Ethylene-dependent ethylene-independent ABA regulation of tomato plants colonized by arbuscular mycorrhiza fungi

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1 Reserh Ethylene-dependent ethylene-independent ABA regultion of tomto plnts olonized y rusulr myorrhiz fungi José Ángel Mrtín-Rodríguez 1, Rfel León-Morillo 1, Horst Vierheilig 1, Jun Antonio Ompo 1, Jutt Ludwig- Müller 2 nd José Mnuel Grí-Grrido 1 1 Deprtment of Soil Miroiology nd Symioti Systems, Estión Experimentl del Zidín (EEZ), CSIC, Clle Profesor Alred no1, 188 Grnd, Spin, 2 Institut für Botnik, Tehnishe Universität Dresden, Zellesher Weg 2, 162 Dresden, Germny Summry Author for orrespondene: José Mnuel Grí Grrido Tel: (ext ) Emil: josemnuel.gri@eez.si.es Reeived: 7 Otoer 21 Aepted: 22 Novemer 21 New (211) 19: doi: /j x Key words: sisi id, rusulr myorrhiz, ethylene, tomto (Solnum lyopersium). We investigted the reltionship etween ABA nd ethylene regulting the formtion of the rusulr myorrhiz (AM) symiosis in tomto (Solnum lyopersium) plnts nd tried to define the speifi roles plyed y eh of these phytohormones in the myorrhiztion proess. We nlysed the impt of ABA iosynthesis inhiition on myorrhiztion y Glomus intrrdies in trnsgeni tomto plnts with n ltered ethylene pthwy. We lso studied the effets on myorrhiztion in sitiens plnts treted with the minoethoxyvinyl glyine hydrohloride (AVG) ethylene iosynthesis inhiitor nd supplemented with ABA. In ddition, the expression of plnt nd fungl genes involved in the myorrhiztion proess ws studied. ABA iosynthesis inhiition qulittively ltered the prmeters of myorrhiztion in ordne with the plnt s ethylene pereption nd ethylene iosynthesis ilities. Inhiition of ABA iosynthesis in wild-type plnts negtively ffeted ll the myorrhiztion prmeters studied, while tomto mutnts impired in ethylene synthesis only showed redued rusulr undne in myorrhizl roots. Inhiition of ethylene synthesis in ABA-defiient sitiens plnts inresed the intensity of myorrhiz development, while ABA pplition resued rusule undne in the root s myorrhizl zones. The results of our study show n ntgonisti intertion etween ABA nd ethylene, nd different roles of eh of the two hormones during AM formtion. This suggests tht dul ethylene-dependent ethylene-independent mehnism is involved in ABA regultion of AM formtion. Introdution The key to understnding the phenomenon of plnt fungus omptiility in rusulr myorrhiz (AM) intertion is to nlyse the reognition mehnisms nd moleules involved in this intertion. In this regrd, there hve een signifint nd relevnt disoveries in reltion to the signls ssoited with the development of AM symiosis. In this respet, group of exudte ompounds (strigoltones) from the plnt root hs reently een identified nd hrterized s hyphl rnhing induers of AM fungi (Akiym et l., 25). Moreover, symioti plnt signl-trnsdution sde is lso tivted in plnt roots fter oth rhizoi nd AM fungi re reognized y the plnt root, nd some genes nd gene produts hve een identified s neessry for fungl penetrtion nd rusule formtion in roots (Prniske, 24, 28). Despite reent progress mde in signlling events, knowledge of the proesses required for AM formtion is still very limited, prtiulrly in reltion to the iohemil nd morphogeneti events tht tke ple fter the fungus enters the plnt root. The estlishment of n AM symioti intertion is suessful strtegy to improve the nutritionl sttus of oth plnt nd fungus. Fungl penetrtion nd estlishment in the host roots involve omplex sequene of events nd intrellulr modifitions (Bonfnte-Fsolo & Ó 211 The Authors New Ó 211 New Trust New (211) 19:

2 194 Reserh New Perotto, 1992; Genre et l., 28; Genre & Bonfnte, 21), leding to n interhnge of minerl nutrient uptke y the fungus in exhnge for plnt rohydrtes. Numerous studies hve supported the hypothesis tht fine lne etween hormones nd nutrient vilility (phosphorus, ron nd nitrogen) is proly importnt for the regultion of myorrhizl formtion nd funtioning (Ludwig-Müller, 2). More reently, myorrhiztion experiments on trnsgeni nd mutnt plnts, ltered in their hormonl iosynthesis nd or pereption, hve shown tht plnt hormones ply n importnt role in the estlishment of funtionl AM symiosis (Isyenkov et l., 25; Herrer- Medin et l., 27). Additionlly, the use of DNA rry methodologies hs dvned our knowledge of ertin