RESEARCH ARTICLE 3997

Development 139, 3997-4006 (2012) doi:10.1242/dev.084079© 2012. Published by The Company of Biologists Ltd

INTRODUCTIONUnder appropriate environmental conditions, legumes can formnodular structures on their roots. Within the nodules, host plantscan obtain a nitrogen source fixed by soil bacteria (rhizobia); inturn, the plants provide a carbon source for the rhizobia. Thismutual interaction between plants and rhizobia is defined as rootnodule symbiosis. During nodule development, plants respond tonodulation (Nod) factors produced by the rhizobia; perception ofthese factors by receptor kinases triggers a signaling cascade in theepidermis of the root. As a result, dedifferentiation of some of thecortical cells is induced; these cells subsequently divide to form thenodule primordia (Szczyglowski et al., 1998; Oldroyd and Downie,2008; Oldroyd et al., 2011). During the course of noduledevelopment, rhizobia invade the dividing cortical cells via atubular structure called the infection thread (Murray, 2011).

A number of genes involved in the positive regulation ofnodulation have been identified through analysis of nodulation-deficient mutants. Additionally, it has been postulated that anothergenetic mechanism, termed autoregulation of nodulation (AON),negatively regulates nodulation to moderate the number of nodulesformed (Caetano-Anollés and Gresshoff, 1991; Oka-Kira and

Kawaguchi, 2006; Ferguson et al., 2010; Kouchi et al., 2010; Reidet al., 2011b). The basis of AON is systemic long-distancesignaling between root and shoot. In Lotus japonicus,HYPERNODULATION ABERRANT ROOT FORMATION 1(HAR1), which encodes a leucine-rich repeat receptor-like kinase,is hypothesized to function in shoots where it recognizes andresponds to root-derived signals involved in the negative regulationof nodulation (Krusell et al., 2002; Nishimura et al., 2002). Amongthe candidates for such signaling molecules in L. japonicus areCLE-ROOT SIGNAL1 (LjCLE-RS1) and LjCLE-RS2 (Okamotoet al., 2009). These two proteins belong to the CLE (CLAVATA3/ESR) family of proteins that play significant roles as signalingmolecules in cell-to-cell communication during a range of plantdevelopmental processes including stem cell maintenance, vascularpatterning and embryo development (Fletcher et al., 1999;Hirakawa et al., 2008; Stahl et al., 2009; Fiume and Fletcher,2012). AON appears to have a conserved molecular mechanismamong leguminous species, as functional counterparts of HAR1and LjCLE-RS1/2 have been identified in Medicago truncatula andGlycine max (Searle et al., 2003; Schnabel et al., 2005; Mortier etal., 2010; Reid et al., 2011a). However, little is known about thesite of AON action in nodule development.

It has been shown that the phytohormones cytokinin and auxinplay fundamental roles in the control of cell proliferation anddifferentiation in many developmental regulatory processes. Theputative cytokinin receptors LOTUS HISTIDINE KINASE 1(LHK1) in L. japonicus and CYTOKININ RESPONSE 1 (CRE1)in M. truncatula are involved in nodulation (Gonzalez-Rizzo et al.,2006; Murray et al., 2007; Tirichine et al., 2007). Nodule formationis impaired in plants carrying loss-of-function mutations of thesegenes, whereas nodule-like organs (termed spontaneous nodules)

1Division of Symbiotic Systems, National Institute for Basic Biology, Okazaki, Aichi444-8585, Japan. 2Department of Basic Biology, School of Life Science, GraduateUniversity for Advanced Studies (SOKENDAI), Okazaki, Aichi 444-8585, Japan.3National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan.4Department of Life Science, Aichi University of Education, Kariya, Aichi 448-8542,Japan.

*Author for correspondence (tsuzaki@nibb.ac.jp)

Accepted 5 July 2012

SUMMARYNodulation is a form of de novo organogenesis that occurs mainly in legumes. During early nodule development, the host plantroot is infected by rhizobia that induce dedifferentiation of some cortical cells, which then proliferate to form the symbiotic rootnodule primordium. Two classic phytohormones, cytokinin and auxin, play essential roles in diverse aspects of cell proliferation anddifferentiation. Although recent genetic studies have established how activation of cytokinin signaling is crucial to the control ofcortical cell differentiation, the physiological pathways through which auxin might act in nodule development are poorlycharacterized. Here, we report the detailed patterns of auxin accumulation during nodule development in Lotus japonicus. Ouranalyses showed that auxin predominantly accumulates in dividing cortical cells and that NODULE INCEPTION, a key transcriptionfactor in nodule development, positively regulates this accumulation. Additionally, we found that auxin accumulation is inhibitedby a systemic negative regulatory mechanism termed autoregulation of nodulation (AON). Analysis of the constitutive activation ofLjCLE-RS genes, which encode putative root-derived signals that function in AON, in combination with the determination of auxinaccumulation patterns in proliferating cortical cells, indicated that activation of LjCLE-RS genes blocks the progress of further corticalcell division, probably through controlling auxin accumulation. Our data provide evidence for the existence of a novel fine-tuningmechanism that controls nodule development in a cortical cell stage-dependent manner.

KEY WORDS: Autoregulation of nodulation, Auxin, CLE, Cytokinin signaling, Lotus japonicus, Nodule development

Positive and negative regulation of cortical cell divisionduring root nodule development in Lotus japonicus isaccompanied by auxin responseTakuya Suzaki1,2,*, Koji Yano1, Momoyo Ito1, Yosuke Umehara3, Norio Suganuma4 and Masayoshi Kawaguchi1,2

DEVELO

PMENT

3998

are formed in the absence of rhizobia in the spontaneous noduleformation2 (snf2) mutant that has a gain-of function mutation ofLHK1. Exogenous application of cytokinin to L. japonicus rootshas been shown to induce the formation of spontaneous nodules(Heckmann et al., 2011). Some response regulators, which areknown to be components of the cytokinin signaling pathway, arereported to be involved in nodulation in both species (Op denCamp et al., 2011). The various reports described above suggestthat activation of cytokinin signaling is a pivotal event in noduleinitiation. Downstream of cytokinin signaling, two putativetranscription factors, NODULE INCEPTION (NIN) andNODULATION SIGNALING PATHWAY2 (NSP2) (Schauser et al.,1999; Heckmann et al., 2006; Murakami et al., 2006), have beensuggested to be involved in nodule organogenesis on the basis thatspontaneous nodule formation in the snf2 mutant is suppressed ifeither of these genes has also been mutated (Tirichine et al., 2007).NIN is thought to be required for nodule organogenesis becausenodule formation is completely blocked in nin mutants (Schauseret al., 1999). Upon rhizobial infection, expression of NIN isstrongly activated both in the epidermis and in the nascent noduleprimordium. Recently, it has also been found that constitutiveexpression of NIN can induce ectopic cortical cell division in theabsence of rhizobia (T. Soyano and M. Hayashi, personalcommunication). Thus, NIN appears to play a crucial role in thededifferentiation and subsequent proliferation of cortical cellsduring nodule development.

