Role of LONELY GUY genes in indeterminate nodulation on ...

Role of LONELY GUY genes in indeterminate nodulation onMedicago truncatula

Virginie Mortier1,2, Anton Wasson3, Pavel Jaworek4, Annick De Keyser1,2, Martijn Decroos1,2, Marcelle Holsters1,2,

Petr Tarkowski4, Ulrike Mathesius3 and Sofie Goormachtig1,2

1Department of Plant Systems Biology, VIB, 9052 Gent, Belgium; 2Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Gent, Belgium; 3Division of Plant Science,

Research School of Biology, The Australian National University, Acton, ACT 0200, Australia; 4Centre of the Region Han�a for the Biotechnological and Agricultural Research, Faculty of

Science, Palack�y University, 783 71 Olomouc, Czech Republic

Author for correspondence:Sofie Goormachtig

Tel: +32 9 3313910Email: [email protected]

Received: 11 September 2013

Accepted: 11 December 2013

New Phytologist (2014) 202: 582–593doi: 10.1111/nph.12681

Key words: cytokinin, LONELY GUY, nodu-lation, nodule primordium, root growth.

Summary

� LONELY GUY (LOG) genes encode cytokinin riboside 5′-monophosphate phosphoribohy-

drolases and are directly involved in the activation of cytokinins.� To assess whether LOG proteins affect the influence of cytokinin on nodulation, we studied

two LOG genes ofMedicago truncatula.� Expression analysis showed that MtLOG1 and MtLOG2 were upregulated during nodula-

tion in a CRE1-dependent manner. Expression was mainly localized in the dividing cells of the

nodule primordium. In addition, RNA interference revealed that MtLOG1 is involved in nod-

ule development and that the gene plays a negative role in lateral root development. Ectopic

expression of MtLOG1 resulted in a change in cytokinin homeostasis, triggered cytokinin-

inducible genes and produced roots with enlarged vascular tissues and shortened primary

roots. In addition, those 35S:LOG1 roots also displayed fewer nodules than the wild-type.

This inhibition in nodule formation was local, independent of the SUPER NUMERIC

NODULES gene, but coincided with an upregulation of theMtCLE13 gene, encoding a CLAV-

ATA3/EMBRYO SURROUNDING REGION peptide.� In conclusion, we demonstrate that in M. truncatula LOG proteins might be implicated in

nodule primordium development and lateral root formation.

Introduction

Cytokinins are essential for legume plants to establish a nitrogen-fixing symbiosis with rhizobia. During this symbiotic interaction,nodules are formed in which the bacteria reside to fix atmo-spheric nitrogen for the plant. Central in nodulation signalingare the bacterially produced Nodulation Factors (NFs) that areperceived by LysM-type receptor kinases at the root hair mem-brane, whereafter a signaling cascade is activated that initiates rhi-zobial infection as well as nodule organogenesis (Oldroyd et al.,2011). In the nodulation process, the essential role played bycytokinin (Dehio & de Bruijn, 1992; Cooper & Long, 1994;Bauer et al., 1996; Fang & Hirsch, 1998; Mathesius et al., 2000;Lohar et al., 2004; Heckmann et al., 2011) has been demon-strated most prominently by the defective nodule primordiumformation in transgenic Medicago truncatula plants, in which theexpression of the cytokinin receptor gene CRE1 is reduced, incre1 mutants, or in knockout mutants for the CRE1 homolog,LOTUS HISTIDINE KINASE1 (LHK1) of Lotus japonicus(Gonzalez-Rizzo et al., 2006; Murray et al., 2007; Plet et al.,2011). Additionally, an L. japonicus gain-of-function mutant forthe LHK1 receptor provoked spontaneous nodules, indicatingthat cytokinin signaling is both necessary and sufficient for

nodule formation (Tirichine et al., 2007). Cytokinin signaling isdetermined by a phosphorelay pathway controlled by A-type andB-type response regulators (RRs). The A-type and B-type RRs,MtRR4 and MtRR1, respectively, are activated by rhizobial inoc-ulation depending on several components of the NF signalingpathway (Gonzalez-Rizzo et al., 2006; Ariel et al., 2012). More-over, in M. truncatula and L. japonicus, additional A-type RRshave been identified that are induced by NF treatment in theroot-susceptible zone and of which gain-of-function mutantstriggered the development of nodule primordia-like structures(Op den Camp et al., 2011).

The cytokinin responses during nodulation have been localizedin the cortex, where cell divisions are activated for primordiumformation (Lohar et al., 2004; Gonzalez-Rizzo et al., 2006; Pletet al., 2011). Cytokinin is sensed by MtCRE1, and via MtRR4 andMtRR1 signaling, stimulates the transcription factors ETHYLENE-RESPONSIVE BINDING DOMAIN FACTOR REQUIREDFORNODULATION1 (ERN1), NODULATION SIGNALINGPATHWAY2 (NSP2) and NODULE INCEPTION (NIN) thatare involved in the transcription of early nodulation (ENOD) genesto induce cortical cell divisions (Tirichine et al., 2007; Frugier et al.,2008; Plet et al., 2011; Ariel et al., 2012). MtCRE1 alsoregulates nodulation-related auxin accumulation by reducing

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PIN-FORMED protein activity and auxin transport regulation (Pletet al., 2011).

Besides their role in nodule primordium development, cytoki-nins might also control the meristem differentiation of indetermi-nate nodules and mediate the root susceptibility for nodulation(Murray et al., 2007; Frugier et al., 2008; Verni�e et al., 2008; Pletet al., 2011). Legume plants regulate the number of nodulesthrough various mechanisms, including autoregulation of nodula-tion (AON), a systemic system that involves root–shoot commu-nication (Mortier et al., 2012b). Central in AON is a shoot-activereceptor-like kinase, designated SUPER NUMERIC NODULES(SUNN) in M. truncatula, similar to various receptors that(potentially) bind CLAVATA3/EMBRYO SURROUNDINGREGION (CLE) peptides (Okamoto et al., 2013).

Cytokinin also plays an important role in root architecturewith a negative effect on the development of lateral roots. Thisprocess has been intensively studied in Arabidopsis thaliana, buthas been reported in M. truncatula as well (Gonzalez-Rizzo et al.,2006; Del Bianco et al., 2013).

