Adaptation of Medicago truncatula to nitrogen limitation is modulated via local and systemic nodule developmental responses

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1 New Reserch Adpttion of Medicgo trunctul to nitrogen limittion is modulted vi locl nd systemic nodule developmentl responses Christin Jeudy 1, Sndrine Ruffel 2, Sndr Freixes 1, Pscl Tillrd 2, Anne Lise Sntoni 1, Sylvin Morel 3, Etienne-Pscl Journet 4,Gérrd Duc 1, Alin Gojon 2, Mrc Lepetit 2 nd Christophe Slon 1 1 Unité Mixte de Recherche en Génétique et Ecophysiologie des Légumineuses, UMR INRA 12, BP 8651, F-2165 Dijon, Frnce; 2 Biochimie et Physiologie Moléculire des Plntes, UMR 54, INRA-CNRS-Sup Agro-UM2, Institut de Biologie Intégrtive des Plntes, 2 Plce Vil, F-346 Montpellier, Frnce; 3 ENESAD, 26, Bd du docteur Petitjen, BP 87999, 2179 Dijon Cedex, Frnce; 4 Lortoire des Interctions Plntes Micro-orgnismes (LIPM), UMR CNRS-INRA , F-3132 Cstnet-Tolosn, Frnce Summry Author for correspondence: Christophe Slon Tel: +33 () Emil: slon@dijon.inr.fr Received: 31 July 29 Accepted: 6 Octoer 29 doi: /j x Key words: hypernodulting mutnt, legume, Medicgo trunctul, nitrogen, nodule development nd growth, nodule N-limittion, root, systemic signling. Adpttion of Medicgo trunctul to locl nitrogen (N) limittion ws investigted to provide new insights into locl nd systemic N signling. The split-root technique llowed chrcteriztion of the locl nd systemic responses of NO 3 ) or N 2 -fed plnts to loclized N limittion. 15 N nd 13 C leling were used to monitor plnt nutrition. Plnts expressing pmtenod11-gus nd the sunn-2 hypernodulting mutnt were used to unrvel mechnisms involved in these responses. Unlike NO 3 ) -fed plnts, N 2 -fixing plnts lcked the ility to compenste rpidly for loclized N limittion y up-regulting the N 2 -fixtion ctivity of roots supplied elsewhere with N. However they displyed long-term response vi growth stimultion of pre-existing nodules, nd the genertion of new nodules, likely through decresed ortion rte of erly nodultion events. Both these responses involve systemic signling. The ltter response is olished in the sunn mutnt, ut the muttion does not prevent the first response. Locl ut lso systemic regultory mechnisms relted to plnt N sttus regulte de novo nodule development in Mt, nd SUNN is required for this systemic regultion. By contrst, the stimultion of nodule growth triggered y systemic N signling does not involve SUNN, indicting SUNN-independent signling. Introduction Although soil nitrte content my, in some griculturl res, show lrge, temporry increses, possily s result of wter pollution (Vitousek et l., 1997), under nturl conditions minerl nitrogen (N) generlly limits plnt growth ecuse of sptil nd temporl fluctutions in its vilility in the soil. Nitrogen is cquired in vriety of forms y most plnt species. Among them, legumes cquire N indirectly from tmospheric N 2, through endosymiosis with N 2 -fixing cteri tht involves the formtion of specific symiotic orgn (nodules) on roots. Dinitrogen-fixing cteri export mmonium to host the plnt, while the plnt supplies cteri with photosynthesis-derived cron metolites (Kouchi & Yoneym, 1984). Nevertheless, even in legumes, ecuse of the high energy cost of N 2 fixtion (Voisin et l., 23) nd the high sensitivity of nodules to environmentl fctors (Sprent et l., 1988; Serrj et l., 1999; Slon et l., 21), N cquisition is often sptilly nd temporlly limited. NO 3 ) -fed plnts cn compenste for locl N limittion nd mintin their N sttus y stimulting NO 3 ) cquisition y roots exposed elsewhere to minerl N (Forde, 22). A short-term dpttion response is medited y rpid increse in oth the cpcity nd the ffinity of the uptke systems present in these roots. High-ffinity NO 3 ) trnsporters involved in these responses were identified in Aridopsis (Cerezo et l., 21; Filleur et l., 21). In the long term, systemic dpttion to N deficit my lso involve chnges in root rchitecture to increse soil explortion nd No clim to originl French government works Journl compiltion Ó New (29) 817

2 818 Reserch New root prolifertion in nitrte-rich zones (Zhng & Forde, 1998; Forde & Lorenzo, 21; Remns et l., 26). In the cse of N 2 -fixing plnts, the suppression of the cteril cpcity to fix N 2 induced y n Ar O 2 tretment is correlted not only to reduction of N 2 cquisition ut lso to locl inhiition of rhizoil nodule nd root growth (Singleton & vn Kessel, 1987). This effect hs een relted to decrese of the lloction of plnt photosynthtes towrd inefficient roots nd nodules. However, to our knowledge it is not known whether there is, in this cse, s in the cse of NO 3 ) s N source, whole-plnt compenstory response to locl N limittion involving long-distnce signling. In previous study we hve shown tht the three min pthwys involved in the cquisition of N (NH 4,NO 3 nd N 2 )inmedicgo trunctul (Mt) re regulted y the N sttus of the whole plnt (Ruffel et l., 28). The uptke ctivities of the three pthwys re repressed y systemic signls when high concentrtion of downstrem N metolites ccumultes in the plnt. Nevertheless, despite the common feture of these regultions, trnscriptomic studies hve shown tht the gene networks trgeted y these systemic signls re highly dependent upon the N source present in the root environment. Evidence of differentil responses of the pthwys of N cquisition to N limittion cme from short-term locl N strvtion. Among the three N sources, only NO 3 ) -fed plnts were found to e le to compenste rpidly for locl N limittion y incresing the N uptke of the roots exposed elsewhere to NO 3 ).By nlogy with the regultion of NO 3 ) cquisition in Aridopsis (Lejy et l., 1999; Gnsel et l., 21), this up-regultion ws explined t oth the physiologicl nd moleculr levels y the relese of systemic repression exerted y downstrem N metolites (Ruffel et l., 28). The sence