In comparison to cytokinin, relatively little is known about the roleof auxin in nodule development. There have been somephysiological studies on auxin, mainly in the genus Medicago. Morethan twenty years ago, for example, it was shown that inhibition ofpolar auxin transport induces the formation of pseudonodules in theabsence of rhizobia in M. sativa (Hirsch et al., 1989), a findingrecently confirmed in M. truncatula (Rightmyer and Long, 2011).Furthermore, it was reported that silencing of the flavonoid pathway,which acts to inhibit auxin transport, causes a reduction in nodulenumber (Wasson et al., 2006). These observations suggest thatlocalized accumulation of auxin at the sites of incipient noduleprimordia might be a key step in nodule development. In addition,expression of some MtPIN genes is upregulated in the cre1 mutantof M. truncatula, suggesting that one role of cytokinin signaling innodulation might be to establish local auxin accumulation throughcontrol of expression of such auxin transporters (Plet et al., 2011).Although some understanding of the behavior of auxin in nodulationhas been achieved, there is still considerable uncertainty as to howand when auxin acts in the various genetic pathways that controlnodule development.

Here we identify the site of auxin action during noduledevelopment in L. japonicus. Our detailed analysis of thespatiotemporal induction pattern of auxin shows that cortical celldivision occurs concurrently with strong induction of auxinaccumulation. We show that auxin accumulation is under thepositive regulation of cytokinin signaling and that NIN functions inthe local accumulation of auxin at cortical cells. Our results furthershow that AON signaling, including HAR1 and LjCLE-RS1/2, actsto inhibit auxin accumulation. Moreover, a simultaneous analysisof the constitutive activation of LjCLE-RS genes in relation toauxin accumulation and cortical cell division patterns shows thatsignaling of these genes negatively regulates nodule development.We suggest that this effect might occur as a result of the blockingof further proliferation of cortical cells, probably by controllingauxin accumulation, although initiation of cell division is unlikelyto be under the same control.

MATERIALS AND METHODSPlant materialsThe Miyakojima MG-20 ecotype of L. japonicus was used in this study.MG-20 plants were mutagenized with ethylmethane sulfonate (EMS) andcyclops-6, nin-9 and har1-8 were isolated from M2 plants. Allelism testsindicated that all three genes were new mutants of cyclops, nin and har1,respectively. The site of mutation in each mutant is shown insupplementary material Table S1. The snf2 mutant, which has an MG-20background, has been reported previously (Miyazawa et al., 2010). TheDR5::GFP-NLS construct was introduced into the mutants by crossing withtransgenic MG-20 plants.

Constructs and transformation of L. japonicusThe primers used for PCR are listed in supplementary material Table S2.To generate the DR5::GFP-NLS construct, a 2.8 kb fragment of theDR5::gateway-cassette (GW) was excised from a vector kindly providedby J. Lohmann (Heidelberg University, Germany) and inserted between theSacI and KpnI sites of pCAMBIA1300. The translational fusion of GFP-NLS in pDONR221 (Invitrogen), which was kindly provided by D. Weigel(Max-Planck Institute for Developmental Biology, Tübingen, Germany),was inserted downstream of DR5 using the LR recombination reaction. Theresulting plasmid was introduced into Agrobacterium tumefaciens strainAGL1 and transformed into MG-20 as described previously (Nishimura etal., 2002).

To obtain the pCYCLOPS::mCherry-NLS construct, GFP was removedusing XhoI from the binary vector pCYCLOPS::GW; p35S::GFP reportedpreviously (Yano et al., 2008). The translational fusion of mCherry-NLS inpDONR221, which was provided by D. Weigel, was inserted downstream ofthe CYCLOPS promoter by the LR recombination reaction. For constitutiveexpression of NIN, the GFP in the pLjUBQ::GW; p35S::GFP binary vector(Maekawa et al., 2008) was removed using XhoI, and PCR-amplified mKO2[Medical and Biological Laboratories (Sakaue-Sawano et al., 2008)] wasinserted into the XhoI site to create the new binary vector pLjUBQ::GW;p35S::mKO2. NIN cDNA was amplified by PCR and cloned into thepENTR/D-TOPO vector (Invitrogen). The NIN cDNA in pENTR/D-TOPOwas inserted downstream of the LjUBQ promoter by the LR recombinationreaction. To create pLjUBQ::LjCLE-RS1; p35S::GFP-LjLTI6b orpLjUBQ::LjCLE-RS2; p35S::GFP-LjLTI6b constructs, first, a GFP fromwhich the stop codon had been deleted was amplified by PCR and clonedinto a pENTR1A (Invitrogen)-based vector (pJL-Blue), which has a multi-cloning site between the attL1 and attL2 sequences; the plasmid was kindlyprovided by D. Weigel. The resulting vector was named pENTR-GFP.

In order to identify a marker for the plasma membrane in L. japonicus(GFP-LjLTI6b), we performed a BLAST search (Altschul et al., 1990) of theL. japonicus genomic sequence database for the chr5.CM0048.40 gene usingas the query the amino acid sequence of Arabidopsis LTI6b, which encodesa plasma membrane-localized protein (Reddy et al., 2004). We designatedthe recovered gene sequence LjLTI6b. The LjLTI6b cDNA was amplifiedwith a short alanine linker sequence by PCR and inserted downstream of theC-terminus of the GFP in pENTR-GFP to produce a translational fusion ofGFP-LjLTI6b. The resulting GFP-LjLTI6b was amplified by PCR and usedto replace the original GFP of the pLjUBQ::GW; p35S::GFP vector to formthe new binary vector pLjUBQ::GW; p35S::GFP-LjLTI6b. The LjCLE-RS1,LjCLE-RS2 and GUS cDNAs in pEDONR221 (Okamoto et al., 2009) wereinserted downstream of the LjUBQ promoter using the LR recombinationreaction. The recombinant plasmids were introduced into A. rhizogenes strainAR1193 and were transformed into roots of DR5::GFP-NLS plants by ahairy root transformation method described previously (Okamoto et al.,2009).

In situ hybridizationThe primers used for PCR are listed in supplementary material Table S2.The in situ hybridization probes for GFP and HISTONE H4 were createdby PCR amplification of 495 bp and 372 bp fragments from each,respectively. The fragments were inserted into a pGEM-T Easy vector byTA cloning (Promega). Probe synthesis, preparation of sections and in situhybridization were performed as described previously (Suzaki et al., 2004).Signals were observed with a light microscope (BX-50, Olympus).

RESEARCH ARTICLE Development 139 (21)

DEVELO

PMENT

Expression analysisThe primers used are listed in supplementary material Table S2. LjTAA1(chr2.CM0008.610) and LjTAR1 (chr2.CM0008.590) were identified by aBLAST search of the L. japonicus genomic sequence database using theamino acid sequence of Arabidopsis TAA1 as the query. Total RNA wasisolated from each plant tissue using the RNeasy Plant Mini Kit (Qiagen).First-strand cDNA was prepared using a QuantiTect Reverse TranscriptionKit (Qiagen). Real-time RT-PCR was performed using an ABI Prism 7000(Applied Biosystems) with THUNDERBIRD SYBR qPCR Mix (Toyobo)according to the manufacturer’s protocol. The expression of ubiquitin wasused as the reference (Takeda et al., 2009). Data show the mean (± s.d.) ofthree biological replicates.

MicroscopyBright-field and fluorescence microscopy were performed with an SZX12stereomicroscope (Olympus) or with an A1 confocal laser-scanningmicroscope (Nikon). Images were acquired and analyzed using DPController (Olympus), NIS Elements (Nikon) or Photoshop (AdobeSystems).