The homeostasis of the major naturally active cytokininsis maintained by a fine-tuned balance between synthesis, catabo-lism and storage of inactive forms (Mok & Mok, 2001; Werneret al., 2003; Sakakibara, 2006). Adenosine phosphate-iso-pentenyltransferases (IPTs) are central in cytokinin synthesisbecause they catalyze the synthesis of N6-(⊿2-isopentenyl)ade-nine (iP) and trans-zeatin (tZ), precursors that are converted intoactive cytokinins (Chen, 1997; Kakimoto, 2001; Takei et al.,2001; Sakamoto et al., 2006; Kurakawa et al., 2007) (SupportingInformation Fig. S1). The direct activation pathway is mediatedby a cytokinin riboside 5′-monophosphate phosphoribohydro-lase, designated LONELY GUY (LOG), that allows immediateconversion to the active cytokinin nucleobases iP and tZ (Kurakawaet al., 2007). IPTs, cytokinin oxidases and LOG proteins areproduced at specific locations and time points, especially at activegrowth regions during plant development (Kuroha et al., 2009;Werner & Schm€ulling, 2009). Cytokinins also act as long-dis-tance signals to control various plant developmental processes(Kudo et al., 2010) and their transport via the phloem is impor-tant for proper root vascular patterning (Bishopp et al., 2011a,b).

The LOG proteins are key players in the release of active cyt-okinins during plant development (Kurakawa et al., 2007;Kuroha et al., 2009), because the ectopic overexpression of theLOG genes reduced apical dominance, retarded leaf senescenceand increased the cell division rate in root vasculature andembryo (Kuroha et al., 2009). Higher-order log mutants hadaltered root and shoot morphologies and a reduced sensitivityto cytokinins during lateral root formation (Kuroha et al., 2009;Tokunaga et al., 2012). The partially overlapping expressionpatterns of the LOG genes, as well as the absence of phenotypesin single vs multiples mutants of LOG in Arabidopsis suggestedredundant functions for the LOG proteins (Kuroha et al.,2009).

Until now, it has remained not well known whether changesin cytokinin homeostasis due to de novo cytokinin synthesis,cytokinin activation or relocation contribute to noduledevelopment. Because of the importance of LOG genes in

cytokinin-dependent developmental processes, we addressed thisquestion by identifying the MtLOG1 and MtLOG2 genesthat show enhanced expression during M. truncatula nodulation.The expression of both genes was analyzed in detail and func-tional analysis suggested an important role during nodulationand lateral root formation.

Materials and Methods

Biological material

Medicago truncatula Gaertn. cv Jemalong A17, J5, as well as thedifferent mutants were grown and inoculated as described(Mergaert et al., 2003). The Sinorhizobium meliloti 1021 strain,the green fluorescent protein (GFP)-tagged Sm2011 strain withthe pHC60 plasmid (Cheng &Walker, 1998), or the monomericred fluorescent protein (mRFP)-tagged Sm2011 strain with thepBHR plasmid (Smit et al., 2005) were grown as described (Mor-tier et al., 2010). For RNAi analysis, the S. meliloti strain E65 wasmaintained on 50 lg ml�1 tetracycline-containing modified Ber-gensen’s medium (Rolfe et al., 1980), the S. meliloti strainWSM1022 was kept on tryptone yeast (TY) medium (Terpolilliet al., 2008) and the Agrobacterium rhizogenes Arqua1 strain(Boisson-Dernier et al., 2001) was preserved on 100 lg ml�1

streptomycin-containing TY medium.Medicago truncatula J5 plants were grown in vitro in square

Petri dishes (129 12 cm) on nitrogen-poor SOLi agar (Blondon,1964). For the temporal expression analysis during nodulation,nodules were harvested at 4–10 d post inoculation (dpi) fromplants grown in pouches, watered with nitrogen-poor SOLimedium and inoculated with Sm1021 pHC60-GFP. Infectionthreads were visible from 4 dpi onward, nodule primordia at6 dpi, small nodules at 8 dpi and slightly enhanced nodules at10 dpi. Tissue was collected by visualizing the GFP-tagged bacte-ria under a stereomicroscope MZFLII (Leica Microsystems,Wetzlar, Germany) equipped with a blue-light source and a LeicaGFP Plus filter set (kex = 480/40; kem = 510 nm longpass barrierfilter). The zones I of uninoculated roots were isolated at thesame developmental stage as the 4-dpi stage. For the MtLOG1expression analysis in cre1-1 mutants, plants were grown in anaeroponic system. Half of the 6-d-old plants were harvested,whereas the other half were inoculated with Sm2011 pBHR-mRFP and harvested 7 d later. For quantitative reverse-transcrip-tion polymerase chain reaction (qRT-PCR) analysis, 1-month-oldchimeric plants bearing one of the constructs 35S:GUS, 35S:MtLOG1, 35S:MtCLE12 or 35S:MtCLE13 were placed in anaeroponic system and grown under nitrogen-rich conditions; theirroots were harvested 2 wk later for qRT-PCR. RNA extraction,cDNA synthesis and qRT-PCR analysis were done as describedpreviously (Mortier et al., 2010).

Generation of 35S:MtLOG1, pMtLOG1 and pMtLOG2constructs and A. rhizogenes-mediated root transformation

A 2-kb region upstream of MtLOG1 and MtLOG2 wasisolated from genomic DNA based on the available genomic

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data (http://www.ncbi.nlm.nih.gov/). The promoters werefused to the b-glucuronidase (GUS)-encoding (uidA) gene inpKm43GWRolDC1 (Karimi et al., 2002). For the 35S:MtLOG1construct, the open reading frame (ORF) of MtLOG1 was iso-lated from genomic DNA. The ORF was cloned behind the 35Spromoter in the pB7WG2D (Karimi et al., 2002). Primers usedfor amplification are presented in Table S1.

The protocol for A. rhizogenes-mediated root transformation,adapted from Boisson-Dernier et al. (2001), was as describedpreviously (Mortier et al., 2010). To measure the effect of 35S:MtLOG1 on root length and lateral root number, uninoculatedroots were aeroponically grown in nitrogen-rich medium until40 d post germination and analyzed by means of the ImageJ pro-gram (http://rsbweb.nih.gov/ij/).

In order to check the long-distance effect of MtLOG1, themain root was kept on the juvenile plant and infected by stabbingthe hypocotyls with a fine needle containing an A. rhizogenes cul-ture. After cotransformation as described above, the plants weregrown for 2 wk at 25°C with a 16-h photoperiod at 70 lmolm�2 s�1 and transferred to an aeroponic system for 7 d. Nodula-tion was analyzed on the main, untransformed root of plantsbearing GFP-positive hairy roots.