of equivlent responses in NH 4 + -fed nd N 2 -fed plnts ws not the result of cron metolite limittion (Ruffel et l., 28) nd suggested tht the olition of systemic repression does not llow the pthwys involved in the cquisition of these sources to dpt rpidly to such constrints. Tken together these dt suggested tht the three cquisition pthwys hve contrsting ilities to medite rpid dpttion of the plnt to N limittion. However, ecuse only short-term responses to N limittion were investigted, the question of developmentl chnges tht my lso contriute to plnt dpttion ws not ddressed. Nodule formtion nd function require s prerequisite low minerl N supply, wheres the ddition of high concentrtions of nitrte or mmonium inhiits the ctivity of preexisting nodules (Streeter & Wong, 1988; Crroll & Mthews, 199; Ferguson & Mthesius, 23; Brulov et l., 27). Vrints mutnts of lflf nd Lotus jponicus which develop spontneous nodules in the sence of rhizoi disply similr nodule development responses to the mount of minerl N supply (Truchet et l., 1989; Tirichine et l., 26), indicting strong plnt sis for this regultion. Evidence suggesting tht control of nodule development y plnt N sttus my involve distinct pthwys involving nitrte itself nd products of its ssimiltion hs een reported (Crroll & Mthews, 199; Brulov et l., 27). A trnscriptomic pproch hs identified N- regulted trnscripts potentilly ssocited with the competence of L. jponicus plnts to perform the nodultion progrm (Omrne et l., 29). Supernodulting mutnts with incresed nodule numer were isolted in severl legume species, including Mt (review y Kinkem et l., 26; Ok- Kir & Kwguchi, 26). These mutnts re impired in the utoregultion of nodule numer (AON) which is the systemic feedck repression of nodultion y the pre-existing nodules (Kosslk & Bohlool, 1984; Cetno-Anollés & Gresshoff, 1991). Different complementtion groups my disply this phenotype, ut until now most of the muttions identified t the moleculr level elong to one group. The corresponding gene, isolted in vrious legume species, (L. jponicus HAR1, pe SYM29, soyen NARK, nd Mt SUNN), encodes LRR-protein kinse closely relted to the Aridopsis CLAVATA 1 protein (Krusell et l., 22; Nishimur et l., 22; Serle et l., 23; Schnel et l., 25). The current model suggests tht following the perception of cteril Nod fctor, root-derived signl is emitted to the shoots tht respond y trnslocting secondry signl to the roots vi the phloem, which ultimtely regultes nodule numer (Ok-Kir & Kwguchi, 26). The phloem signl is lcking in supernodulting mutnts (Crroll et l., 1985; Delves et l., 1992; Jing & Gresshoff, 22). Interestingly, the AON defect in the Mt sunn mutnts nd in other relted mutnts of soyen nd Lotus hs een shown to e ssocited with the ility of roots to form nodules in the presence of high concentrtions of minerl N, conditions where nodule formtion is inhiited in the wild-type, suggesting tht the AON pthwy shres common elements with the control of nodule development y whole-plnt N sttus (Kinkem et l., 26 nd references therein). Recently, Nod fctor nitrte-induced CLE genes identified in L. jponicus hve een proposed s cndidtes for the root-derived signl driving systemic regultion of nodultion upstrem of HAR1 (Okmoto et l., 29), thus giving further support to the hypothesis of cross-tlk etween the two pthwys (Gresshoff et l., 29). In this study, we hve chrcterized the long-term dpttion strtegy of Mt N 2 -fixing plnts to loclized suppression of N cquisition nd compred this with the strtegy of the NO 3 ) -fed plnt. To mimic locl N stress, hlf of the root smples were deprived of N 2 (Ar O 2 insted of ir) in split-root systems. Systemic effects of tretments were investigted on the other hlves of the root systems tht remined supplied with the N source (ir). The durtion of the tretment (up to 18 d) ws intended to revel ny significnt chnges in the process of functionl nodule formtion, in contrst to erlier short-term studies (Singleton & No clim to originl French government works Journl compiltion Ó New (29)

3 New Reserch 819 vn Kessel, 1987; Ruffel et l., 28). Plnt growth, root growth, nodule growth nd nitrogen fluxes were mesured, llowing us to distinguish etween the dynmics of the responses ffecting the structures involved in N cquisition (root, nodule) nd their N retrievl ctivities nd to chrcterize the functionl significnce of these responses with regrd to plnt nutrition. Trnsgenic plnts expressing the reporter gene pmtenod11-gus were used to investigte the effect of N limittion on erly nodultion events. Photossimilte lloction during N strvtion ws monitored y 13 C isotopic leling. The role of the AON pthwy in the dpttion of N 2 -fixing plnts to N deficit ws further evluted using the Mt sunn-2 mutnt. Mterils nd Methods Plnt growth conditions nd experimentl design Seeds of Mt genotype A17, hypernodulting sunn-2 mutnt (1 ck-cross; Schnel et l., 25), nd trnsgenic line L416 expressing the pmtenod11-gus construct (Chrron et l., 24) were scrified in H 2 SO 4 99% (8 min), rinsed with wter, plced on lotting pper in Petri dishes nd cold-treted t 5 C (12 h). Seeds were then germinted in the drk. After 4 d, once primry roots were c. 3 4 cm long, the root tips were cut, which promoted root rnching. As soon s plnts hd produced c. 1 secondry roots (3-wk-old plnts), they were plced in hydroponic culture tnks (Brker et l., 26). The plnts were rised in glsshouse with tempertures of 2 : 22 C (night : dy) nd photoperiod of 14 h, nd supplementry rtificil light ws provided when necessry to complement photosynthetic ctive rdition. Hydroponic culture tnks contined nutrient solution erted permnently with n qurium pump (Schemel & Goetz, Offench m Min, Germny). Plnts were supplied with nutrient solution contining minerl N (5 mm KNO 3, 1 mm KH 2 PO 4, 1 mm MgSO 4,.25 mm K 2 SO 4,.25 mm CCl 