RESULTSAuxin accumulates predominantly in dividingcortical cells during nodule developmentIn order to determine the precise distribution of auxin duringnodule development in L. japonicus, we created stable transgenicplants that express a GFP and nuclear localization signal (NLS)fusion protein (GFP-NLS) under the control of DR5, which is ahighly active synthetic auxin-responsive element (Ulmasov et al.,1997). Strong GFP expression was observed in the root apexincluding the putative quiescent center, and during lateral rootdevelopment, as has been reported in other plants (supplementarymaterial Fig. S1A-C) (Benková et al., 2003). In addition, GFPexpression was induced by exogenous application of auxin(supplementary material Fig. S1D). Our analysis showed that auxindistribution patterns during nodule development could be indirectlymonitored in DR5::GFP-NLS transgenic plants.

The Mesorhizobium loti strain MAFF303099, whichconstitutively expresses DsRED, was used to visualize rhizobia indeveloping nodules following infection of plant roots. Three daysafter inoculation (dai) with rhizobia, infection threads had startedto form in some root hairs, and GFP expression was observed in asmall number of cortical cells beneath root hairs with infectionthreads (Fig. 1A,E). The nuclei in the cortical cells immediatelybefore cell division appeared larger than those of surrounding cells.In roots at 5 dai, variable degrees of cortical cell proliferation hadoccurred and this cell division was coincident with strong GFPexpression (Fig. 1B,C,F,G). Strong GFP expression continued untilthe actively dividing cortical cells produced the initial bulge of thenodule primordia, which was invaded by rhizobia via the growinginfection threads (Fig. 1D,H). After rhizobial colonization of thedeveloping nodule primordia, the strong GFP expression halted inthe infected region of the nodule and was restricted to thesurrounding tissues (Fig. 1I). This pattern was maintained after thenodules enlarged and the rhizobia expanded their region ofinfection (Fig. 1J). At this stage, GFP expression was alsoobserved at the vascular bundle (lenticels).

Next, we carried out an in situ hybridization analysis to confirmthe GFP expression patterns described above. This analysis showedthat GFP transcripts were detectable in proliferating cortical cells(Fig. 1K); a similar expression pattern was found for the S-phase-specific marker HISTONE H4 (Fig. 1L). In developing noduleswith rhizobial colonies, GFP transcripts were distributed aroundthe region of infection (Fig. 1M). These distribution patterns were

consistent with the results of the GFP fluorescence analysisdescribed above.

It has been reported that the CYCLOPS gene is specificallyexpressed in dividing cortical cells during nodulation in L.japonicus (Yano et al., 2008). We performed hairy roottransformation of DR5::GFP-NLS transgenic plants in order toobtain expression of mCherry-NLS under control of the CYCLOPSpromoter. A co-localization analysis of DR5::GFP-NLS andpCYCLOPS::mCherry-NLS showed that GFP and mCherry areexpressed in the same cells during nodule development (Fig. 1N-P). This suggests that auxin accumulation occurs in cells in whichthe nodulation-related gene is expressed. We therefore concludethat auxin accumulation during nodule development occurspredominantly in proliferating cortical cells.

During nodule development, infection thread formation andcortical cell proliferation are closely related (Murray, 2011). Tofurther investigate the relationship between infection threadformation and auxin accumulation, we examined auxin distributionpatterns in a cyclops mutant (Yano et al., 2008). In contrast to wildtype (WT), in which the infection threads grew through root hairstowards the cortical cells, they did not reach the cortical cells in thecyclops-6 mutant (Fig. 2A,B). This failure of infection threadgrowth is the likely cause of the premature arrest of noduledevelopment in the mutant (Fig. 2C,D). DR5::GFP-NLS/cyclops-6 plants were produced by crossing DR5::GFP-NLS transgenicplants with cyclops-6 plants. During nodulation in DR5::GFP-NLS/cyclops-6 plants, however, induction of GFP expression wasobserved in cortical cells below the root hairs where the defectiveinfection threads were located (Fig. 2F). GFP was still expressedin the developmentally arrested nodules formed in the mutant (Fig.2G). These results suggest that it is not necessary for the infectionthreads to reach the cortical cells for the local accumulation ofauxin to occur.

Cytokinin signaling regulates auxin accumulationduring nodule developmentIn L. japonicus plants carrying the dominant snf2 mutation, theconstitutive activation of the cytokinin signaling pathway causesspontaneous nodule formation in the absence of rhizobia (Fig. 2E)(Tirichine et al., 2007). To clarify the relationship between auxinaccumulation and cytokinin signaling during nodule development,auxin distribution patterns were analyzed in snf2 mutant plants.DR5::GFP-NLS/snf2 plants were produced by crossingDR5::GFP-NLS transgenic plants with snf2 plants. Duringspontaneous nodule formation in DR5::GFP-NLS/snf2, inductionof GFP expression was observed in the cortical cells that wereproliferating to form the primordia of spontaneous nodules (Fig.2H). GFP expression was maintained in the inner region ofgrowing spontaneous nodules even when they were almost as largeas normal nodules formed following rhizobial infection. In thelatter, GFP expression is excluded from the inner regions colonizedby the rhizobia (Fig. 1J; Fig. 2I). Thus, auxin accumulation patternsduring spontaneous nodule development in the snf2 mutant suggestthat cytokinin signaling positively regulates auxin accumulationand that rhizobia might have a role in the exclusion of auxin frominfected cells.

NIN is involved in the local accumulation of auxinNIN encodes an RWP-RK type transcription factor that acts in thedownstream part of the cytokinin signaling pathway (Schauser etal., 1999; Tirichine et al., 2007; Madsen et al., 2010). To elucidatethe relationship between NIN and auxin, we analyzed auxin

3999RESEARCH ARTICLEAuxin involvement in nodulation

DEVELO

PMENT

4000

distribution patterns in a nin mutant and in the roots of transgenicplants that constitutively express NIN. In the nin-9 mutant,nodulation was completely inhibited, although excessive curling ofroot hairs was observed, as has been reported previously for othernin alleles (Schauser et al., 1999). DR5::GFP-NLS/nin-9 plantswere produced by crossing DR5::GFP-NLS transgenic plants withnin-9 plants. No specific distribution of GFP was observed in thecortical cells beneath the curled root hairs in DR5::GFP-NLS/nin-9 (Fig. 3A). When NIN was expressed under the control of the L.japonicus ubiquitin promoter (pLjUBQ) in the hairy root ofDR5::GFP-NLS transgenic plants, ectopic division of cortical cellswas induced. The cortical cells formed nodule- and lateral root-likeorgans in the absence of rhizobia (Fig. 3B,C; T. Soyano and M.Hayashi, personal communication). During formation of these

structures, localized expression of GFP was observed in the cortexof DR5::GFP-NLS roots that constitutively expressed NIN (Fig.3D). This pattern of GFP expression continued in the bulge formedby the nodule/lateral root-like organs (Fig. 3E,F). Thus, ourobservations indicate that NIN plays a role in the localaccumulation of auxin in the cortical cells of roots and that thisprobably leads to the activation of cortical cell division.