Generation of theMtLOG1 RNAi vector and transforma-tion by A. rhizogenes

A 218-bp fragment of the MtLOG1 gene of M. truncatula wasamplified with the primers listed in Table S1. The attB siteswere inserted into the pDONOR vector (Invitrogen, Carlsbad,CA, USA) and finally into the RNAi vector pHellsgate8 (Helli-well et al., 2002), which drives the hairpin RNA expression withthe 35S promoter. The inserts were verified by restrictiondigests. The verified constructs were transformed into theA. rhizogenes strain Arqua1 with the freeze–thaw transformationmethod (H€ofgen & Willmitzer, 1988). M. truncatula cv Jem-along A17 seeds were scarified on sand paper, sterilized in 6%(w/v) sodium hypochlorite for 10 min and thoroughly rinsed insterile water. Seeds were vernalized at 4°C overnight and germi-nated at 25°C in the dark. M. truncatula was transformed withA. rhizogenes as described (Boisson-Dernier et al., 2001), usingthe empty pHellsgate8 vector as a negative control. Compositeplantlets were grown on 15-cm diameter Petri dishes filled withslanted modified F�ahraeus medium-containing agar (F�ahraeus,1957) supplemented with 25mg l�1 kanamycin (Boisson-Dernieret al., 2001) and grown at 20°C for 7 d, before transfer to25°C. A 16-h photoperiod at 150 lmol m�2 s�1 light intensitywas used throughout. Afterward, plants were transferred toplates containing F�ahraeus medium without kanamycin andincubated for one more week before inoculation with a freshculture of S. meliloti strain E65 (OD600 = 0.2), which constitu-tively expresses NodD3 (Fisher et al., 1988) or with S. melilotistrain WSM1022. Individual roots (one per plantlet) weremarked on the plate and inoculated with 10 ll of bacterialculture. Plates were incubated for 2 wk at 25°C with a 16-hphotoperiod at 150 lmol m�2 s�1 light intensity, whereaftereach of the marked roots was analyzed for nodule number, root

length and lateral root numbers. Three lots of 8–10 roots foreach treatment were excised, frozen in liquid nitrogen and usedfor qRT-PCR. Primers used to test the expression of all sixMtLOG homologs are listed in Table S1.

Statistical analysis

A generalized linear model was fitted to nodule number, lateralroot number, lateral root density data, which partitioned thephenotypic variation into fixed genotype and experiment effectsand random error effects. Because the data followed a Poissondistribution, a logarithmic base e link function was incorpo-rated. Root length was analyzed by one-way ANOVA with thegenotype as fixed term. For the qRT-PCR data involving onlya genotype effect, a one-way ANOVA model was fitted to thelog2-transformed DD cycle threshold (Ct) values with genotypeas fixed term and a random error term. The three biologicalrepeats were added as a random effect, nested under genotypeto account for dependencies between samples originating fromthe same genotype. For the qRT-PCR experiments involvingtwo factors (genotype and time), a two-way ANOVA modelwas fitted to the data with genotype and time as fixed factorsin the model and the interaction term genotype9 time and arandom error term. The three biological repeats were added asa random effect nested under the interaction term. To estimateall-pairwise comparisons, post-hoc tests were done. The Tukeymethod was applied to adjust for multiple comparisons. Differ-ences were declared significant when P < 0.05. For all analyses,except for the qRT-PCR, the GenStat software (http://www.vsni.co.uk/software/genstat/) was utilized. Differential expres-sion of qRT-PCR was analyzed with the mixed procedure asdescribed in the SAS/Enterprise Guide 5.1 (SAS Institute Inc.,Cary, NC, USA).

Histochemistry and microcopy

GUS activity in cotransformed roots and nodules was analyzedwith 5-bromo-4-chloro-3-indolyl-b-D-glucuronic acid as sub-strate (Van den Eede et al., 1992). Roots and nodules werevacuum infiltrated for 20 min and subsequently incubated inGUS buffer at 37°C. Incubation lasted 4 h for pMtLOG1:GUSand overnight for pMtLOG2:GUS. After staining, roots androot nodules were fixed, dehydrated, embedded with the Tech-novit 7100 kit (Heraeus Kulzer, Wehrheim, Germany), accord-ing to the manufacturer’s instructions, and sectioned with amicrotome (Reichert-Jung, Nussloch, Germany). The 3-lmthick sections were mounted on coated slides (Sigma-Aldrich).For tissue-specific staining, sections were submerged in a0.05% (w/v) ruthenium red solution (Sigma-Aldrich), washedin distilled water and dried. Finally, sections were mountedwith Depex (BDH Chemicals, Poole, UK). Photographs weretaken with a Diaplan microscope equipped with bright-fieldand dark-field optics (Leica).

For analysis of the nodule infection of RNAi-transformedroots of MtLOG, root segments inoculated with the GFP-taggedSm2011 pHC60 (Gage et al., 1996) were embedded in 3% (w/v)

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agarose, cross-sectioned at 150-lm thickness on a 1000plusvibratome (Vibratome Company, St Louis, MO, USA), andviewed under an epifluorescence microscope DMLB (Leica) withan UV excitation filter (excitation maximum 488 nm, 515 nmlongpass filter). Images were taken with a mounted charge-coupled device camera (RT Slider; Diagnostic Instruments, Ster-ling Heights, MI, USA).

Cytokinin measurements

Composite plants carrying 35S:GUS or 35S:MtLOG1 roots weregrown under aeroponic conditions in SOLi medium. After 30 dof growth, 10 g of total root material was taken. Samples weredivided into five technical replicates (1000 mg). Cytokinins wereextracted and separated, essentially as outlined by Nov�ak et al.(2003). Quantification was done by the isotope dilution method(ultra-high performance liquid chromatography–mass spectrom-etry) according to Nov�ak et al. (2008).

Results

Identification of two nodulation-induced LOG genes inM. truncatula

Based on the sequences of the nine identified members of theArabidopsis LOG protein family (Kurakawa et al., 2007), homo-logs were identified in the genome of M. truncatula by tBLASTxanalysis against theM. truncatula genomic data (Mt3.5) and theirexpression patterns were analyzed with the Medicago GeneExpression Atlas (http://mtgea.noble.org/v2/). In total, six LOGhomologs were found in the M. truncatula genomic data (Fig.S2; Table S2). As the expression of two of them, designatedMtLOG1 (IMGA|Medtr7 g101290.1) and MtLOG2 (IMGA|Medtr1 g064260.1), was upregulated when compared to uninoc-ulated control roots, we concentrated our analyses on these twogenes. The similarities between the MtLOG and AtLOG geneswere calculated with the supermatcher tool wEMBOSS (Sarachu

(a) (b)

(c) (d) (e)

(f) (g) (h) (i)

