallythesameresultswhenQC46andstem fasciation and growth arrest, in addition to abnormalQC184(Sabatini etal.,2003),other QC markers,were usedphyllotaxy,wereseenduringthereproductivegrowthphase(Figure S1).2011TheAuthorsThePlantJournal2011Blackwell PublishingLtd,ThePlantJournal,(2011),68,657-669
apices (the stop-and-go phenotype; Figure 1j, arrow). The phyllotaxy of the floral buds was also distorted (Figure 1k). The abnormal developmental pattern of the lateral organs as well as the frequently observed stem fasciation in the mdo1-1 plants strongly suggested that the mutant SAM is impaired structurally and/or functionally. Thus, we focused upon the mutational effect on apical meristem functioning. SAM phenotype of mdo1-1 To determine whether or not the structure of the SAM is affected by the mdo1-1 mutation, we inspected the mutant shoot apices histologically. Because severe phenotypes, stem fasciation and growth arrest, in addition to abnormal phyllotaxy, were seen during the reproductive growth phase, inflorescence meristems in the early reproductive growth phase were used for the following analysis. Scanning electron microscopic analysis indicated that the wild-type SAM showed a dome-shaped structure with a smooth surface (Figure 2a).We did not observe such a regular structure in the mdo1-1 shoot apex: the apical region of mdo1-1, a region surrounded by floral buds, was expanded and covered with a lot of small convex structures (Figure 2b). Longitudinal sections revealed that the mdo1-1 shoot apex was flat, enlarged laterally and was not covered by a canonical surface layer structure, i.e. the L1 and L2 layers, as is usually observed in the wild-type SAM (Figure 2c,d). In the mutant apical region, especially in the surface area, we observed a lot of enlarged cells, suggesting that the cells had started to differentiate. To clarify this further, we carried out in situ hybridization analysis of the STM and AINTEGUMENTA (ANT) genes, the transcripts of which can mark undifferentiated and differentiating cells, respectively (Elliott et al., 1996; Long et al., 1996). STM expression was detected in the central region of the wild-type SAM and downregulated in the incipient floral buds (Figure 2e), as has been reported previously (Long et al., 1996). In the mdo1-1 shoot apex, STM expression was reduced in the surface cells (Figure 2f), or, in some samples, multiple STM expression domains could be observed (Figure 2g). Although ANT expression was downregulated in the wild-type central region (Figure 2h), it was detected in a wide range of cells in the mdo1-1 shoot apex (Figure 2i). These results thus suggest that the structural organization of the SAM in mdo1-1 is disrupted by the failure of maintaining the undifferentiated state of stem cells. RAM phenotype of mdo1-1 The mdo1-1 mutation also affected root growth. The root elongation rate was substantially decreased in the mdo1-1 mutant (Figure 3a), suggesting a functional defect of the mutant RAM. To examine this further, we investigated the expression of marker genes specifying several types of stem cell niche cells. QC25 expression was observed in QC cells in the wild type (Figure 3b; Sabatini et al., 2003), whereas QC25 expression was reduced in some QC cells or was totally absent in mdo1-1 (Figure 3c,d). This suggested that the maintenance of QC function is, at least partially, impaired by the mdo1-1 mutation. In the wild type, columella initials located directly below the QC were maintained in an undifferentiated state so that they did not accumulate starch granules that were visualized by Lugol staining (Willemsen et al., 1998; Figure 3b). In contrast, cells just beneath the QC cells accumulated starch granules in mdo1-1 (Figure 3c,d), indicating a failure in the maintenance of the undifferentiated state of columella initials, presumably because of a malfunction of the QC cells and/or columella initials per se. We obtained essentially the same results when QC46 and QC184 (Sabatini et al., 