2, 3 lm H 3 BO 3, 1.6 lm MnSO 4,.7 lm ZnSO 4, 3.2 lm CuSO 4, 1 lm N 2MoO 4,.5 mm N-Fe-EDTA, ph 6.8). For experiments with nodulted plnts, roots of plnts t the eighth lef stge were inoculted nd grown for two dditionl weeks with the Sinorhizoium meliloti strin 211 in the sme nutrient solution without minerl N, renewed every week. Typiclly, nodules ppered t 4 6 d fter inocultion nd were fully functionl fter 2 wk. Nitrogen deprivtion tretments were chieved y removing minerl N from the nutrient solution in the cse of NO ) 3 -fed plnt, wheres in the cse of N 2 -fixing plnts, the ir ws replced y n Ar : O 2 (8 : 2) mixture. plnts were quntified with 13 CO 2 nd 15 N 2 (Wremourg & Roumet, 1989; Voisin et l., 23). Leling experiments were conducted in gs-proof chmer, llowing control of the shoot nd root tmospheres using Dsyl softwre (SM2i, Villiers St Frédéric, Frnce). The environmentl prmeters of this chmer were s follows: 8 : 16 h, light : drk cycle, 22 C, 6% hygrometry, 4 6 lmol photon m )2 s )1 (ottom nd top of cnopy, respectively) photosynthetic ctive rdition, nd 35 ll l )1 CO 2 concentrtion. Light ws provided y four 4 W metl hlide lmps (Mzd MAIH, APPRO5-2185, Sint Apollinire, Frnce) on ech side of the enclosure nd two 1 W high-pressure sodium vpor lmps (Osrm Nv-T, APPRO5-2185, Sint Apollinire, Frnce) ove it. Aeroponic split-root systems were inserted in the leling chmer 3 d efore experiments for cclimtion of plnts. Shoots were exposed to 13 C-enriched tmosphere (5% 13 C 12 C) for 7.5 h; the ir CO 2 concentrtion ws continuously mesured with n infrred gs nlyzer (Cirs, PP Systems, Montigny le Bretonneux, Frnce) nd mintined y utomtic CO 2 injection using mssic flow meters (Fluxmtec-936, Aulny sous Bois, Frnce). Symiotic N fixtion ws mesured y enriching the root tmosphere with 15 N 2 (5% 15 N 14 N) for 24 h. Isotopic compositions of gses in the leling chmer were controlled y mss spectrometry throughout the experiment. Plnts were hrvested t the end of the experiment, nd seprted into shoots, roots, nd nodules. Dry mtter ws determined fter oven drying t 8 C (48 h). 15 N nd 13 C content ws nlyzed using continuous-flow isotope rtio mss spectrometer (Sercon, Crewe, UK) coupled to C-N elementl nlyzer (Thermo Electron NC25, Courtoeuf, Frnce). The quntities of C (Q C )ndn(q N ) in shoot, roots or nodules cquired during the leling period were clculted s follows: Q C ¼ DM ð%c=1þ ðec orgn EC control Þ=EC source EC control Þ Q N ¼ DM ð%n=1þ ððen orgn EN control Þ=EN source EN control ÞÞ EC orgn nd EN orgn re the 13 C nd 15 N undnces, respectively, of the plnt orgn of the leled plnt or nonleled control plnt; EC source nd EN source re the 13 C nd 15 N enrichment, respectively, of the leled shoot nd root tmosphere; DM is dry mtter; nd %C nd %N re the percentges of C nd N in dry mtter (w w), respectively. Leling experiments The mounts of C entering the plnt through photosynthesis nd N eing reduced through symiosis on whole intct Anlysis of nodule nd root development nd growth To nlyze the response of nodule nd root growth to locl deprivtion of individul plnts, reltive increments of root No clim to originl French government works Journl compiltion Ó New (29)

4 82 Reserch New nd nodule iomss present in comprtments of the splitroot systems (Fig. 1) were estimted. Nitrogen limittion ws generted y removing the N source on one side of roots of split-root plnts (see Fig. 1). At the end of the tretment, nodules nd roots were collected nd their dry weights were determined. The initil nodule nd root iomss present efore the initition of the N-limittion tretment on the plnt were estimted nondestructively y imge nlysis: preliminry experiment (Supporting Informtion, Fig. S1) ws conducted on nodulted plnts similr to those used t the initition of the tretments to estlish the reltionships etween vlues estimted from imge nlysis (root nodule surfce, pprent nodule numer) nd vlues mesured y destructive methods (root dry weight, nodule dry weight, nodule numer). Imges of roots were otined using n Epson GT 15 scnner (Seiko-Epson Corportion, Nogno, Jpn) nd nlyzed (ARCGIS 9 softwre; ESRI, Redlnds, CA, USA) to determine root nodule surfce nd the numer of visile nodules. To detect erly nodultion events, trnsgenic L416 roots were stined for -glucuronidse (GUS) ctivity s in Lgrde et l.(1996). Nodultion events were scored on high-resolution imges nd ssigned to three clsses ccording to their developmentl stge (1, root infection sites, including those ssocited with nodule primordium; 2, young nodules ssocited with visile root outgrowth; 3, well-protruding N 2 N 2 N 2 -N Control N-lilimited Fig. 1 Description of the split-root system used to investigte the effect of long-term locl N 2 limittion on nitrogen (N) cquisition. Aeroponiclly grown Medicgo trunctul plnts fixing N 2 (nodulted in the presence of Rhizoium meliloti strin 211) were sujected to two contrsting N regimes. In control plnts, oth sides of the root system (C roots, drk gry) were supplied with nutrient solution erted with ir s the sole N source. In N-limited plnts, the N source ws removed from one side of the root system ()N roots, medium gry) y suppressing N 2 from the root tmosphere (replcement of norml ir y 8% : 2% Argon : O 2 mixture), the other side (L roots, light gry) eing mintined in the sme conditions s the control. Therefore, lthough C nd L roots elong to control nd N-limited plnts, respectively, they were continuously directly exposed to the sme locl environment during the tretment. A similr system ws used to investigte the effect of locl nitrte limittion (see Fig. 2). nodules with sphericl or elongted shpe (see Figs S2, 2e,f in Journet et l., 21). Sttistics Sttisticl nlyses were performed using the GLM procedure of SAS (SAS Institute, 1987). Mens were compred using the Student Newmn Keuls test t the.5 proility