Fig. 1 Expression analysis ofMtLOG genes during nodulation. (a) Relative expression ofMtLOG1. Expression levels were measured by qRT-PCR on cDNAsamples of zone-I root tissues of uninoculated (NI)Medicago truncatula plants (NI) and at 4, 6, 8 and 10 d post inoculation (dpi). Data are means� SE ofthree biological repeats. (b) Relative expression ofMtLOG2 as measured by qRT-PCR. Data are means� SE of three biological repeats. Asterisks indicatestatistically significant differences compared to NI. (c) pMtLOG2:GUS activity in a developing nodule. (d) pMtLOG1:GUS activity in a nodule primordium.(e) Bright-field picture of a longitudinal section through the nodule primordium presented in (d). Expression is observed in the upper dividing cells of theprimordium (arrow), but not in the dividing cells close to the root vasculature (asterisk) nor in the outer cortical cells (hash). (f) Bright-field picture of alongitudinal section through the young nodule on pMtLOG1:GUS-expressing roots. Expression is observed throughout the dividing cells (arrow), but not incells of the outer cortex layers (hash). (g) pMtLOG1:GUS activity in a mature nodule. (h) Bright-field picture of a longitudinal section through the nodulepresented in (g). A weak expression is observed in the meristem (m, arrow) and early differentiating cells, but not in the infection zone (inf), nor in theouter cortical cells of the nodule (hash). (i) pMtLOG1:GUS expression in a lateral root primordium. All sections were counterstained with 0.05% rutheniumred. Bars: (c, d, g, i) 1 mm; (e, f, h) 0.5mm.





& Colet, 2005). MtLOG1 and MtLOG2 shared 87.6% similar-ity. MtLOG1 was most homologous with AtLOG3 (78.7%) andshared between 60.6% and 78.0% similarity with the otherAtLOG genes, whereas MtLOG2 was also most homologous withAtLOG3 (78.5%) and shared between 57.3% and 77.5% similar-ity with the other AtLOG genes. Phylogenetic analysis clearlyindicated that MtLOG1 and MtLOG2 are related to each otherand to AtLOG3, AtLOG4 and AtLOG6 (Fig. S2).

In order to confirm the expression pattern, the transcript levelswere analyzed in nodulated root tissues at 4, 6, 8 and 10 dpi andcompared to the expression levels in uninoculated control roots.The elongation zone of uninoculated roots was used as the refer-ence tissue. Whereas the MtLOG1 transcript level steadilyincreased from 4 dpi on (Fig. 1a), that of MtLOG2 was low at4 dpi, but increased at 6 dpi, and remained high until 10 dpi(Fig. 1b). The spatial expression of MtLOG1 and MtLOG2 wasinvestigated by promoter:GUS analysis in developing nodules.Transcriptional activation of the uidA gene was visualized byGUS staining. For pMtLOG2:GUS, no GUS staining wasobserved except very faintly in the center of developing nodules(Fig. 1c). In uninoculated pMtLOG1:GUS roots, no GUS stain-ing was seen in the nodulation-susceptible root zone. At 3 dpi,when incipient nodule primordia were present, pMtLOG1:GUSwas visible in the nodule primordium cells (Fig. 1d). Microscopicanalysis revealed GUS staining in cells in the center of the noduleprimordium and, to a much lesser extent, in dividing cells at thebase of the nodule primordium (Fig. 1d,e). At a slightly laterstage, GUS staining was detected throughout the cells of the nod-ule primordium, but not in the outer cortex cells (Fig. 1f). Inmature nodules, the expression of pMtLOG1:GUS was visible atthe nodule apex, more specifically in the meristem and early dif-ferentiating nodule cells (Fig. 1g,h), but did not occur in theouter cortical cells and in cells of the mature infection zone(Fig. 1h). MtLOG1:GUS expression was observed in lateral rootprimordia (Fig. 1i) and in root tips (data not shown). Together,these data imply a tissue-specific expression of two of theMtLOGgenes during nodulation and root development.

MtLOG1 andMtLOG2 expression in cre1-1mutants

As nodule primordium development depends on the CRE1 func-tion and because theMtLOG1 andMtLOG2 expression had beencorrelated with nodule primordium development, we investi-gated the transcript levels in the cytokinin receptor-affected cre1-1 mutant by qRT-PCR, before and after inoculation (Fig. 2).Upon inoculation of the wild-type M. truncatula, the expressionof MtLOG1 and MtLOG2 was significantly increased comparedto control roots (Fig. 2a–d), whereas this increase was not seen incre1-1 roots (Fig. 2a–c). These results suggest that the expressionofMtLOG1 andMtLOG2 is only activated late in the nodulationsignaling cascade, downstream of CRE1. To confirm these data,we generated transgenic roots of pMtLOG1:GUS in a wild-typeand cre1-1 mutant background, and analyzed the root-susceptiblezones harboring infection threads at 5 dpi. pMtLOG1:GUSexpression at the nodule primordium stage was observed only inwild-type plants and not in cre1-1 (Fig. 2c), but still in lateral

root primordia (Fig. 2c). These results indicate that during nodu-lation the expression of MtLOG1 and MtLOG2 depends onCRE1, which is essential for nodule organ formation, althoughMtLOG1 does not need CRE1 for expression in lateral root pri-mordia.

Influence of ectopic expression ofMtLOG1 on cytokininhomeostasis

In order to test whether LOG1 is a true cytokinin riboside5′-monophosphate phosphoribohydrolase, composite plants with35S:MtLOG1-expressing roots were made by A. rhizogenestransformation and concentrations of different cytokinin metabo-lites were measured and compared between 35S:MtLOG1 and35S:GUS roots. qRT-PCR confirmed the ectopic overexpressionof the 35S:MtLOG1 constructs (Fig. 3a). The concentrations ofthe LOG enzyme substrates, the cytokinin nucleotides N6-(⊿2-isopentenyl)adenosine-5′-monophosphate) (iPMP) and tZriboside-5′-monophosphate (tZMP), as well as N6-(⊿2-isopente-nyl)adenosine) (iPR), tZ riboside (tZR) and tZ were statisticallylower in 35S:MtLOG1 roots than those in 35S:GUS roots(Fig. 3b; Table S3), but the concentrations of iP did not signifi-cantly differ. In addition, the concentration of cis-zeatin (cZ),which is not produced by a LOG-mediated pathway, and mostof its metabolites did not differ significantly (data not shown).The reduced concentrations of iPMP and tZMP suggest metabo-lization by MtLOG1. Because the measurements did not showunequivocally the accumulation of active cytokinins, we followedthe expression of a subset of M. truncatula A-type RRs that areinduced by exogenous cytokinin or activated in transgenic roots

(a)

(c)

(b)

Fig. 2 AbolishedMtLOG expression in the cre1-1mutant. (a) and (b)Relative expression analysis ofMtLOG1 andMtLOG2 by qRT-PCR oncDNA samples of zone-I root tissues ofMedicago truncatulawild-typeplants (A17) and cre1-1mutants before inoculation (light gray bars) and at7 d post inoculation (dark grey bars), respectively. Data are means� SE ofthree biological repeats. Asterisks indicate statistically significantdifferences compared to NI. (c) pMtLOG1:GUS expression in lateral rootsand nodule primordia in a cre1-1mutant, as compared to wild-type plants(A17) (n = between 15 and 20 for each genotype and observation).Expression in the lateral root primordia was observed in every plantanalyzed. Bars, 1 mm.