2003), other QC markers, were used (Figure S1). (a) (f) (i) (j) (k) (l) (g) (h) (b) (c) (d) (e) Figure 1. Shoot phenotypes of the mdo1-1 mutant. (a–c) Morphology of 7-day-old seedlings. Plants were grown on MS gellan gum plates with 16-h light/8-h dark fluorescent illumination at 22C. Col (a) and mdo1-1 mutants showing delayed emergence of the first pair of leaves (b) and the second leaf (c) are shown. Scale bars: 5 mm. (d, e) Col (d) and mdo1-1 (e) seedlings (10 days old) are shown. The arrow and arrowheads indicate the first leaf, and second, third and fourth leaves, respectively. Scale bars: 5 mm. (f–h) Morphology of 26-day-old rosettes. Plants were grown on MS gellan gum plates for 10 days and then on rockfiber with 16-h light/8-h dark fluorescent illumination at 22C. Inflorescence stems were removed. Col (f), mdo1-1 (g) and the mdo1-1 carrying genomic MDO1 allele (h) are shown. Scale bars: 1 cm. (i–l) Inflorescence phenotypes of 35-day-old plants. Plants were grown on MS gellan gum plates for 10 days and then on rock fiber with 16-h light/8-h dark fluorescent illumination at 22C. Col (i), mdo1-1 (j, k) and mdo1-1 carrying the genomic MDO1 allele (l) are shown. The arrow in panel (j) indicates an inflorescence showing the stop-and-go phenotype. Scale bars: 1 cm. DNA damage and stem cell maintenance 659 ª 2011 The Authors The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 68, 657–669
660 Yuma Hashimura andChiharu Ueguchi(a)(a)cF(c)(d)OL910732678Daysaftergermination(b)Cd(hFigure3.Rootphenotypes of themdo1-1mutant(a)Thekinetics ofrootgrowth.Plantsweregerminated and grown onthesurface of MS gellan gum plates with 16-h light/8-h dark fluorescentillumination at 22°C. The length of main roots was determined every day.Each value represents an average, with standard error, for 20 plants. Thestrains used were: Col (red circles); mdo1-1 (blue triangles); and mdo1-1carrying genomic MDO1 allele (green squares).Figure 2. Shoot apical meristem (SAM) phenotypes of the mdo1-1 mutants.(bd)ExpressionofQC25and accumulationof starchgranules inroottips(a, b) SEM images of 28-day-old Col (a) and mdo1-1 (b) shoot apices. ScaleBlue and purple represent QC25-expressing cells and accumulated starchbars:100um.granules, respectively. QC25 (b) and QC25 mdo1-1(c, d) Longitudinal plastic sections of 28-day-old Col (c) and mdo1-1 (d) shoot(c,d) seedlings(7-daysold) were subjected to GUS stainingfollowed by Lugolapices.Scale bars: 50 μm.staining. The arrowheads indicate the position of columella initials. Scale(e-g) Expression of the STM gene in 14-day-old Col (e) and mdo1-1bars: 50 μm.(f,g)shootapices.AccumulationofSTMmRNAwasanalyzed byin situ(e-g) Expression of SCRpro:GFP-TIP. Col (e) and mdo1-1hybridization.The arrowhead in panel (e) indicates an incipient leaf primor-(f, g) seedlings (7-days old) were counterstained with propidium iodide (PI).dium. Scale bars: 50 μm.Thearrowhead inpanel (f) indicates acellfile showingreduced expression of(h, i) Expression of the ANT gene in 28-day-old Col (h) and mdo1-1 (i) shootSCRpro:GFP-TIP.The arrowhead and arrow in panel g) indicate reduced andapices. The accumulation of ANT mRNA was analyzed by in situ hybridization.discontinuous expression,and ectopic expression of SCRpro:GFP-TiP,Scale bars: 50 μm.respectively. Scale bars: 100 μm.AsdemonstrateduponexpressionofaSCARECROW(Figure 3f,g). It should be noted that the emergence of(SCR)promoter-GFP fusion gene(SCR:GFP-TIP;Saitoectopicexpressionof SCR:GFP-TIPinthemutantsteleswasetal.,2005),SCRisexpressed inQCcells,cortical/endoder-accompanied by the disappearance of expression of themal initials and endodermis cell layers in the wild typefusion gene in the endodermal cell files (Figure 3g).This(Figure 3e). In the mdo1-1 root tips, the SCR:GFP-,TIPfinding suggested that the function of cortical/endodermalexpression was significantlyreduced in someendodermisinitialsand/orthecell fateofendodermalcellfilesarenotcell layers, or was abolished in a discontinuous mannermaintained properly in the mdo1-1roots.2011TheAuthorsThePlantJournal2011BlackwellPublishingLtd,ThePlantJournal,(2011),68,657-669