level. Results N 2 -fixing plnts disply long-term compenstory responses tht enhnce N 2 cquisition in response to N limittion To detect long-term dptive responses tht my involve chnges in root nd or nodule development, experiments were conducted with either NO ) 3 -fed or N 2 -fixing plnts up to 14 d of N limittion. The systemic responses to N limittion were investigted y compring the untreted roots of N-limited plnts tht remined supplied with N, with the roots of control plnts homogeneously supplied with N during the tretment (Figs 1,2). In NO ) 3 -fed plnts, removing the N source from hlf of the root system did not result in ny overll growth reduction fter 14 d (Fig. 2), with little impct on plnt totl N concentrtion (Fig. 2c). This indictes tht the untreted roots efficiently incresed their NO ) 3 uptke, fully compensting for the suppression of NO ) 3 cquisition y treted roots. This dptive response to compenste for the N deficit is explined y oth the rpid up-regultion of NO ) 3 trnsport systems (Ruffel et l., 28) nd specific stimultion of growth of the untreted roots s compred with oth treted roots of N-limited plnts nd roots of control plnts (Fig. 2d). Conversely, growth nd totl N concentrtion (Fig. 3,) of N-limited plnts, were reduced y 2% s compred with the N 2 -fixing control plnts. Tking into ccount the fct tht control plnts cquired c. 5% of their iomss nd N content during the lst 14 d, the N limittion of hlf of the root system resulted in c. 4 5% reduction of growth nd N cquisition. This grees with, nd extends, our previous results showing lck of up-regultion of N 2 fixtion fter 4 d of tretment (Ruffel et l., 28). However, mesurements of N intke t the end of the 14-d period showed tht the untreted roots of N-limited plnts displyed 7% increse in net 15 N 2 cquisition s compred with their counterprts in control plnts (Fig. 3c). This showed tht n up-regultion of N cquisition y the plnt lso occurred in untreted roots of N 2 -fixing, N-limited plnts, proly with mrked dely nd not s efficiently s for NO ) 3 -fed plnts, to mintin plnt growth under locl N limittion. Becuse this response ws No clim to originl French government works Journl compiltion Ó New (29)

5 New Reserch 821 () 4 (d) ()??? NO 3 NO 3 NO 3?N -N Control N-limited Plnt iomss (mg DW per plnt) (c) N content (mmol N g 1 DW) Root iomss per comprtment (mg DW) c Control + N -N N-limited Control N-Limited Fig. 2 Adpttion of NO 3 ) -fed plnts to locl nitrogen (N) limittion. Nonnodulted Medicgo trunctul plnts (fourth to fifth lef stge) grown hydroponiclly in nutrient solution contining 1 mm NO 3 ) s the N source were trnsferred for 14 d in split-root systems shown in () (N limittion ws initited y supplying the )N comprtment of the N-limited plnt with N -free medium). The whole plnt iomss (), plnt N content (c), nd iomss of the vrious components of split-root systems (d) were mesured in control (lck rs) nd N-limited plnts (medium gry light gry rs) t the end of the experiment. Vlues re the mens of five to 1 replictes. Letters denote significnt differences y pired t-test, P <.5. Verticl rs indicte SE. oserved in the untreted roots of N-limited plnts, we concluded tht it ws the result of systemic signling exerted y the plnt N sttus. The systemic response of N 2 -fixing plnts to N 2 limittion specificlly involves nodule growth nd development Specific N 2 -fixtion ctivity ws clculted s the rtio of 15 N 2 ssimiltion per unit of nodule iomss. Specific N 2 - fixtion ctivity ws similr for roots of control plnts (C roots in Fig. 1, 131 ± 11 lmol N g )1 nodules h )1 ) nd untreted roots of N-limited plnts (L roots in Fig. 1, 15 ± 7.5 lmol N g )1 nodules h )1 ) exposed to the sme locl environment during the tretment. Hence specific N 2 - fixtion ctivity ws not instrumentl in the up-regultion of N, N cquisition y the plnt oserved fter 14 d of loclized N strvtion (Fig. 3c). Indeed, this response resulted entirely from strong stimultion of nodule growth s compred with control plnts oserved fter only 11 d of tretment (Fig. 4). In the untreted roots of N-limited plnts, nodule growth ccelerted mrkedly, while it lmost cesed in the Ar O 2 - treted roots. This indictes tht the N-limittion tretment triggered oth locl nd systemic signls, modulting nodule development to fvor N cquisition y the untreted side of the root system, still supplying plnts with N 2 fixtion products; lthough, to lesser extent, similr response ws lso oserved on roots, suggesting tht locl nd systemic signls re lso regulting root development (Fig. 4). The stimultion of nodule growth in the untreted root side of N-limited plnts my hve resulted from the enlrgement of lredy developed nodules nd or the initition nd development of new nodules. Well-protruding nodules (clss 3; Mterils nd Methods) were scored in ll plnts to determine whether ny chnge in numers occurred in response to N limittion (Fig. 5). After 11 d of N limittion, little chnge in nodule numer ws oserved in oth sides of the root system of control nd N-limited plnts (Fig. 5). Consequently, s men nodule weight in oth control plnts nd untreted roots of N-limited plnts incresed (Fig. 5), the increment of nodule iomss during this period resulted lmost exclusively from the enlrgement of pre-existing nodules. However, fter 18 d of locl N strvtion, significnt increse (+ 2%) in nodule numer ws oserved on the untreted roots of the N-limited plnts s compred with their treted roots nd roots of control plnts (Fig. 5). This suggests tht prolonged N limittion period stimulted nodule formtion specificlly in roots of N-limited plnts tht remined exposed to N 2. Hence the overll dptive response of nodule growth to N limittion in Mt (Fig. 4) my e the sum of two successive processes: growth stimultion of pre-existing nodules followed y n incresed genertion of new nodules. Nodule formtion includes successive phses where suset of the erly symiotic structures my e rrested in their development. Therefore, the lte stimultion of No clim to originl French government works Journl compiltion Ó New (29)