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expressing the Mt35S:CRE1*(L267F) construct (Gonzalez-Rizzoet al., 2006; Op den Camp et al., 2011). The mRNA levels of thecytokinin and nodulation-induced RR4 were significantly higherin 35S:MtLOG1 plants than those in control plants, as was alsothe case for the expression levels of RR5, RR9 and RR11, but notfor RR8 (Fig. 4).

Next, we reasoned that the absence of active cytokininaccumulation in the 35S:MtLOG1 plant could be due to arapid turnover or conjugation. In fact, the expression of theonly studied cytokinin oxidase (CKX1) gene (Ariel et al.,2012) was not enhanced (Fig. S3a) and, additionally, amongthe cytokinin conjugates, the concentrations of N6-(⊿2-isopen-tenyl)adenine 7-glucoside (iP7G), N6-(⊿2-isopentenyl)adenine9-glucoside (iP9G) and tZ 7-glucoside (tZ7G) did not differsignificantly between 35S:MtLOG1 and 35S:GUS roots,whereas the concentrations of tZ 9-glucoside (tZ9G) and tZriboside O-glucoside (tZROG) were statistically reduced in

35S:MtLOG1 (Fig. S3b). Thus, these data revealed that cyto-kinin homeostasis was modified and that cytokinin signalingwas activated in 35S:MtLOG1 plants.

Effect ofMtLOG1 RNAi on root and nodule development

In order to unravel the role of MtLOG1, an RNAi hairpin con-struct was created that targeted 218 bp of the MtLOG1 sequence.Due to the homology between MtLOG1 and the other MtLOGgene candidates, this region also produced identical overlaps with

(a)

(b)

Fig. 3 Effects of the ectopic expression ofMtLOG1 on cytokininconcentrations. (a) Relative expression ofMtLOG1 in 35S:MtLOG1 rootscompared to 35S:GUS roots. Expression levels were measured by qRT-PCRon cDNA samples of uninoculatedMedicago truncatula root tissues madeby Agrobacterium rhizogenes transformation with either 35S:GUS or 35S:MtLOG1, 45 d after germination. Data are means� SE of three biologicalrepeats. Asterisks indicate statistically different expression levels (P < 0.05).(b) Average amount (pg g�1 FW) of various cytokinin metabolites in 35S:GUS (dark gray bars) and 35S:MtLOG1 (light gray bars) roots. iP, N6-(⊿2-isopentenyl)adenine); iPR, N6-(⊿2-isopentenyl)adenosine); iPMP, N6-(⊿2-isopentenyl)adenosine-5′-monophosphate; tZ, trans-zeatin; tZR,trans-zeatin riboside; tZMP, trans-zeatin riboside-5′-monophosphate.Averages are shown from five repeated measurements. Numeric valuescan be found in Supporting Information Table S3. Data are means� SE.Asterisks indicate a statistically significant difference when compared to35S:GUS transgenic roots (P < 0.001).

(a) (b)

(c) (d)

(e) (f)

Fig. 4 Effect of ectopicMtLOG1 expression on the expression of cytokininRR genes. Expression levels were measured by qRT-PCR on cDNA samplesofMedicago truncatula uninoculated root tissues made by Agrobacteriumrhizogenes transformation with either 35S:GUS or 35S:MtLOG1, 45 dafter germination. Data are means� SE of three biological repeats. (a–f)Relative expression ofMtLOG1, RR4, RR5, RR8, RR9 and RR11 in 35S:MtLOG1 roots when compared to 35S:GUS roots, respectively. Asterisksindicate statistically different expression levels (P < 0.05).





a maximal length of 29, 23, 25, 12 and 17 bp of MtLOG2,Medtr4 g058740.1, Medtr1 g105240.1, Medtr1 g015830.1 andMedtr3 g113710.2, respectively. Composite plants were inocu-lated with S. meliloti strain WSM1022 and the nodule numberwas assessed at 14 dpi. The number was reduced from an average

of 9.2 nodules per root in empty-vector hairy roots to 4.7 nod-ules in MtLOG1 RNAi hairy roots. (P < 0.005) (Fig. 5a).A significant reduction in nodule numbers was also seen afterinfection of MtLOG1 RNAi roots with S. meliloti E65 comparedto controls (data not shown). qRT-PCR analysis showed that theRNAi vector significantly decreased the MtLOG1 expression to c.44% of the empty-vector controls, but that it did not alter signifi-cantly the expression of any of the other five MtLOG homologs(Fig. 5b). After microscopic analysis of the vibratome sections, novisible difference in nodule structure and infection with GFP-producing rhizobia was observed between MtLOG1-silencedroots and empty-vector controls (Fig. 5c,d).

When the root architecture between control and MtLOG1RNAi plants was compared, on average, the root length was72.00� 9.05 mm (Fig. 6a), the number of lateral roots3.65� 1.02 (Fig. 6b), giving a lateral root density of 0.50� 0.074lateral roots cm�1 in control plants (Fig. 6c) vs 56.82� 4.79 mmroot length, 4.96� 0.60 lateral roots and 1.0� 0.1 lateralroots cm�1 in the MtLOG1 RNAi roots (Fig. 6a–c). Thus, thelateral root density of MtLOG1 RNAi roots was two-fold higherthan that of the control roots (P = 0.02), in agreement with a nega-tive role for cytokinins in lateral root development.

Effect of ectopic expression ofMtLOG1 on root develop-ment

We examined the potential role of LOG proteins in root andnodule development by analyzing 35S:MtLOG1 roots in detailfor root structure, lateral root development and nodulation. Thelengths of 35S:MtLOG1-expressing roots of composite plantsmade by A. rhizogenes transformation were measured 38 d posttransformation. On average, the length of control roots was40.52� 3.55 cm vs 22.88� 3.55 cm for 35S:MtLOG1 roots(Fig. 7a). In accordance, the lateral root number for control 35S:GUS and 35S:MtLOG1 transgenic roots was on average40.07� 3.31 and 18.23� 2.45 (Fig. 7b), resulting in a lateralroot density of 1.36� 0.13 and 1.38� 0.14 lateral roots cm�1,respectively (Fig. 7c). Except for the lateral root density, all differ-ences in measurements were statistically significant (P < 0.001).