Autoregulation of nodulation controls auxinaccumulationAutoregulation of nodulation (AON) is a presumptive systemicregulatory mechanism that controls nodule number (Oka-Kira andKawaguchi, 2006). We investigated the interaction between auxinand key components of AON, specifically, the HAR1 and LjCLE-

RESEARCH ARTICLE Development 139 (21)

Fig. 1. Auxin accumulation patterns during wild-type nodule development. (A-J)Auxin accumulation patterns during nodule developmentare indirectly shown by GFP expression (green) in DR5::GFP-NLS L. japonicus plants. Fluorescence and nodule formation were observed from front(A-D) and side (E-J) views of infected roots. Red areas indicate the presence of rhizobia. (K-M)In situ localization of GFP (K,M) and HISTONE H4 (L)in DR5::GFP-NLS plants. Arrow indicates the region of rhizobial colonization. (N,O)DR5::GFP-NLS plant that has transgenic hairy roots containingthe pCYCLOPS::mCherry-NLS construct. (P)A merged image of N and O. Yellow signals indicate co-localization of GFP (N, green) and mCherry (O,red). Plants were inoculated with M. loti MAFF303099 that constitutively expressed DsRED (A-M) or with M. loti MAFF303099 (N-P). Roots wereobserved 3 (A,E), 5 (B,C,F,G,K,L,N-P), 8 (D,H), 10 (I) and 12 (J) days after inoculation (dai) of rhizobia. For fluorescence analysis, at least 20 plants (A-J) or 15 plants having transgenic hairy roots (N-P) were analyzed at each developmental stage in three independent experiments. Scale bars:100m.

DEVELO

PMENT

RS genes. Compared with the WT, the number of nodules increasedwithin a much larger nodulation zone in har1 mutants (Fig. 2C;Fig. 4A). DR5::GFP-NLS/har1-8 plants were produced by crossingDR5::GFP-NLS transgenic plants with har1-8 plants. In 5-dai rootsof DR5::GFP-NLS/har1-8, GFP was not only strongly expressedbut also expressed more widely than in DR5::GFP-NLS/WT plants(Fig. 1C,G; Fig. 4B,C). The GFP expression pattern in the har1mutant suggests that excessive induction of cortical cell divisionleads to the increased number of nodules and that this is associatedwith a considerable increase in auxin accumulation in the mutant.Overall, our findings suggest that HAR1 might function tonegatively regulate the accumulation of auxin.

LjCLE-RS1 and LjCLE-RS2, which encode small CLE peptides,are presumed to act as root-derived negative regulatory signals thatfunction via receptor complexes, including HAR1, duringnodulation (Okamoto et al., 2009; Kouchi et al., 2010). Althoughthe expression of LjCLE-RS1 and LjCLE-RS2 is significantlyincreased in roots immediately after rhizobial infection, constitutiveexpression of either gene can suppress nodule formation (Okamotoet al., 2009). We therefore sought to identify the site of action ofthe negative regulation resulting from activation of the LjCLE-RSgenes and to investigate its relationship to auxin accumulation. Inorder to monitor cell division patterns, we made use of LjLTI6b(the putative L. japonicus ortholog of Arabidopsis LTI6b) because

LTI6b has been reported to be localized at the plasma membrane(see Materials and methods) (Reddy et al., 2004). The plasmamembrane of host plants could be labeled by expressing a GFP-LjLTI6b fusion protein under control of the 35S promoter (Fig. 5A).

Nodulation phenotypes were examined in DR5::GFP-NLS plantsthat carried either pLjUBQ::LjCLE-RS1 or LjCLE-RS2;p35S::GFP-LjLTI6b in their hairy roots. In comparison to the hairyroots of control plants that expressed the GUS gene, constitutiveexpression of either of the LjCLE-RS genes reduced the number ofnodules (Fig. 4D-F; supplementary material Fig. S2A) (Okamotoet al., 2009). Infection thread formation was observed in almost allhairy roots of control plants (19/20; Fig. 4D; Fig. 5A). Importantly,although nodule formation was suppressed by constitutiveexpression of LjCLE-RS genes, infection thread formation wasobserved in 85% of the plants (17/20; Fig. 4E; Fig. 5F), suggestingthat the rhizobial infection process was unaffected by activation ofthe LjCLE-RS genes.

Comparison of cortical cell division patterns and auxindistribution in control roots showed that auxin accumulation wasclosely related to the progress of bulge formation in the noduleprimordia (Fig. 5A-C). In hairy roots of plants constitutivelyexpressing LjCLE-RS genes, auxin accumulation patternsimmediately before the initiation of cortical cell proliferation wereindistinguishable from those of control hairy roots (Fig. 5A,F).Furthermore, we noticed that some initial cortical cell divisionswere accompanied by auxin accumulation in the roots (Fig. 5G;supplementary material Fig. S2B). Initial cortical cell division wasobserved in 21/25 pLjUBQ::LjCLE-RS1 plants and 17/20

4001RESEARCH ARTICLEAuxin involvement in nodulation

Fig. 2. Auxin accumulation patterns in cyclops and snf2 mutants.(A,B)Infection thread formation in wild-type (WT) (A) and cyclops-6 (B)L. japonicus plants at 7 dai. (C,D)Nodules in WT (C) and cyclops-6 (D)at 14 dai. (E)Spontaneous nodule in snf2 35 days after germination(dag) in the absence of rhizobia. (F,G)GFP expression patterns duringnodule formation in DR5::GFP-NLS/cyclops-6 at 7 (F) and 14 (G) dai.(H,I)GFP expression patterns during spontaneous nodule formation inDR5::GFP-NLS/snf2 at 14 (H) and 28 (I) dag in the absence of rhizobia.Fluorescence, nodules and spontaneous nodule formation wereobserved using side views of the root (F-I). Arrows indicate prematuregrowth arrest of infection threads. Arrowhead indicates prematurearrest of nodule formation. For fluorescence analysis, at least 20 plantswere analyzed at each developmental stage in three independentexperiments. Scale bars: 100m in A,B,F-I; 1 mm in C-E.

Fig. 3. Relationship between NIN expression and auxinaccumulation. (A)GFP expression patterns in DR5::GFP-NLS/nin-9 L.japonicus plants at 7 dai. (B,C)Non-symbiotic root phenotype inDR5::GFP-NLS plants that have transgenic hairy roots, in which NIN andmKO2 are constitutively expressed. Transgenic roots were identified bymeans of mKO2 expression (C). Arrowheads indicate nodule/lateralroot-like organs. (D-F)GFP expression patterns during development ofthe nodule/lateral root-like organs formed in DR5::GFP-NLS plants thathave transgenic hairy roots constitutively expressing NIN in the absenceof rhizobia. Arrow indicates locally accumulated GFP signal in corticalcells. Fluorescence was observed using side views of the root (A,D-F).For fluorescence analysis, at least 15 plants (A) or 15 plants havingtransgenic hairy roots (B-F) were analyzed at each developmental stagein three independent experiments. Scale bars: 100m in A,D-F; 1 mmin B,C.

DEVELO

PMENT

4002

pLjUBQ::LjCLE-RS2 plants. These results suggest that althoughthe numbers of nodules decreased, initial cortical cell division stilloccurred in the presence of constitutive activation of LjCLE-RSgenes. However, we also frequently observed vestiges of celldivision with diminished auxin accumulation (Fig. 5H-J;supplementary material Fig. S2C,D). Such vestiges were present in22/25 pLjUBQ::LjCLE-RS1 plants and 18/21 pLjUBQ::LjCLE-RS2plants. This implied that premature arrest of cortical cell divisionsoccurred in hairy roots with constitutively activated LjCLE-RSgenes. In control hairy roots, vestiges of cell division were alsoobserved in 15/20 plants, although most cortical cell divisionsproceeded to form nodule primordia (Fig. 5C-E). Thus, arrest ofcortical cell divisions can also occur in WT nodule development inwhich the AON mechanism is anticipated to be fully functional.