6 822 Reserch New () Dry weight per plnt (mg per plnt) () Reltive increment of nodule iomss (% of estimted DW t the eginning of N restriction) () c cd e d e N content (µmol N g 1 plnt DW) (c) Net N intke (µmol N g 1 roots nd nodules DW h 1 ) Control nodule formtion in response to N limittion (Fig. 5) might e the result of either n incresed initition rte of infection nodultion events or decrese in the ortion rte of such erly events. To distinguish etween these two hypotheses, we used trnsgenic Mr plnts expressing the - glucuronidse (GUS) reporter gene under the control of the promoter of the erly nodulin MtENOD11 gene (Journet et l., 21; Chrron et l., 24) to score erly nodultion events (root infection sites possily ssocited with underlying nodule primordi; clss 1; Mterils nd Methods). The N limited Fig. 3 Long-term effect of locl nitrogen (N) limittion on the N cquisition of N 2 -fixing plnts. Dry weights () nd plnt N concentrtion () of control nd N-limited Medicgo trunctul plnts were mesured during the leling experiment in eroponiclly grown plnts fter 14 d in split-root systems descried in Fig. 1. Nitrogen intkes of control nd N-limited plnts t the end of the 14-d period were mesured using 15 N leling on C nd L roots (c). Vlues for control (lck rs) nd N-limited (gry rs) plnts. Vlues re the mens of 1 replictes. Letters denote significnt differences y pired t-test, P <.5. Verticl rs indicte SE. Reltive increment of root iomss (% of estimted DW t the ginning of N restriction) () c frequency of erly events detected y this method ws expressed s rtio of the totl numer of infection sites nd nodules present. Four dys fter the initition of the tretment, the reltive numers of erly nodultion events were similr in roots of control plnts nd N-limited plnts (Fig. 6). After 11 d, lot fewer erly nodultion events were scored on the treted side of N-limited plnts s compred with the other roots, showing tht the tretment itself my loclly ffect nodule initition. Although the reltive numer of erly nodultion events t dy 11 ws similr for the untreted roots of N- limited plnts nd the roots of control plnts(fig. 6), these roots displyed mrkedly different rtes of nodule formtion etween dys 11 nd 18 (Fig. 5). Thus, nothing indictes tht the higher frequency of well-protruding nodules oserved in the untreted roots of N-limited plnts t dy 18 resulted from enhnced nodule initition 1 wk erlier. Insted, this would support the hypothesis tht reduction of ortion rte of erly nodultion events is responsile for this developmentl response. c Fig. 4 Response of nodule nd root dry weights to long term locl nitrogen (N)-limittion. For nlysis of nodule nd root development nd growth, reltive increment of nodule dry weight () nd roots dry weight () of the C, L, )N roots of the Medicgo trunctul plnts grown in hydroponics (descried in Fig. 1) were mesured fter 4 d, 11 d nd 18 d. of N limittion. Color legends for the rs re similr to those in Fig. 1. Vlues re the mens of 1 replictes. Letters denote significnt differences y pired t-test, P <.5. Verticl rs indicte SE. c No clim to originl French government works Journl compiltion Ó New (29)

7 New Reserch 823 Reltive increment of nodule numer (% of estimted numer t the eginning of N restriction of weight pe er nodule ed weight t the f N restriction n) Reltiv ve increment (% of estimte eginning of 3 () () c cd e e Dys fter N restriction Fig. 5 Response of nodule numer nd men nodule weight to long-term locl nitrogen (N) limittion. Reltive increment of nodule numer () nd men nodule weight () of the C, L, nd )N roots were mesured fter 4, 11 nd 18 d of N limittion. Medicgo trunctul plnts were cultivted in the experimentl hydroponic system descried in Fig. 1. Only well-protruding nodules (clss 3; see the Mterils nd Methods section) were considered. Vlues re the mens of 1 replictes. Letters denote significnt differences y pired t-test, P <.5. Verticl rs indicte SE. Chnge in C ssimilte prtitioning is n erly response to locl N strvtion The response of nodule growth oserved fter 11 d in N- limited plnts, with strong stimultion on the untreted side nd complete rrest on the treted side (Fig. 4), suggested tht mjor chnge in C ssimilte prtitioning occurred in these plnts. To detect erly chnges in C flow triggered y locl N limittion, oth control nd N-limited plnts were exposed for 1 d to 13 CO 2 tmosphere 4 d fter the eginning of the N-limittion tretment. The orgn sink strength ws evluted y the rtio of 13 C llocted to the orgn y its iomss. Interestingly, significnt chnge in C lloction within the plnt ws reveled erly fter the eginning of the N-limittion tretment (Fig. 7). In oth control nd N-limited plnts, nodules were the plnt orgn displying the highest sink strength. Nodule sink strength ws highest in the untreted roots of N-limited plnts d numer totl nodule n % of t Dys fter N restriction Fig. 6 Erly nodultion events detected using MtENOD11-GUS plnts. The reltive frequency of erly nodule initition on the roots of trnsgenic plnts ws determined following -glucuronidse (GUS) stining fter 4 nd 11 d of N limittion. Plnts were cultivted in the experimentl hydroponic system descried in Fig. 1. Trnsgenic C, L, nd )N roots were compred. Reltive frequency vlues were clculted s the occurrence rtio of clss 1 structures to totl symiotic structures present (clsses 1 3; see the Mterils & Methods section). Color legends for rs re similr to those in Fig. 1. Vlues re the mens of 1 replictes. Letters denote significnt differences y pired t-test with n = 1 experiment, P <.5. Verticl rs indicte SE. (Fig. 7), indicting tht suppressing N 2 fixtion loclly for 4 d enefited the roots still le to fix N 2 for photosynthte lloction. This chnge in C lloction occurred efore ny significnt chnge in nodule development nd or ctivity ws detected. % (µmol C h 1 per plnt) / g orgn DW 1.6 c Leves Roots Nodules Fig. 7 Cron lloction to plnt prts fter 4-d locl N 2 -limittion. Leves, roots nd nodules sink strength were estimted s the percentge of the cron ssimilted during the 13 C leling experiment y the plnt tht ws cquired y these orgns (leves, roots nd nodules) divided y their dry mss. Color legends for rs re similr to those in Fig. 1. Vlues re the mens of 1 replictes. Letters denote significnt differences y pired t-test, P <.5. Verticl rs indicte SE. No clim to originl French government works Journl compiltion Ó New (29)