As demonstrated upon expression of a SCARECROW (SCR) promoter-GFP fusion gene (SCR:GFP-cTIP; Saito et al., 2005), SCR is expressed in QC cells, cortical/endodermal initials and endodermis cell layers in the wild type (Figure 3e). In the mdo1-1 root tips, the SCR:GFP-cTIP expression was significantly reduced in some endodermis cell layers, or was abolished in a discontinuous manner (Figure 3f,g). It should be noted that the emergence of ectopic expression of SCR:GFP-cTIP in the mutant steles was accompanied by the disappearance of expression of the fusion gene in the endodermal cell files (Figure 3g). This finding suggested that the function of cortical/endodermal initials and/or the cell fate of endodermal cell files are not maintained properly in the mdo1-1 roots. 2 3 4 5 6 0 1 1 2 3 4 5 6 7 8 9 10 Days after germination Root length (cm) (a) (b) (c) (d) (e) (f) (g) Figure 3. Root phenotypes of the mdo1-1 mutant. (a) The kinetics of root growth. Plants were germinated and grown on the surface of MS gellan gum plates with 16-h light/8-h dark fluorescent illumination at 22C. The length of main roots was determined every day. Each value represents an average, with standard error, for 20 plants. The strains used were: Col (red circles); mdo1-1 (blue triangles); and mdo1-1 carrying genomic MDO1 allele (green squares). (b–d) Expression of QC25 and accumulation of starch granules in root tips. Blue and purple represent QC25-expressing cells and accumulated starch granules, respectively. QC25 (b) and QC25 mdo1-1 (c, d) seedlings (7-days old) were subjected to GUS staining followed by Lugol staining. The arrowheads indicate the position of columella initials. Scale bars: 50 lm. (e–g) Expression of SCRpro:GFP-cTIP. Col (e) and mdo1-1 (f, g) seedlings (7-days old) were counterstained with propidium iodide (PI). The arrowhead in panel (f) indicates a cell file showing reduced expression of SCRpro:GFP-cTIP. The arrowhead and arrow in panel (g) indicate reduced and discontinuous expression, and ectopic expression of SCRpro:GFP-cTIP, respectively. Scale bars: 100 lm. (a) (b) (c) (d) (e) (f) (g) (h) (i) Figure 2. Shoot apical meristem (SAM) phenotypes of the mdo1-1 mutants. (a, b) SEM images of 28-day-old Col (a) and mdo1-1 (b) shoot apices. Scale bars: 100 lm. (c, d) Longitudinal plastic sections of 28-day-old Col (c) and mdo1-1 (d) shoot apices. Scale bars: 50 lm. (e–g) Expression of the STM gene in 14-day-old Col (e) and mdo1-1 (f, g) shoot apices. Accumulation of STM mRNA was analyzed by in situ hybridization. The arrowhead in panel (e) indicates an incipient leaf primordium. Scale bars: 50 lm. (h, i) Expression of the ANT gene in 28-day-old Col (h) and mdo1-1 (i) shoot apices. The accumulation of ANT mRNA was analyzed by in situ hybridization. Scale bars: 50 lm. 660 Yuma Hashimura and Chiharu Ueguchi ª 2011 The Authors The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 68, 657–669
DNAdamageandstemcell maintenance661BILITY1(ATBRCA1)genes.The products of these genesCell death observed in the mdo1-1stem cell nicheshave been demonstrated to be involved in DNA repair, andAs shown byFigure 3(f.g),we observed that some cells weretheir expression is induced by DSBs (Klimyuk and Jones,stained with propidium iodide (Pl). PI stains the cell walls of1997;Deveaux etal.,2000; Lafarge and Montane,2003)living cells but is also usedto detectdead cellsthat havelostTotal RNAwaspreparedfromshootapextissuesandthentheir membrane integrity (Truernit and Haseloff, 2008)subjected to quantitative real-time RT-PCR(qRT-PCR)anal-WhenroottipswerestainedwithPlalone,cells instemcellysis. It was revealed that, in mdo1-1, the transcript levels ofniches,QC cells,several initials and the early descendantsthesegenes wereincreased2-2.5-foldcomparedwith thosein the wild type (Figure 5a).Furthermore, we checked thewere stained preferentially in mdo1-1, but not in the