Overall, these findings indicate that activation of LjCLE-RS genesmay act to inhibit further cortical cell divisions; the initial corticalcell divisions appear to avoid this negative regulation.

The expression of a TAA-like gene is induced uponrhizobial infectionThe analyses described above identify the site of auxinaccumulation and place this in a genetic context with respect to theregulation of nodule development. To determine the relationshipbetween auxin production and nodulation, we examined theexpression patterns of genes involved in auxin biosynthesis duringnodulation. We focused on the L. japonicus homologs ofTRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA)and its paralog TRYPTOPHAN AMINOTRANSFERASE RELATED

RESEARCH ARTICLE Development 139 (21)

Fig. 4. Auxin accumulation patterns in the har1 mutant and the effects of constitutive activation of LjCLE-RS genes on nodulation.(A)Nodulation phenotype of har1-8 L. japonicus plants at 14 dai. (B,C)GFP expression patterns during nodule development of DR5::GFP-NLS/har1-8 plants at 5 dai. Fluorescence and nodule formation were observed using front (B) or side (C) views of infected roots. For fluorescence analysis, atleast 20 plants were analyzed at each developmental stage in three independent experiments. (D,E)Nodulation phenotype of DR5::GFP-NLS plantsthat have transgenic hairy roots constitutively expressing both GUS and GFP-LjLTI6b (D), or both LjCLE-RS1 and GFP-LjLTI6b (E). Transgenic rootswere identified by their widespread distribution of GFP signals, derived from GFP-LjLTI6b, compared with those of GFP-NLS that are localized inspecific regions of the root. Infection threads appear as red spots in the roots. (F)Nodule numbers at 16 dai in DR5::GFP-NLS plants that havetransgenic hairy roots constitutively expressing LjCLE-RS genes (n13-17). Error bars indicate s.d. Scale bars: 1 mm in A,D,E; 100m in B,C.

Fig. 5. The effects of constitutive activation of LjCLE-RS1 on cortical cell division and auxin accumulation. (A-E)Infected DR5::GFP-NLS L.japonicus plants that have transgenic hairy roots containing pLjUBQ::GUS and p35S::GFP-LjLTI6b. (F-I)Infected DR5::GFP-NLS plants that havetransgenic hairy roots containing pLjUBQ::LjCLE-RS1 and p35S::GFP-LjLTI6b. (J)Magnified image of I. Fluorescence and cortical cell division wereobserved using front (E,G,I,J) or side (A-D,F,H) views of the roots. Plant plasma membrane was visualized by the expression of GFP-LjLTI6b. Greendots indicate auxin accumulation. Arrows indicate the regions where vestiges of cell division with diminished auxin accumulation were observed.Roots were at 6 (A,F) or 12 (B-E,G-J) dai. At least 20 plants having transgenic hairy roots were analyzed at each developmental stage in threeindependent experiments. Scale bars: 100m. D

EVELO

PMENT

(TAR) because the TAA family produces indole-3-pyruvic acid(IPA), which is converted to indole-3-acetic acid (IAA) by theYUCCA family; this IPA pathway is thought to be the main routefor IAA biosynthesis (Mashiguchi et al., 2011; Won et al., 2011).During nodulation, we found that expression of LjTAR1 wastransiently increased at 3 dai, which is when local auxinaccumulation started to occur, whereas LjTAA1 expression did notchange following rhizobial infection (Fig. 1A; Fig. 6A,B).

DISCUSSIONIn this study, we focused on the patterns of auxin accumulationduring early nodule development, that is, the developmental stagefrom the initiation of cortical cell proliferation to the formation ofnodule primordia. We show here that auxin predominantlyaccumulates in dividing cortical cells. The observed auxindistribution patterns are similar to those reported previously usingthe GH3 promoter (Pacios-Bras et al., 2003; Takanashi et al.,2011a); however, we have also been able to correlate auxinaccumulation patterns with various genetic factors that are key tothe formation of nodules.

Leguminous plants have two major and morphologically distincttypes of nodule: indeterminate and determinate (Ferguson et al.,2010). In indeterminate nodules, such as those of Medicago andPisum, the nodule meristem can be distinguished at the tip ofnodules and this meristem stays active until the nodule becomessenescent. By contrast, in determinate nodules, such as those inLotus, the activity of the nodule meristem appears to cease duringearly nodule development, although the identity and location of thenodule meristem is poorly characterized. Our observations applyparticularly to the development of determinate nodules. Afterrhizobial colonization of developing nodules, the accumulation ofauxin becomes restricted to the region surrounding the infectedcells. This region appears to correspond to the nodule parenchyma,which has previously been reported to show expression of organdifferentiation markers such as ENOD2 (van de Wiel et al., 1990;Niwa et al., 2001). As the size of a determinate nodule continuesto increase by cell proliferation and elongation even afterdifferentiation, it is likely that the meristematic activities aremaintained within the nodule. Since the auxin distribution patternduring nodule development in Lotus resembles that of meristematiccells, the nodule parenchyma might be a candidate for the nodulemeristem. Further analysis, for example defining the cell division

patterns in the nodule, will be needed to clarify the identity andlocation of the nodule meristem in determinate nodules. Auxinaccumulation occurs not only in dividing cortical cells but also inthe vascular bundle (lenticels) at later stages of noduledevelopment. The formation of lenticels is inhibited by auxintransport inhibitors (Takanashi et al., 2011a; Takanashi et al.,2011b).

During spontaneous nodule formation in the snf2 mutant, auxinaccumulation was observed at the inner nodular regions; bycontrast, these inner regions are occupied by rhizobia in normalnodules. In comparison to normal nodules, some of thespontaneous nodules formed in the snf2 mutant were distorted inshape (data not shown). Thus, it is possible that persistent auxinaccumulation in the inner region causes excess cell division andleads to the distorted shape of some spontaneous nodules.

Many of the genes that have been shown to be involved in thepositive regulation of nodulation function in the epidermis to signalthe occurrence of infection (Kouchi et al., 2010). However,nodulation-related cytokinin receptors (LHK1 in L. japonicus andCRE1 in M. truncatula) are specifically involved in noduledevelopment (Gonzalez-Rizzo et al., 2006; Murray et al., 2007;Tirichine et al., 2007). The RWP-RK type transcription factor NINacts in the downstream part of the cytokinin signaling pathway(Schauser et al., 1999; Tirichine et al., 2007; Madsen et al., 2010).Recently, NIN has also been shown to be able to initiate corticalcell division: constitutive activation of NIN induces ectopicdivision of the cells in the absence of rhizobia (T. Soyano and M.Hayashi, personal communication). Here, we investigated therelationship between auxin accumulation patterns and theexpression of genes that encode positive regulators of noduledevelopment. Our results show that auxin accumulation wasinduced when cytokinin signaling was constitutively activated.Furthermore, in the roots of transgenic plants that constitutivelyexpressed NIN, local auxin accumulations were observed. Basedon these results, we propose a model for the molecular mechanismthat regulates cortical cell division through control of auxinaccumulation (Fig. 7). In the model, infection signals from theepidermis ultimately activate cytokinin signaling in the cortex,which causes a downstream transcription factor, NIN, to establishlocal auxin accumulation in some cortical cells, which in turntriggers division of these cortical cells. Since auxin accumulates inthe dividing cortical cells until the formation of nodule primordia,then maintenance of auxin accumulation is required for noduleorganogenesis. The establishment of local auxin accumulationappears to be related to the inhibition of polar auxin transport;previous studies have shown that inhibition of polar auxin transportin Medicago roots induces the formation of pseudonodules (Hirschet al., 1989; Rightmyer and Long, 2011) and that expression ofsome auxin transporters is negatively regulated by cytokininsignaling (Plet et al., 2011). Here, however, we found thatexpression of a gene involved in auxin biosynthesis wascontemporaneous with the beginning of local auxin accumulation.This observation suggests that inhibition of polar auxin transportin conjunction with de novo auxin production might contribute tothe establishment of local auxin accumulation. Furtherinvestigation to identify the interactions of genes involved in thepositive regulation of nodule development, polar auxin transportand auxin biosynthesis would clarify the molecular mechanismresponsible for the local accumulation of auxin at the sites ofincipient nodule primordia.