8 824 Reserch New WT NH4NO3 1 mm -N KNO3.5 mm 14 d fter inocultion The sunn-2 mutnt is impired in the systemic control of nodule formtion y plnt N sttus As the increse of nodule iomss in the plnt response to N limittion is derived in prt from stimultion of nodule formtion y whole-plnt N deficit (Fig. 5), the question of the control of AON pthwy y the N sttus of the plnt ws investigted. Split-root experiments (Fig. 8) were designed to compre the ility of roots elonging to either N-sufficient or N-limited wild-type nd sunn-2 mutnt plnts to form nodules in the sence of minerl N using n experimentl system similr to tht in Ruffel et l. (28). Both types of genotypes hd hlf of their root system trnsferred to N-free nutrient solution ()N roots), nd the other hlf supplied with minerl N (+N roots): high N (1 mm NH 4 NO 3 ) for N-sufficient plnts nd low N (.5 mm NO 3 ) ) for N-limited plnts. A mixed N source ws chosen for the high N tretment to ensure tht whole plnts were fully N-sufficient, s NH 4 NO 3 is more efficient N source thn NO 3 ) or NH 4 + lone (Krouk et l., 26 nd references therein). In wild-type Mt plnts, high locl N provision in N-sufficient plnts triggered strong systemic inhiition of nodule formtion in oth +N nd )N roots (Fig. 8, Tle S1), demonstrting tht nodule formtion ws drmticlly feedck-regulted y systemic signls relted to the wholeplnt N sttus. However, nodule formtion ws clerly more strongly inhiited in +N roots (no visile nodules) thn in )N roots (few smll nd white nodules, Fix - phenotype), confirming tht locl inhiitory effect of NH 4 NO 3 1 mm on nodule formtion lso exists. Comprison of the size nd the color of the nodules present in the )N roots of N-sufficient nd N-limited plnts suggested tht plnt N sttus inhiited not only nodule formtion ut lso nodule function. Interestingly, the inhiition of nodule formtion y plnt N sttus ws no longer oserved in the sunn-2 mutnt, which hd mny visile nodules in oth the +N nd )N roots of either N-limited or N-sufficient plnts (Fig. 8, Tle S1). The SUNN pthwy ws indeed strongly involved in the systemic control of nodule formtion y the N sttus of the whole plnt. Nevertheless, creful comprison of nodules on the )N roots of N-sufficient plnts nd N-limited plnts indicted tht N sttus of the plnt lso hd strong effect on the size nd the color (smll nd white nodules in N-sufficient plnts vs lrge pink nodules nd N-sufficient plnts) of the sunn-2 nodules. Thus other mechnisms, regulting nodule development nd function in reltion to the plnt N sttus, ut independent of SUNN, remined efficient in the mutnt. sunn Fig. 8 Locl nd systemic effect of minerl nitrogen (N) on nodultion in wild-type (WT) nd sunn-2 mutnt plnts. Hydroponiclly grown Medicgo trunctul plnts fed with 1 mm NO 3 ) (sixth lef stge) were sujected to two contrsting nitrogen (N) regimes in split-root systems. Tretments consisted of mintining hlf of the root system of these plnts in nutrient solution contining either high concentrtion (1 mm NH 4 NO 3, N-sufficient plnts) or low concentrtion (.5 mm NO 3 ), N-limited plnts) of minerl N, the other roots eing exposed in oth cses to minerl N-free nutrient solution. After 4 d, roots of N-sufficient nd N-limited plnts were then exposed to Sinorhizoium meliloti 211. Root nodule morphology ws oserved 14 d fter inocultion. The sunn-2 mutnt is le to respond to N deficiency y rpid increse in N 2 fixtion We hve shown previously (Ruffel et l., 28) tht nodule N 2 fixtion-specific ctivity is under the control of systemic repressive signl relted to the N sttus of the plnt. Nevertheless, ttempts to relieve this systemic repression y preventing N 2 fixtion on one-hlf of the split-root system (Ar O 2 tretment) filed to up-regulte N 2 fixtion-specific ctivity in the other hlf of the root system, oth in shortterm (4 d; Ruffel et l., 28) nd long-term (14 d) experiments. One hypothesis to explin this prdox ws tht nodule N 2 fixtion-specific ctivity ws lredy fully de-repressed nd t its mximum cpcity in control plnts, nd thus could not e further incresed y the N-limittion tretment. Interestingly, the sunn-2 mutnt offered the opportunity to directly test this hypothesis. Indeed, No clim to originl French government works Journl compiltion Ó New (29)