The 35S:MtLOG1-expressing roots seemed to be more robustthan the 35S:GUS roots. To analyze this observation in moredetail, we sectioned transversally the root tissue located 1–2 cmabove the root tip of 1-month-old plants. The vascular tissue ofMtLOG1 transgenic roots was more expanded than that of con-trol roots (Fig. 7d–f). The diameter of the vascular bundle,

(a) (b)

(c) (d)

Fig. 5 Effects on nodulation ofMtLOG1 knockdown. (a) Nodule numbersin hairy roots at 14 d post inoculation (dpi) with Sinorhizobium melilotistrain WSM1022.Medicago truncatulawild-type (A17) plants weretransformed with an empty vector (control) or an RNAi construct targetingMtLOG1 and inoculated 3 wk post transformation (n = 85). Data aremeans� SE. Asterisks indicate statistically significant differences incomparison to empty-vector control roots (P < 0.005). (b) Expression ratioofMtLOG genes inMtLOG1 RNAi-transformed hairy roots of wild-type(A17) plants (dark gray) compared to empty-vector controls (light gray)determined by qRT-PCR. The PROTODERMAL FACTOR2 (PDF2) genewas used as the housekeeping gene. Values are means� SD of threebiological replicates. Each biological replicate contained 8–10 roots andwas harvested at 14 dpi (time of nodule counting). The asterisk indicates astatistically significant difference in comparison to the empty-vectorcontrol root (P < 0.005). (c, d) Cross-sections of nodules formed in theempty vector control (c) andMtLOG1 RNAi-silenced plants (d) 2 wk postinoculation. Sections were viewed under blue excitation (excitationmaximum 488 nm, 515 nm longpass filter) to visualize GFP constitutivelytranscribed in S. meliloti. Green autofluorescence in the nodule peripheryis due to the presence of flavonoids in the tissue. Bars, 100 lm.

(a) (b) (c)

Fig. 6 Effects ofMtLOG1 RNAi on lateralroot formation. Root length, lateral rootnumbers and lateral root density in hairyroots onMedicago truncatulawild-type(A17) plants, transformed with an emptyvector (control) or an RNAi constructtargetingMtLOG1 (n = between 70–99).Data are means� SE. Asterisks markstatistically significant differences incomparison to control roots (P < 0.05).



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NewPhytologist588

comprising xylem tissue, phloem tissue, pericycle and endoder-mal cell layer, was measured. On average, the diameter of 35S:MtLOG1 roots was 276.2� 6.0 lm vs only 182.0� 5.0 lm forcontrol roots (P < 0.001, Student’s t-test) (Fig. 7f). Careful analy-sis of the sections indicated enlarged, but also numerous, cells inthe vascular tissues of 35S:MtLOG1 (Fig. 7e,f). The same mea-surements done on the cortical tissue of these roots revealedchanges neither in size nor in cell number in radial sections of35S:MtLOG1 roots when compared to control roots. The datasuggest that the thickening of transformed 35S:MtLOG1 resultsfrom vascular tissue expansion.

Effect of 35S:MtLOG1 on nodule development

Analysis of nodulation of the 35S:MtLOG1 roots revealed thatectopic expression of MtLOG1 resulted in only a few nodulescompared to those counted on control roots (Fig. 8a). The struc-ture of these limited nodules was examined by microscopy on 3-lm–thick Technovit-embedded sections of 14-d-old nodules.Infection threads were clearly distinguishable on sections of 35S:GUS and 35S:MtLOG1 transgenic nodules (Fig. 8b–e), indicativefor normal rhizobial infection. However, the meristematic tissueof 35S:MtLOG1-transformed nodules seemed to disappear andthe fixation zone was disorganized (Fig. 8b–e).

Because of the reduced number of nodules in 35S:MtLOG1roots, we wondered whether the observed phenotype might inter-act with AON, a long-distance, nodule number-controllingmechanism. Previously, MtCLE13, a CLE peptide gene thatputatively activated the AON pathway, had been shown to beinduced by cytokinin (Mortier et al., 2010). Therefore, theexpression of MtCLE13 was analyzed by qRT-PCR on cDNA

samples of uninoculated root tissues transformed by A. rhizogeneswith 35S:MtLOG1. In addition, MtCLE12 was tested, whichdoes not show increased expression in response to cytokinin(Mortier et al., 2010). The MtCLE13 expression was indeedhigher in 35S:MtLOG1 roots than that in control roots, but notsignificant for MtCLE12 (Fig. 9a,b). To investigate whetherMtCLE13 overexpression had an effect on MtLOG1 expression,we analyzed the cDNA of roots ectopically expressing MtCLE13by qRT-PCR and found no consistent differential expression ofMtLOG1 and MtLOG2 between control and 35S:MtCLE12 or35S:MtCLE13 roots (Fig. S4). Together, these data might implythat MtLOG1 expression contributes to the MtCLE13 expressionduring nodulation.

Previously, nodulation had been found to be totally inhibitedon 35S:MtCLE13 roots and that this effect involved long-dis-tance mechanisms (Mortier et al., 2010). To test whether theectopic expression of MtLOG1 might also result in long-distanceeffects, we generated composite plants with small 35S:GUS or35S:MtLOG1 transgenic roots in addition to the wild-type mainroot. At 7 dpi with the Sm2011 carrying the pBHR-mRFP con-struct, the nodule number was assessed on the primary wild-typeroot. On average, 17.36� 1.72 nodules were counted on theprimary root of the wild-type 35S:GUS plants vs 14.62� 1.59nodules on that of 35S:MtLOG1 plants (Fig. 9c). Regressionanalysis (Poisson distribution, logarithmic link) by means of theGenStat software revealed that these differences were not statisti-cally significant (P > 0.05). Thus, because ectopic MtLOG1expression could only reduce the nodule number in the roots inwhich the construct was expressed and not in nontransgenic rootsof composite plants that additionally carried transgenic 35S:LOG1 roots, the effect of the ectopic overexpression of MtLOG1

(a) (b) (c)

(d) (e) (f)

Fig. 7 Effects of the ectopic expression ofMtLOG1 on root formation. (a–c)Medicago truncatula average root length, average lateral root number andaverage lateral root density of transgenic roots of composite plants (n = 29–35) measured 40 d after germination on 35S:GUS or 35S:MtLOG1,respectively. Asterisks indicate statistically significant differences in comparison to transgenic 35S:GUS roots (P < 0.001). Data are means� SE. The totalmean of two biological repeats is presented. (d, e) Toluidine blue-stained transversal sections through young root segments ectopically expressing 35S:GUS

and 35S:MtLOG1, respectively. Arrows indicate the endodermis with Casparian strips. (f) Average diameter of the vascular tissue, including theendodermal cell layer, identified by the presence of Casparian strips measured in root material harvested from 1-month-old chimeric 35S:GUS and 35S:

MtLOG1 plants (n = 3). Asterisk indicates a statistically significant difference when compared to transgenic 35S:GUS roots (P < 0.001; one-tailed, unpairedStudent’s t-test). Data are means� SE. e, endodermis; p, pericycle; ph, phloem; xy, xylem. Bars, 0.25mm.





on nodulation had to be local and not systemic. This observationis in contradiction with what had been seen for MtCLE13, wherethe ectopic expression affected nodulation not only on the trans-genic roots, but also on the nontransgenic roots of the same plant(Mortier et al., 2010).