wildtype (Figure 4a,b). To confirm cell death in the mdo1-1expressionofCYCB1;1becauseits expression isalsoknownmutant, root tips were stained with Sytox Orange,anotherto be upregulated by irradiation, a typical treatmentcell death marker (Truernit and Haseloff,2008).As shown inresulting in DSBs (Culligan et al., 2006). Plants carrying aFigure 4(d), we observed a similar staining pattern in theCYCB1;1:GUS fusion containinga mitotic destruction boxmdo1-1 roots:that is, cells in the stem cell niches were(Colon-Carmona etal.,1999)were subjected to GUS staining.TheGUSactivitywas stronglyenhanced bythemdo1-1preferentially stained with Sytox Orange.mutation in both shoot apices and root tips (Figure 5c,e).DNA damage in the mdo1-1 mutantThis enhancement neither results from enhanced cell divi-sion activity norfrom cellcycle arrest at the G2/M boundaryIt was reported recently that the cells in plant stem cellnichesarehypersensitivetoDNAdamage,leadingtoDSBsbecause,comparedwiththewild-typebackground,expres-sion of CYCB1;2:GUS containing a destruction box, the(Fulcherand Sablowski,2009).Themdo1-1rootphenotypedescribed above led us to theidea that the mdo1-1mutationexpression of which is induced by the duration of the G2/Mcauses DNA damage in the cells.To examine this idea, wetransition,but not by DNA damage responses (Culliganfirstexaminedtheexpressionof theRAD51,GAMMAetal.,2006),wasnotdrasticallyenhanced in themutantRESPONSE1(ATGR1)andBREASTCANCERSUSCEPTIshootapex,and was instead slightly reduced in themutantroot tips (Figure 5g,i). These results thus strongly suggestthat the mdo1-1 cells are exposed to DNA damage consti-tutively,even without externalgenotoxic stress.(a)(b)It is known thatATMactivatedin responseto DSBsmediates the transcriptional activation of several genes,including RAD51,ATGR1,ATBRCA1and CYCB1;1(Garciaetal.,2003;Culligan etal.,2006).Therefore, it would beexpected that the level of DNA damage including DSBs isenhanced in mdo1-1 cells. To examine this directly, DSBswereanalyzedusingtheterminaldeoxynucleotidetransfer-ase-mediated dUTP nick-end labeling (TUNEL)method(Gavrieli et al., 1992), which can detect 3'-OH break ends.Labeled samples were further stained with 4',6-diamidino-2-phenylindole (DAPl) to detect nuclei. In the wild-type shootapex,apparent TUNELstaining was not detected (Figure5j).(cdIncontrast,weobserved a lotof TUNEL-stained nuclei inawide range of cells in the mutant apex (Figure 5k). Essen-tiallythe same results were obtained forthemutant roottips(Figure 5m). It should be noted that the TUNEL-stainednuclei weredetectednot onlyin stemcellnichesbut also indifferentiating cells, such as cells in growing leaves and inthe differentiation zone of the root tip (Figure 5k,m).Theseresults strongly suggest that a lesion of the MDO1functionresultsinincreasedlevelsofDSBs.Wethen examined whether or not mdo1-1plants aresensitive toDNA-damaging agents.Seedlings grown in MSmediumfor8dayswerefurthergrownfor2weeksinthesame medium containing various concentrations of bleo-Figure 4.Dead cells detected in mdo1-1root tipsmycin,a chemical agent leading to DSBs.As shown inCol (a, c) and mdo1-1 (b, d) seedlings (7-days old) were stained withpropidium iodide (a, b) and Sytox Orange (c, d). Scale bars: 100 μm.Figure6,thegrowthofthemdo1-1plants wasaffectedmore2011TheAuthorsThePlantJournal2011Blackwell PublishingLtd,ThePlantJournal,(2011),68,657-669
Cell death observed in the mdo1-1 stem cell niches As shown by Figure 3(f,g), we observed that some cells were stained with propidium iodide (PI). PI stains the cell walls of living cells but is also used to detect dead cells that have lost their membrane integrity (Truernit and Haseloff, 2008). When root tips were stained with PI alone, cells in stem cell niches, QC cells, several initials and the early descendants were stained preferentially in mdo1-1, but not in the wild type (Figure 4a,b). To confirm cell death in the mdo1-1 mutant, root tips were stained with Sytox Orange, another cell death marker (Truernit and Haseloff, 2008). As shown in Figure 4(d), we observed a similar staining pattern in the mdo1-1 roots: that is, cells in the stem cell niches were preferentially stained with Sytox Orange. DNA damage in the mdo1-1 mutant It was reported recently that the cells in plant stem cell niches are hypersensitive to DNA damage, leading to DSBs (Fulcher and Sablowski, 2009). The mdo1-1 root phenotype described above led us to the idea that the mdo1-1 mutation causes DNA damage in the cells. To examine this idea, we first examined the expression of the RAD51, GAMMA RESPONSE 1 (ATGR1) and BREAST CANCER SUSCEPTIBILITY 1 (ATBRCA1) genes. The products of these genes have been demonstrated to be involved in DNA repair, and their expression is induced by DSBs (Klimyuk and Jones, 1997; Deveaux et al., 2000; Lafarge and Montane´, 2003). Total RNA was prepared from shoot apex tissues and then subjected to quantitative real-time RT-PCR (qRT-PCR) analysis. It was revealed that, in mdo1-1, the transcript levels of these genes were increased 2–2.5-fold compared with those in the wild type (Figure 5a). Furthermore, we checked the expression of CYCB1;1 because its expression is also known to be upregulated by c irradiation, a typical treatment resulting in DSBs (Culligan et al., 2006). Plants carrying a CYCB1;1:GUS fusion containing a mitotic destruction box (Colo´ n-Carmona et al., 1999) were subjected to GUS staining. The GUS activity was strongly enhanced by the mdo1-1 mutation in both shoot apices and root tips (Figure 5c,e). This enhancement neither results from enhanced cell division activity nor from cell cycle arrest at the G2/M boundary because, compared with the wild-type background, expression of CYCB1;2:GUS containing a destruction box, the expression of which is induced by the duration of the G2/M transition, but not by DNA damage responses (Culligan et al., 2006), was not drastically enhanced in the mutant shoot apex, and was instead slightly reduced in the mutant root tips (Figure 5g,i). These results thus strongly suggest that the mdo1-1 cells are exposed to DNA damage constitutively, even without external genotoxic stress. It is known that ATM activated in response to DSBs mediates the transcriptional activation of several genes, including RAD51, ATGR1, ATBRCA1 and CYCB1;1 (Garcia et al., 2003; Culligan et al., 2006). Therefore, it would be expected that the level of DNA damage including DSBs is enhanced in mdo1-1 cells. To examine this directly, DSBs were analyzed using the terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) method (Gavrieli et al., 1992), which can detect 3¢-OH break ends. Labeled samples were further stained with 4¢,6-diamidino- 2-phenylindole (DAPI) to detect nuclei. In the wild-type shoot apex, apparent TUNEL staining was not detected (Figure 5j). In contrast, we observed a lot of TUNEL-stained nuclei in a wide range of cells in the mutant apex (Figure 5k). Essentially the same results were obtained for the mutant root tips (Figure 5m). It should be noted that the TUNEL-stained nuclei were detected not only in stem cell niches but also in differentiating cells, such as cells in growing leaves and in the differentiation zone of the root tip (Figure 5k,m). These results strongly suggest that a lesion of the MDO1 function results in increased levels of DSBs. We then examined whether or not mdo1-1 plants are sensitive to DNA-damaging agents. Seedlings grown in MS medium for 8 days were further grown for 2 weeks in the same medium containing various concentrations of bleomycin, a chemical agent leading to DSBs. As shown in Figure 6, the growth of the mdo1-1 plants was affected more (a) (b) (c) (d) Figure 4. Dead cells detected in mdo1-1 root tips. Col (a, c) and mdo1-1 (b, d) seedlings (7-days old) were stained with propidium iodide (a, b) and Sytox Orange (c, d). Scale bars: 100 lm. DNA damage and stem cell maintenance 661 ª 2011 The Authors The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 68, 657–669