9 New Reserch 825 lthough hypernodulting mutnts form mny more nodules thn wild-type plnts, this is generlly not ssocited with incresed N 2 cquisition, ecuse of lower specific N 2 - fixtion ctivity in these mutnts thn in the wild-type (Crroll et l., 1985; Schuller et l., 1988). Thus, it is possile tht N 2 -fixtion ctivity is not t its mximum in the sunn- 2 mutnt, nd therefore could respond to the N-limittion tretment. To test this prediction, sunn-2 nd wild-type N 2 -fixing plnts were sujected to N limittion for 4 d y tretment with Ar O 2 exposure on hlf of their root system, while the other hlf ws flushed with mixture of 15 N 2 O 2, in n experiment similr to the one descried in Fig. 1. As expected, the specific N 2 -fixtion ctivity of sunn-2 nodules ws much lower thn tht of the wild-type in control conditions (Fig. 9). After 4 d of N limittion, oth specific N 2 - fixtion ctivity nd totl N intke in the untreted side of the root system remined unchnged in wild-type plnts (Fig. 9,). However, in the sme conditions (Fig. 9), the sunn-2 genotype incresed specific N 2 -fixtion ctivity in the untreted roots (+ 6%) nd incresed whole-plnt totl Nodule N fixtion ctivity (µmol N g 1 nodules DW h 1 ) Net N intke (µmol N g 1 roots nd nodules DW h 1 ) () () c c A17 Sunn Genotype Fig. 9 Comprison of the short-term responses of N 2 -fixing plnts to locl nitrogen (N) limittion in wild-type (WT) nd sunn-2 mutnt plnts. Split-root systems were used to compre the responses to N limittion of wild-type A17 nd the hypernodulting sunn mutnt of Medicgo trunctul. For oth genotypes, hlf of the root system of the nodulted plnt ws sujected to 4 d Ar O 2 tretment while the other hlf ws exposed to ir in n experiment similr to tht descried in Fig. 1. Nitrogen (N) intkes of plnts t the end of the 4 d period were mesured using 15 N leling. The nodule-specific ctivities () nd N intke () of control nd N-limited plnts were determined in oth the wild-type nd the SUNN mutnt. Vlues re the mens of 1 replictes. Letters denote significnt differences y pired t-test, P <.5. Verticl rs indicte SE. N cquisition (+ 3%). These results indicted tht sunn-2 mutnt plnts trigger quicker response of N 2 fixtion thn the wild-type to the vritions of the whole-plnt N sttus, ecuse they re le to de-repress specific N 2 -fixtion ctivity, while wild-type plnts cnnot. As consequence, this supports the hypothesis tht specific N 2 -fixtion ctivity ws indeed lredy fully de-repressed in control wild-type plnts. This lso shows tht the systemic N signling pthwy controlling nodule N 2 fixtion-specific ctivity is ctive in the sunn-2 mutnt, nd therefore tht this pthwy is independent of the SUNN gene. Discussion The ility of NO 3 ) -fed plnts to rect to N deprivtion of one portion of its root system y incresing the NO 3 ) uptke of the other roots hs een documented in severl species (Burns, 1991; Liné et l., 1995). It involves the upregultion of NO 3 ) uptke systems (Cerezo et l., 21; Gnsel et l., 21) nd the stimultion of root growth (Drew, 1975; Roinson, 1994; Zhng & Forde, 1998; Zhng et l., 1999). Following our previous report concerning NO 3 ) uptke systems (Ruffel et l., 28), this work showed tht this generl scheme holds true for M. trunctul. When NO 3 ) is the N source, the suppression of N cquisition in hlf of the roots is efficiently compensted y the other hlf, nd results in unltered growth of the whole plnt. Equivlent dpttion ws not found in N 2 -fed legume plnts since the locl suppression of N 2 fixtion in split-root Mt plnts did not result, fter either 4 d (Ruffel et l., 28) or 14 d (this study), in ny increse in specific N 2 -fixtion ctivity in the untreted roots. Nevertheless, in the long term, N 2 -fixing plnts compensted prtilly for the detrimentl effect of locl N limittion y stopping nodule growth in treted roots nd y stimulting nodule development in untreted roots. Locl reduction of nodule growth nd rhizoi prolifertion in response to Ar O2 tretment hs een reported in soyen, nd hs een interpreted s snction mechnism exerted y the plnt on n inefficient symiotic prtner (Singleton & vn Kessel, 1987; Kiers et l., 23). In this study we showed tht this locl response ws ssocited with systemic stimultion of nodule development in roots of N-limited plnts still exposed to N 2. However, the dely required for the full ctivtion of new nodules genertes temporry N deficit, nd plnt growth restriction. In the untreted roots of N-limited plnts, the responses of nodule development to systemic N signling involved two different nd successive processes. First, the men nodule weight of pre-existing nodules incresed. Second, new nodules were generted. We used trnsgenic MtENOD11- GUS plnts to investigte more precisely the effect of such N limittion on erly nodule development. The increse in nodule numer oserved fter 18 d ws not ssocited with No clim to originl French government works Journl compiltion Ó New (29)

10 826 Reserch New previous stimultion of de novo erly nodultion events, suggesting tht other steps, downstrem of root infection nodule initition, re likely to e trgeted y N signling. Therefore, the locl nd systemic effects induced y the sme Ar O 2 tretment re likely to regulte different steps of nodule development, suggesting tht different pthwys re involved in these two responses. The mechnisms involved in the regultion of nodule development s function of the N sttus of the whole plnt remin to e chrcterized. In our study, incresed cron lloction towrds efficient nodules efore ny growth response ecme oservle ppered s fst response to locl N limittion in N 2 -fixing plnts. These dt re consistent with previous studies in soyen, indicting tht Ar O 2 tretments triggered reduction of C lloction towrds inefficient nodules (Singleton & vn Kessel, 1987). Although nodules constitute reltively smll comprtment of the plnt in terms of iomss, they disply one of the highest sink strengths for cron. The vilility of cron metolites nd their utiliztion s key fctor governing nodule growth nd ctivity is very populr hypothesis (review y Vnce & Heichel, 1991 nd references therein). Our dt provide some support for this hypothesis, suggesting tht developmentl responses to N limittion my e triggered y modified cron lloction within the plnt. However, whether the stimulted cron lloction to nodules is prt of the signling mechnisms involved in the developmentl responses, or is n erly consequence of the initition of these responses remins unknown. To dte, AON involving the SUNN gene in Mt (Schnel et l., 25) is the unique signling mechnism identified s responsile for systemic control of nodultion. AON nd N signling my e connected ecuse the