In legumes, AON is known as a genetic mechanism that controlsthe number of nodules via long-distance communication between

4003RESEARCH ARTICLEAuxin involvement in nodulation

Fig. 6. LjTAA-like gene expression patterns during nodulation.Real-time RT-PCR analysis of (A) LjTAA1 and (B) LjTAR1 expression inWT non-inoculated (0), 1, 3, 5 and 7 dai roots. Each cDNA wasprepared from total RNA derived from the whole root. Fold changes inexpression are shown relative to roots at 0 dai. Error bars indicate s.d. D

EVELO

PMENT

4004

the shoot and the root. Mutation or knockdown of the genesinvolved in this feedback mechanism cause hypernodulationphenotypes (Wopereis et al., 2000; Krusell et al., 2002; Nishimuraet al., 2002; Miyazawa et al., 2010; Mortier et al., 2012). In AON,it has been proposed that root-generated signals are induced andtransported to the shoot upon infection by rhizobia. Two LjCLE-RS peptides have been identified as possible candidates for thesignals in L. japonicus (Okamoto et al., 2009). In M. truncatula,cytokinin treatment induces the expression of MtCLE13, afunctional counterpart of the LjCLE-RS genes, and the effects areabolished in nin mutants (Mortier et al., 2012). This suggests thatactivation of AON-related CLE genes occurs downstream of NIN.The root-derived signals are thought to be perceived by a receptorcomplex that includes HAR1, which is a putative receptor-likekinase, in the shoot; certainly, constitutive activation of LjCLE-RSgenes has no effect on nodulation in har1 mutant plants (Nishimuraet al., 2002; Okamoto et al., 2009). The output of LjCLE-HAR1signaling, an unidentified shoot-derived inhibitor, might betransported to the root to negatively regulate nodulation (Kouchi etal., 2010; Reid et al., 2011b). Here, we showed that, compared withthe WT, the har1 mutant has increased and more widespreadaccumulation of auxin in cortical cells. This indicates that auxinaccumulation prior to cortical cell division is controlled by theAON mechanism (including HAR1) and that it determines not onlythe auxin accumulation level but also the site of accumulation (Fig.7; supplementary material Fig. S3).

Although constitutive expression of AON-related CLE genesin legumes causes inhibition of nodulation (Okamoto et al.,2009; Mortier et al., 2010; Reid et al., 2011a), the site of thenegative regulation has remained elusive. Our simultaneousobservation of cortical cell division and auxin accumulationpatterns shows that early auxin accumulation and the initiationof cell division can occur even in the presence of constitutivelyactivated LjCLE-RS genes. Thus, the developmental process doesnot seem to be affected by constitutive expression of LjCLE-RSgenes. By contrast, we frequently observed vestiges of corticalcell division, implying its premature arrest, accompanied byreduced levels of auxin accumulation in transgenic roots.Importantly, these cell division vestiges were also observed incontrol roots, in which the LjCLE-RS genes were appropriatelyactivated by rhizobial infection. Thus, the premature arrest ofcortical cell division was not a secondary effect of constitutiveactivation of LjCLE-RS genes, as it is probable that such arrestoccurs normally. Overall, we presume that activation of LjCLE-RS genes has a negative regulatory effect on cortical cell divisionafter its initiation, possibly through inhibiting the maintenanceof auxin accumulation (Fig. 7; supplementary material Fig. S3).Further investigation needs to be carried out to determinewhether the link between activation of LjCLE-RS genes andauxin accumulation is direct or indirect.

Both nodules and lateral roots are formed as lateral organs ofroots, and the regulatory mechanisms for these two organs seem tohave some components in common and others that are organspecific (Desbrosses and Stougaard, 2011). On the basis of theresults obtained here, we propose the existence of a novelmolecular mechanism that controls cortical cell division in adevelopmental stage-specific manner during nodule development.This fine-tuning mechanism for regulation of cortical cell divisionis reminiscent of lateral root development, in which organogenesisinitiation in the founder cells, the formation of primordia and lateralroot emergence are regulated by distinct factors involved in auxinsignaling (Benková and Bielach, 2010). In order to achieve agreater understanding of the characteristic features of noduleorganogenesis, the identification of stage-dependent regulators ofcortical cell division is required.

AcknowledgementsWe thank Jan Lohmann for the DR5 construct; Detlef Weigel for the GFP-NLSand mCherry-NLS constructs; Makoto Hayashi for the pCYCLOPS construct andfor M. loti MAFF303099 expressing DsRED; Satoru Okamoto for LjCLE-RS1/2constructs; Jens Stougaard for A. rhizogenes strain AR1193 and the nin-2 andhar1-3 mutants; and Takashi Soyano and M. Hayashi for sharing unpublisheddata. Confocal images were acquired at the Spectrography and BioimagingFacility, NIBB Core Research Facilities.

FundingThis research was supported by Grants-in-Aid for Scientific Research from theMinistry of Education, Culture, Sports, Science and Technology of Japan[22870035 and 23012038 to T.S., 22128006 to M.K.], grants from TheNOVARTIS Foundation (Japan) for the Promotion of Science (to T.S.) and grantsfrom The Sumitomo Foundation (to T.S.).

Competing interests statementThe authors declare no competing financial interests.

Supplementary materialSupplementary material available online athttp://dev.biologists.org/lookup/suppl/doi:10.1242/dev.084079/-/DC1

ReferencesAltschul, S. F., Gish, W., Miller, W., Myers, E. W. and Lipman, D. J. (1990).

Basic local alignment search tool. J. Mol. Biol. 215, 403-410.

RESEARCH ARTICLE Development 139 (21)

Fig. 7. Model for the regulation of cortical cell division and thesite of auxin action in nodule development. The light-blue boxindicates the developmental stage of cortical cell division that isnegatively regulated by AON. The darker blue box beneath, whichpartially overlaps the light-blue box, indicates the developmental stagewhen maintenance of auxin accumulation occurs in all proliferatingcortical cells. Green and red regions of cortical cells indicate thepresumed sites of auxin accumulation and of rhizobia, respectively.CCD, cortical cell division; SDI, shoot-derived inhibitor. See text forexplanation of the model.

DEVELO

PMENT

Benková, E. and Bielach, A. (2010). Lateral root organogenesis-from cell toorgan. Curr. Opin. Plant Biol. 13, 677-683.