This negative effect of 35S:MtCLE13 on nodulation is abol-ished in sunn-4 mutants that are affected in the AON pathwaydue to a mutation in a leucine-rich repeat receptor-like proteinkinase, possibly perceiving CLE peptides (Mortier et al., 2012a).To study the link between MtCLE13 and MtLOG1, we testedthe effect of ectopic overexpression of MtLOG1 on nodulation ofa sunn-4 mutant. Nodulation was assessed at 7 dpi with Sm2011pBHR-mRFP and resulted in 41.50� 4.41 nodules on 35S:GUSroots (control) and in 8.67� 1.75 nodules on 35S:MtLOG1roots (Fig. 9d). These differences were statistically significant(P < 0.001) in the regression analysis (Poisson distribution, loga-rithmic link) by means of the GenStat software. 35S:MtLOG1expression caused a similar decrease in nodule numbers in sunn-4mutant plants compared to the wild-type (compare Fig. 9d withFig. 8a). These results indicate that the MtCLE13 peptide signal-ing might be involved in the nodule number reduction caused bythe 35S:MtLOG1 expression, but that the inhibition is local andindependent of SUNN.

Discussion

Many experiments have shown that cytokinin signaling is essen-tial for the development of the nodular organ. Here, we identifiedtwo LOG genes in Medicago truncatula that showed increasedexpression upon nodulation. The two MtLOG genes share a highdegree of similarity with AtLOG1, AtLOG2, AtLOG3, AtLOG4,AtLOG5, AtLOG7 and AtLOG8 that are involved in the release

of the active cytokinin nucleobases from the cytokinin riboside5′-monophosphates, as reported for the LOG gene of rice(Oryza sativa) (Kurakawa et al., 2007; Kuroha et al., 2009). Mea-surements of cytokinin metabolites have shown that 35S:MtLOG1 roots contain fewer cytokinin nucleotides than 35:GUSroots, indicating that MtLOG1 might be a true 5′-monophos-phate phosphoribohydrolase, but without increase in free iP andtZ. This lack of increase might be due to activation of negativefeedback mechanisms or to a rapid turnover for a tight control ofthe cytokinin levels (Sun et al., 2003; Miyawaki et al., 2006);however, no elevated expression of a gene encoding a cytokinin-degrading oxidase was measured, nor were elevated concentra-tions of metabolites detected, indicative for the activation ofconjugating mechanisms. By contrast, a decrease in some glycosy-lated cytokinin forms in the 35S:MtLOG1 plants was observed,implying a compensatory action of other parts of the cytokininpool. Whatever the reason for these results, the cytokininsignaling increased in 35S:MtLOG1 roots because severalcytokinin-responsive genes were activated.

Expression of MtLOG1 and MtLOG2 had been shown todepend on the cytokinin receptor CRE1. Accordingly, the earliestMtLOG1 and MtLOG2 expression occurred in cells of developingnodule primordia. Hence, we propose that the cytokinins releasedvia MtLOG1 and MtLOG2 might probably not act as a primarycytokinin pool that is sensed by MtCRE1 to reinitiate cell divisionin the cortex for nodule development, but rather are part of apositive feedback to trigger cell division. Indeed, silencing ofMtLOG1 expression reduced the nodule number, supporting thepositive effect of cytokinins on nodulation as suggested previously(Lohar et al., 2004; Gonzalez-Rizzo et al., 2006; Murray et al.,2007; Tirichine et al., 2007; Plet et al., 2011), although the nod-ules that were formed did not differ structurally from those

(a)

(c) (d) (e)

(b)

Fig. 8 Effects of ectopic expression ofMtLOG1 on nodulation. (a)Medicagotruncatula average nodule number.Transgenic roots of composite plants (n = 29–35) were measured at 7 d post inoculation on35S:GUS or 35S:MtLOG1. The total mean oftwo biological repeats is shown. Asteriskindicates statistically significant difference incomparison to transgenic 35S:GUS roots(P < 0.001). Data are means � SE. (b)Longitudinal section through a noduleectopically expressing 35S:MtLOG1. Nodense cells, indicative of meristematic tissue,are seen. (c) Detail of the meristem andinfection zone of the nodule shown in (b). (d)Detail of the meristem and infection zone ofthe 35S:GUS control nodule shown in (e).The meristematic tissue and infection zoneare distinguishable. (e) Longitudinal sectionthrough a nodule ectopically expressing 35S:

GUS. The sections were stained withtoluidine blue. f, fixation zone; i, infectionzone; m, meristem. Bars: (b, e) 5 mm; (c, d)0.5 mm.



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NewPhytologist590

observed on control nodules. Thus, the cytokinins possibly pro-duced by the MtLOG proteins might account, at least partly, forthe cytokinin pool needed to maintain nodule formation (Fig. 10).

The MtLOG1 gene also contributes to the cytokinin effect onroot development, similarly to the AtLOG genes that negativelycontrol lateral root emergence (Kuroha et al., 2009). Indeed,pMtLOG1:GUS activity was detected in lateral root primordiaand the lateral root density was increased on MtLOG1 RNAiplants. Moreover, on transgenic 35S:MtLOG1 roots, root lengthwas reduced, indicative of a possibly enhanced production ofroot growth-inhibiting cytokinins. In Arabidopsis, the length ofthe primary root in plants ectopically expressing an AtLOG genewas similar to that of control plants (Kuroha et al., 2009). Possi-bly, morphological or molecular differences between Arabidopsisand M. truncatula, but also between the activities of the LOGproteins, might explain this discrepancy. Alternatively, the mod-ified auxin sensitivity of transgenic roots generated byA. rhizogenes could influence the cytokinin response of the root.However, as observed for the ectopic expression of LOG genesin Arabidopsis (Kuroha et al., 2009), the root vascular tissue hadexpanded.