inhiition of nodule formtion y high minerl N concentrtion is suppressed in hypernodulting mutnts (Kinkem et l., 26). This repression opertes t erly stges of nodultion (Mlik et l., 1987; Heidstr et l., 1994), nd some of the erly steps of the Nod fctor signling cscde re locked y high NO 3 ) (Heidstr et l., 1997). Whether the AON pthwy is involved in direct inhiition of nodultion y minerl N or y orgnic N metolites is still mtter of dete (Cetno-Anolles & Gresshoff, 199; George & Roert, 1991; Kuppusmy et l., 24; Tirichine et l., 26). Differentil inhiitory effects of NO 3 ) nd mmonium on nodultion in L. jponicus (Brulov et l., 27) suggested tht NO 3 ) nd NH 4 + my ct on different regultory mechnisms. The involvement of AON in the regultion of nodule development y the N sttus of the plnt hs een poorly investigted, especilly in Mt. In this pper, the effect of locl high minerl N provision on distnt nodule formtion nd growth ws demonstrted, indicting tht not only locl ut lso systemic regultory mechnisms relted to the plnt N sttus re regulting nodule development in Mt. This oservtion does not confirm the sttement of Cho & Hrper (1991) tht, in soyen, the site of N ppliction primrily controls the site of nodultion inhiition. However, creful nlysis of Cho & Hrper s dt revels tht systemic effect of N ppliction ws lso present in the soyen experiment ut ws not discussed in the pper; locl ppliction of 5 mm NO 3 ) to one side of split-root system did reduce twofold the numer of nodules present on the untreted roots t the other side (Tle S1, cf. lines 1 nd 3). The role of SUNN in nodule development responses to systemic repression triggered y high N supply ws investigted. Although Brulov et l. (27) recently hypothesized tht inhiition of erly steps of nodultion y high concentrtions of minerl N in L. jponicus might e medited y HAR1-dependent nd independent pthwys, they were not le to seprte locl nd systemic mechnisms nd did not ddress the question of nodule growth, since L. jponicus nodules re determinte. The outcome of our Medicgo experiments is tht the nodule growth response to systemic signling of plnt N sttus involves oth SUNNdependent nd SUNN-independent processes. The SUNN pthwy is clerly required for the systemic repression of nodule formtion in response to high N supply. However, the stimultion of nodule growth triggered y systemic N signling does not involve SUNN, s the increse of nodule size in the untreted roots of N-limited plnts ws mintined in the mutnt. We lso showed tht the systemic regultion of the N 2 fixtion-specific ctivity y the N sttus of the plnt is independent of SUNN, since the sunn-2 mutnt ws le to up-regulte this ctivity in response to loclized N strvtion. The sunn-2 mutnt remined le to sense its N sttus nd to trigger severl dptive responses to N limittion, such s increse in nodule growth nd stimultion of N 2 -fixtion ctivity. A low specific N 2 -fixtion ctivity hs een recorded in hypernodulting mutnts (Schuller et l., 1988). In this pper we found tht, in sunn-2 plnts, this ctivity my e up-regulted in response to systemic signling triggered y locl N limittion. This oservtion suggests tht the low specific N 2 -fixtion ctivity in sunn results from repression y systemic N signling, which compenstes for the de-regulted nodule prolifertion. Therefore, the sence of upregultion of this ctivity in control wild-type plnts in response to N limittion is likely to e ecuse it hs reched its mximum cpcity rther thn ecuse of lck of control of this ctivity y plnt N sttus. Why nodule N 2 -fixtion cpcity would e so tightly limited in wildtype plnts remins to e determined. The flux of cron metolites delivered to the nodules hs een determined to e limiting fctor for N 2 fixtion (Bcnmwo & Hrper, 1997; Slon et l., 21). However, other fctors hve to e involved, s stimulted cron lloction to untreted roots of N-limited plnts is No clim to originl French government works Journl compiltion Ó New (29)

11 New Reserch 827 not ssocited with n increse in nodule-specific N 2 -fixtion ctivity. Interestingly, previous studies hve pointed out tht M. trunctul A17 plnts inoculted with the clssicl Sm211 generlly used y the scientific community re N-stressed when symiotic N 2 fixtion is the min N source (Moreu et l., 28), nd tht severl other Rhizoium strins my promote higher fixtion ctivity when ssocited with Medicgo (Mhdhi et l., 25). Whether plnts ssocited with these strins disply the sme or different responses s when ssocited with Sm211 deserves to e investigted. This my help to clrify the role of the microsymiont in the modultion of N 2 fixtion in response to N limittion. Acknowledgements We would like to thnk Richrd Thompson for criticl nd constructive reding of the mnuscript. This work ws supported y the Sixth Frmework Progrmme Grin Legume Integrted Project of the Europen Union (postdoctorl grnts to SF nd SR), y the AgroBI incittive ction of INRA. References Bcnmwo M, Hrper JE Response of hypernodulting soyen mutnt to incresed photosynthte supply. Plnt Science 124: Brulov A, Rogto A, d Apuzzo E, Omrne S, Chiurzzi M. 27. Differentil effects of comined N sources on erly steps of the nod fctor dependent trnsduction pthwy in Lotus jponicus. Moleculr Plnt-Microe Interctions 2: Brker DG, Pfff T, Moreu D, Groves ED., Ruffel S, Lepetit M, Whitehnd S, Millet F, Nir RM, Journet E-P. 26. Growing M. trunctul: choice of sustrtes nd growth conditions. In: Mthesius U, Journet EP, Sumner LW, eds. The Medicgo trunctul hndook. ISBN Burns IG Short-term nd long-term effects of chnge in the sptil-distriution of nitrte in the root zone on N uptke, growth nd root development of young lettuce plnts. Plnt, Cell & Environment 14: Cetno-Anolles G, Gresshoff PM Erly induction of feedck regultory responses governing nodultion in soyen. 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