Benková, E., Michniewicz, M., Sauer, M., Teichmann, T., Seifertová, D.,Jürgens, G. and Friml, J. (2003). Local, efflux-dependent auxin gradients as acommon module for plant organ formation. Cell 115, 591-602.

Caetano-Anollés, G. and Gresshoff, P. M. (1991). Plant genetic control ofnodulation. Annu. Rev. Microbiol. 45, 345-382.

Desbrosses, G. J. and Stougaard, J. (2011). Root nodulation: a paradigm forhow plant-microbe symbiosis influences host developmental pathways. Cell HostMicrobe 10, 348-358.

Ferguson, B. J., Indrasumunar, A., Hayashi, S., Lin, M. H., Lin, Y. H., Reid, D.E. and Gresshoff, P. M. (2010). Molecular analysis of legume noduledevelopment and autoregulation. J. Integr. Plant Biol. 52, 61-76.

Fiume, E. and Fletcher, J. C. (2012). Regulation of Arabidopsis embryo andendosperm development by the polypeptide signaling molecule CLE8. Plant Cell24, 1000-1012.

Fletcher, J. C., Brand, U., Running, M. P., Simon, R. and Meyerowitz, E. M.(1999). Signaling of cell fate decisions by CLAVATA3 in Arabidopsis shootmeristems. Science 283, 1911-1914.

Gonzalez-Rizzo, S., Crespi, M. and Frugier, F. (2006). The Medicago truncatulaCRE1 cytokinin receptor regulates lateral root development and early symbioticinteraction with Sinorhizobium meliloti. Plant Cell 18, 2680-2693.

Heckmann, A. B., Lombardo, F., Miwa, H., Perry, J. A., Bunnewell, S.,Parniske, M., Wang, T. L. and Downie, J. A. (2006). Lotus japonicusnodulation requires two GRAS domain regulators, one of which is functionallyconserved in a non-legume. Plant Physiol. 142, 1739-1750.

Heckmann, A. B., Sandal, N., Bek, A. S., Madsen, L. H., Jurkiewicz, A.,Nielsen, M. W., Tirichine, L. and Stougaard, J. (2011). Cytokinin induction ofroot nodule primordia in Lotus japonicus is regulated by a mechanism operatingin the root cortex. Mol. Plant Microbe Interact. 24, 1385-1395.

Hirakawa, Y., Shinohara, H., Kondo, Y., Inoue, A., Nakanomyo, I., Ogawa,M., Sawa, S., Ohashi-Ito, K., Matsubayashi, Y. and Fukuda, H. (2008). Non-cell-autonomous control of vascular stem cell fate by a CLE peptide/receptorsystem. Proc. Natl. Acad. Sci. USA 105, 15208-15213.

Hirsch, A. M., Bhuvaneswari, T. V., Torrey, J. G. and Bisseling, T. (1989). Earlynodulin genes are induced in alfalfa root outgrowths elicited by auxin transportinhibitors. Proc. Natl. Acad. Sci. USA 86, 1244-1248.

Kouchi, H., Imaizumi-Anraku, H., Hayashi, M., Hakoyama, T., Nakagawa, T.,Umehara, Y., Suganuma, N. and Kawaguchi, M. (2010). How many peas ina pod? Legume genes responsible for mutualistic symbioses underground. PlantCell Physiol. 51, 1381-1397.

Krusell, L., Madsen, L. H., Sato, S., Aubert, G., Genua, A., Szczyglowski, K.,Duc, G., Kaneko, T., Tabata, S., de Bruijn, F. et al. (2002). Shoot control ofroot development and nodulation is mediated by a receptor-like kinase. Nature420, 422-426.

Madsen, L. H., Tirichine, L., Jurkiewicz, A., Sullivan, J. T., Heckmann, A. B.,Bek, A. S., Ronson, C. W., James, E. K. and Stougaard, J. (2010). Themolecular network governing nodule organogenesis and infection in the modellegume Lotus japonicus. Nat. Commun. 1, 1-12.

Maekawa, T., Kusakabe, M., Shimoda, Y., Sato, S., Tabata, S., Murooka, Y.and Hayashi, M. (2008). Polyubiquitin promoter-based binary vectors foroverexpression and gene silencing in Lotus japonicus. Mol. Plant MicrobeInteract. 21, 375-382.

Mashiguchi, K., Tanaka, K., Sakai, T., Sugawara, S., Kawaide, H., Natsume,M., Hanada, A., Yaeno, T., Shirasu, K., Yao, H. et al. (2011). The main auxinbiosynthesis pathway in Arabidopsis. Proc. Natl. Acad. Sci. USA 108, 18512-18517.

Miyazawa, H., Oka-Kira, E., Sato, N., Takahashi, H., Wu, G. J., Sato, S.,Hayashi, M., Betsuyaku, S., Nakazono, M., Tabata, S. et al. (2010). Thereceptor-like kinase KLAVIER mediates systemic regulation of nodulation andnon-symbiotic shoot development in Lotus japonicus. Development 137, 4317-4325.

Mortier, V., Den Herder, G., Whitford, R., Van de Velde, W., Rombauts, S.,D’Haeseleer, K., Holsters, M. and Goormachtig, S. (2010). CLE peptidescontrol Medicago truncatula nodulation locally and systemically. Plant Physiol.153, 222-237.

Mortier, V., De Wever, E., Vuylsteke, M., Holsters, M. and Goormachtig, S.(2012). Nodule numbers are governed by interaction between CLE peptides andcytokinin signaling. Plant J. 70, 367-376.

Murakami, Y., Miwa, H., Imaizumi-Anraku, H., Kouchi, H., Downie, J. A.,Kawaguchi, M. and Kawasaki, S. (2006). Positional cloning identifies Lotusjaponicus NSP2, a putative transcription factor of the GRAS family, required forNIN and ENOD40 gene expression in nodule initiation. DNA Res. 13, 255-265.

Murray, J. D. (2011). Invasion by invitation: rhizobial infection in legumes. Mol.Plant Microbe Interact. 24, 631-639.

Murray, J. D., Karas, B. J., Sato, S., Tabata, S., Amyot, L. and Szczyglowski,K. (2007). A cytokinin perception mutant colonized by Rhizobium in the absenceof nodule organogenesis. Science 315, 101-104.

Nishimura, R., Hayashi, M., Wu, G. J., Kouchi, H., Imaizumi-Anraku, H.,Murakami, Y., Kawasaki, S., Akao, S., Ohmori, M., Nagasawa, M. et al.

(2002). HAR1 mediates systemic regulation of symbiotic organ development.Nature 420, 426-429.

Niwa, S., Kawaguchi, M., Imazumi-Anraku, H., Chechetka, S. A., Ishizaka,M., Ikuta, A. and Kouchi, H. (2001). Responses of a model legume Lotusjaponicus to lipochitin oligosaccharide nodulation factors purified fromMesorhizobium loti JRL501. Mol. Plant Microbe Interact. 14, 848-856.

Oka-Kira, E. and Kawaguchi, M. (2006). Long-distance signaling to control rootnodule number. Curr. Opin. Plant Biol. 9, 496-502.

Okamoto, S., Ohnishi, E., Sato, S., Takahashi, H., Nakazono, M., Tabata, S.and Kawaguchi, M. (2009). Nod factor/nitrate-induced CLE genes that driveHAR1-mediated systemic regulation of nodulation. Plant Cell Physiol. 50, 67-77.