Surprisingly, despite the positive regulation of nodulation bycytokinins, the number of nodules of 35S:MtLOG1 roots wasmuch lower than that of controls and the nodule meristem disap-peared in the few produced nodules. These observations are incontrast with a positive cytokinin role in nodule developmentand might result from a secondary effect caused by changesoccurring in the root. Indeed, it is well known that hormones actin a strict spatiotemporal pattern (Vanstraelen & Benkov�a, 2012)and the general ectopic expression of MtLOG1 might influencethe hormonal landscape in the root so as to inhibit nodule devel-opment. Other studies reported similar unexpected effects, suchas the RNAi of RR9, an A-type RR that negatively influencescytokinin signaling, reduced nodulation, in spite of the expectedopposite result (Op den Camp et al., 2011). Ectopic expressionof RR9 formed primordia that could either be arrested lateralroots or de novo nodule primordia (Op den Camp et al., 2011).Interestingly, the expression of RR9, together with other A-typeRRs, was enhanced in 35S:MtLOG1 roots, but no ectopic pri-mordia were observed in our study.

The MtCLE13 peptides might act as a downstream factor ofMtLOG1, because the expression of the corresponding gene washigher in transgenic 35S:MtLOG1 roots than that in controlroots. MtCLE13 negatively regulates nodulation and interactswith the AON pathway, which systemically controls the nodulenumber and is activated at the onset of primordium formation(Li et al., 2009; Okamoto et al., 2009; Mortier et al., 2010; Reidet al., 2011). Although this hypothesis is tempting, the nodula-tion phenotype of roots overexpressing MtLOG1 was milder thanthat of the overexpressed CLE genes, because, instead of a totalinhibition, the nodule number was only reduced and, in addi-tion, the effect of 35S:MtLOG1 was local and independent ofSUNN. Thus, the inhibitory effect seen on the nodulation ofMtLOG1 plants might be (in part) due to the upregulation of

Nod factors

Nod factor signaling

Cytokinins

CRE

MtCLE13

MtLOG

Nodule organogenesis

Fig. 10 Model of theMtLOG position in the nodulation signaling cascade.MtLOG1 andMtLOG2 (MtLOG) are activated downstream of thecytokinin receptor CRE1 inside the cells of the nodule primordium. RNAiresulted in a reduction in nodule development indicating that the genesmight be involved in nodule organogenesis. By contrast, induction ofMtCLE13 by 35S:MtLOG1might hint at a possible role in the nodulenumber control.

(a) (b)

(c) (d)

Fig. 9 Interaction ofMtLOG1with autoregulation of nodulation (AON). (a,b) Relative expression ofMtCLE12 andMtCLE13, respectively. Expressionlevels weremeasured by qRT-PCR on cDNA samples ofMedicago

truncatula uninoculated root tissues transformed byAgrobacteriumrhizogeneswith either 35S:GUS or 35S:MtLOG1, 45 d after germination.Data are means� SE of three biological repeats. Asterisks indicatestatistically different expression levels (P < 0.05). (c) Long-distance effect ofroots expressingMtLOG1 onwild-type root nodulation of compositeplants. Average nodule number at 7 d post inoculation (dpi) on the wild-typemain root of composite plants bearing additional transgenic 35S:GUS(control) or 35S:MtLOG1 roots (n = 28–31). Data are means� SE.Regression analysis by means of the GenStat software revealed nostatistical differences (P > 0.05). The total mean of two biological repeats ispresented. (d) Effect of ectopic overexpression ofMtLOG1 on nodulationin a sunn-4mutant background. Average nodule number at 7 dpi ontransgenic 35S:GUS (control) or 35S:MtLOG1 roots (n = between 26–28).Note that the nodule number of the sunn-4mutants is higher than that ofthe wild-type due to defective AON. Data are means� SE. Statisticaldifferences were evaluatedwith a regression analysis bymeans of theGenStat software. Asterisk marks a statistically significant difference incomparison to 35S:GUS transgenic roots (P < 0.001). The total mean of twobiological repeats is presented.





MtCLE13. As many aspects are still to be resolved, it would beinteresting to control the localized expression of MtCLE13 in the35S:MtLOG1 plants. Moreover, the analysis of the MtCLE13RNAi in 35S:MtLOG1 roots might reveal whether the CLE pep-tides play an active role in reducing the nodulation induced by35S:MtLOG1.

In summary, the MtLOG genes might substantiate the propernodule primordium development and they might negatively reg-ulate lateral root formation. Further studies will reveal the indi-vidual contributions of both MtLOG proteins studied here, butstable mutants will be necessary. Moreover, via the MtCLE13activation,MtLOG1 might link to the AON pathway mechanismthat is well known to be activated when the first cell divisionsappear in the cortex (Li et al., 2009). Nevertheless, additionalexperiments are required to clearly demonstrate the potential linkbetween cytokinin production and the AON mechanism.

Acknowledgements

The authors thank Rene Geurts (Wageningen University,Wageningen, the Netherlands), Pascal Gamas (Institut de laRecherche Agronomique, Toulouse, France), Doug Cook (Uni-versity of California, Davis, CA, USA), Giles Oldroyd (John In-nes Institute, Norwich, UK), Florian Frugier (Institut desSciences du V�eg�etal, Centre National de la Recherche Scientifi-que, Gif-sur-Yvette, France), Sharon Long (Stanford University,Stanford, CA, USA) and Julia Frugoli (Clemson University,Clemson, SC, USA) for S. meliloti strains and M. truncatulamutants, their colleagues Christa Verplancke and GuangLingCui for skillful assistance, Wilson Ardiles for sequence analysis,Marnik Vuylsteke and V�eronique Storme for help with the statis-tical analysis and Stephane Rombauts for help with the bioinfor-matics. This work was supported by grants from the EuropeanCommission Marie Curie International Research Staff ExchangeScheme (IRSES) (grant no. PIRSES-GA-2008-230830), theMinisterie van de Vlaamse Gemeenschap (grant no. CLO/IWT/020714), and the Research Foundation-Flanders (grant nos.G.0350.04N and G.0066.07N) (to M.H. and S.G.), the Centreof the Region Han�a for the Biotechnological and AgriculturalResearch, Faculty of Science (ED 0007/01/01) (to P.T.), and aFuture Fellowship from the Australian Research Council(FT100100669) (to U.M.).

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Supporting Information

Additional supporting information may be found in the onlineversion of this article.

Fig. S1 Simplified overview of iP and tZ synthesis and the role ofLOG enzymes in the synthesis pathway.

Fig. S2 Phylogenetic tree of the Arabidopsis and M. truncatulaLOG genes.

Fig. S3 Analysis of cytokinin conjugates or cytokinin turnover in35S:MtLOG1 roots.

Fig. S4 Interaction ofMtLOG1 with AON.

Table S1 List of primers

Table S2Nomenclature of Arabidopsis and M. truncatula LOGgenes

Table S3 Average concentration of various cytokinin metabolitesin 35S:MtLOG1 and 35S:GUS roots

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