Oldroyd, G. E. and Downie, J. A. (2008). Coordinating nodule morphogenesiswith rhizobial infection in legumes. Annu. Rev. Plant Biol. 59, 519-546.

Oldroyd, G. E., Murray, J. D., Poole, P. S. and Downie, J. A. (2011). The rulesof engagement in the legume-rhizobial symbiosis. Annu. Rev. Genet. 45, 119-144.

Op den Camp, R. H., De Mita, S., Lillo, A., Cao, Q., Limpens, E., Bisseling, T.and Geurts, R. (2011). A phylogenetic strategy based on a legume-specificwhole genome duplication yields symbiotic cytokinin type-A response regulators.Plant Physiol. 157, 2013-2022.

Pacios-Bras, C., Schlaman, H. R., Boot, K., Admiraal, P., Langerak, J. M.,Stougaard, J. and Spaink, H. P. (2003). Auxin distribution in Lotus japonicusduring root nodule development. Plant Mol. Biol. 52, 1169-1180.

Plet, J., Wasson, A., Ariel, F., Le Signor, C., Baker, D., Mathesius, U., Crespi,M. and Frugier, F. (2011). MtCRE1-dependent cytokinin signaling integratesbacterial and plant cues to coordinate symbiotic nodule organogenesis inMedicago truncatula. Plant J. 65, 622-633.

Reddy, G. V., Heisler, M. G., Ehrhardt, D. W. and Meyerowitz, E. M. (2004).Real-time lineage analysis reveals oriented cell divisions associated withmorphogenesis at the shoot apex of Arabidopsis thaliana. Development 131,4225-4237.

Reid, D. E., Ferguson, B. J. and Gresshoff, P. M. (2011a). Inoculation- andnitrate-induced CLE peptides of soybean control NARK-dependent noduleformation. Mol. Plant Microbe Interact. 24, 606-618.

Reid, D. E., Ferguson, B. J., Hayashi, S., Lin, Y. H. and Gresshoff, P. M.(2011b). Molecular mechanisms controlling legume autoregulation ofnodulation. Ann. Bot. 108, 789-795.

Rightmyer, A. P. and Long, S. R. (2011). Pseudonodule formation by wild-typeand symbiotic mutant Medicago truncatula in response to auxin transportinhibitors. Mol. Plant Microbe Interact. 24, 1372-1384.

Sakaue-Sawano, A., Kurokawa, H., Morimura, T., Hanyu, A., Hama, H.,Osawa, H., Kashiwagi, S., Fukami, K., Miyata, T., Miyoshi, H. et al. (2008).Visualizing spatiotemporal dynamics of multicellular cell-cycle progression. Cell132, 487-498.

Schauser, L., Roussis, A., Stiller, J. and Stougaard, J. (1999). A plant regulatorcontrolling development of symbiotic root nodules. Nature 402, 191-195.

Schnabel, E., Journet, E. P., de Carvalho-Niebel, F., Duc, G. and Frugoli, J.(2005). The Medicago truncatula SUNN gene encodes a CLV1-like leucine-richrepeat receptor kinase that regulates nodule number and root length. Plant Mol.Biol. 58, 809-822.

Searle, I. R., Men, A. E., Laniya, T. S., Buzas, D. M., Iturbe-Ormaetxe, I.,Carroll, B. J. and Gresshoff, P. M. (2003). Long-distance signaling innodulation directed by a CLAVATA1-like receptor kinase. Science 299, 109-112.

Stahl, Y., Wink, R. H., Ingram, G. C. and Simon, R. (2009). A signaling modulecontrolling the stem cell niche in Arabidopsis root meristems. Curr. Biol. 19, 909-914.

Suzaki, T., Sato, M., Ashikari, M., Miyoshi, M., Nagato, Y. and Hirano, H. Y.(2004). The gene FLORAL ORGAN NUMBER1 regulates floral meristem size inrice and encodes a leucine-rich repeat receptor kinase orthologous toArabidopsis CLAVATA1. Development 131, 5649-5657.

Szczyglowski, K., Shaw, R. S., Wopereis, J., Copeland, S., Hamburger, D.,Kasiborski, B., Dazzo, F. B. and de Bruijn, F. J. (1998). Nodule organogenesisand symbiotic mutants of the model legume Lotus japonicus. Mol. PlantMicrobe Interact. 11, 684-697.

Takanashi, K., Sugiyama, A. and Yazaki, K. (2011a). Involvement of auxindistribution in root nodule development of Lotus japonicus. Planta 234, 73-81.

Takanashi, K., Sugiyama, A. and Yazaki, K. (2011b). Auxin distribution andlenticel formation in determinate nodule of Lotus japonicus. Plant Signal. Behav.6, 1405-1407.

Takeda, N., Sato, S., Asamizu, E., Tabata, S. and Parniske, M. (2009).Apoplastic plant subtilases support arbuscular mycorrhiza development in Lotusjaponicus. Plant J. 58, 766-777.

Tirichine, L., Sandal, N., Madsen, L. H., Radutoiu, S., Albrektsen, A. S., Sato,S., Asamizu, E., Tabata, S. and Stougaard, J. (2007). A gain-of-functionmutation in a cytokinin receptor triggers spontaneous root noduleorganogenesis. Science 315, 104-107.

Ulmasov, T., Murfett, J., Hagen, G. and Guilfoyle, T. J. (1997). Aux/IAAproteins repress expression of reporter genes containing natural and highlyactive synthetic auxin response elements. Plant Cell 9, 1963-1971.

4005RESEARCH ARTICLEAuxin involvement in nodulation

DEVELO

PMENT

4006

van de Wiel, C., Scheres, B., Franssen, H., van Lierop, M. J., van Lammeren,A., van Kammen, A. and Bisseling, T. (1990). The early nodulin transcriptENOD2 is located in the nodule parenchyma (inner cortex) of pea and soybeanroot nodules. EMBO J. 9, 1-7.

Wasson, A. P., Pellerone, F. I. and Mathesius, U. (2006). Silencing the flavonoidpathway in Medicago truncatula inhibits root nodule formation and preventsauxin transport regulation by rhizobia. Plant Cell 18, 1617-1629.

Won, C., Shen, X., Mashiguchi, K., Zheng, Z., Dai, X., Cheng, Y., Kasahara,H., Kamiya, Y., Chory, J. and Zhao, Y. (2011). Conversion of tryptophan to

indole-3-acetic acid by TRYPTOPHAN AMINOTRANSFERASES OF ARABIDOPSISand YUCCAs in Arabidopsis. Proc. Natl. Acad. Sci. USA 108, 18518-18523.

Wopereis, J., Pajuelo, E., Dazzo, F. B., Jiang, Q., Gresshoff, P. M., De Bruijn, F.J., Stougaard, J. and Szczyglowski, K. (2000). Short root mutant of Lotusjaponicus with a dramatically altered symbiotic phenotype. Plant J. 23, 97-114.

Yano, K., Yoshida, S., Müller, J., Singh, S., Banba, M., Vickers, K.,Markmann, K., White, C., Schuller, B., Sato, S. et al. (2008). CYCLOPS, amediator of symbiotic intracellular accommodation. Proc. Natl. Acad. Sci. USA105, 20540-20545.

RESEARCH ARTICLE Development 139 (21)

DEVELO

PMENT