plnt hormonl pthwys, suh s gierellins nd ABA, tivted during myorrhiztion (Fiorilli et l., 29; Grí-Grrido et l., 21). Thus, vritions in the myorrhiztion hrteristis of wild-type nd ABA-defiient tomto roots re ompnied y speifi trnsriptomi ltertions ssoited with differenes in myorrhiztion sttus ording to the ABA ontent in the roots (Grí-Grrido et l., 21). Anlysis of AM oloniztion in the sitiens ABA-defiient tomto mutnt hs shown tht ABA is neessry in order to omplete the rusule formtion proess nd its funtionlity nd to promote sustined oloniztion of the plnt root (Herrer-Medin et l., 27). Comprtive nlysis of two ABA-defiient tomto mutnts showed oth quntittive nd qulittive differenes in the pttern of AM oloniztion. The highly limited fungl oloniztion of sitiens mutnts in terms of myorrhizl intensity nd rusule formtion losely orrelted with their inpity in ABA iosynthesis. Notilis plnts, whose ABA defiieny in roots ws less ffeted, showed lower myorrhizl intensity only t the end of the myorrhiztion proess (Mrtín-Rodríguez et l., 21). In ddition, ABA defiieny indued ethylene prodution, nd it hs een suggested tht one of the mehnisms used y ABA to determine suseptiility to fungl infetion is through negtive modultion of the ethylene pthwy (Herrer- Medin et l., 27; Mrtín-Rodríguez et l., 21). The ABA signlling pthwy interts ntgonistilly with the ethylene signlling pthwy nd vie vers, in order to modulte plnt development (Beudoin et l., 2; Ghssemin et l., 2) nd plnt disese resistne (Anderson et l., 24). However, no speifi role hs een demonstrted for ethylene during AM symiosis, lthough reent results suggest its prtiiption in the regultion of the AM (Penmets et l., 28; Riedel et l., 28; Zsögön et l., 28), nd our understnding of how the ABA pthwy interts with ethylene pthwys during AM formtion is very limited. The ojetive of this study ws to nlyse the role plyed y the reltionship etween ABA nd ethylene in regulting the formtion of AM symiosis in tomto plnts. Using geneti tools, suh s plnt mutnts defetive in hormone prodution or pereption, in omintion with histohemil nd moleulr iology tehniques for the nlysis of AM formtion, we studied the mehnism used y ABA to determine suseptiility to AM fungl root oloniztion. Our results suggest tht the redution in rusulr undne in the myorrhizl roots of sitiens mutnts is diretly ssoited with their ABA iosynthesis defiieny. The umultion of ethylene in the roots of these mutnt plnts s result of their ABA defiieny minly ffets myorrhizl intensity. A dul ethylene-dependent ethyleneindependent mehnism hs een suggested to explin ABA regultion of the AM formtion. As ABA is neessry for rusule formtion, ABA defiieny hs diret negtive effet on the perentge of rusules in myorrhizl roots. ABA defiieny enhnes ethylene ontent, thus ting s negtive regultor of myorrhizl intensity. Mterils nd Methods Plnt growth nd AM inoultion Seeds of Solnum lyopersium L. (Mill.) wild-type Rheinlnds Ruhm (Aession LA535) nd ABA-defiient mutnt (Tylor et l., 1988) sitiens (ession LA575) were otined from the Tomto Genetis Resoure Centre (TGRC) of the University of Cliforni. Trnsgeni tomto seed lines overexpressing the Never Ripe (NR) ethylene reeptor (NRO plnts) (Cirdi et l., 2), LeETR6 ethylene reeptor ntisense lines (ETR6-AS plnts) (Kevny et l., 27), ethylene trnsgeni tomto lines expressing teril 1-minoylopropne-1-roxylte (ACC) deminse (ACCDexpressing plnts) (Klee et l., 1991) nd their isogeni wild-type vs Flor-Dde nd UC82B were kindly provided y Dr H. Klee (University of Florid). Tomto seed steriliztion, AM fungi inoultion nd plnt growth were rried out s previously desried y Herrer-Medin et l. (27). Plnt growth nd tretments were rried out in growth hmer (16 : 8 h, 25 : 19 C, dy : night yle; reltive humidity 5%). Inoultion with Glomus intrrdies (DAOM ), relssified s Glomus irregulre ording to Stokinger et l. (29), ws rried out in 2 ml pots. Eh seedling ws grown in seprte pot nd inoulted with piee of monoxeni ulture in Gel-Gro (ICN Biohemils, Auror, OH, USA) medium ontining 5 G. intrrdies spores nd infeted rrot roots. The monoxeni ulture (G. intrrdies nd rrot roots) ws produed ording to the method desried y Chot et l. (1992). In the noninoulted tretment, the plnts were pplied with piee of Gel-Gro medium ontining only uninfeted rrot roots. One week fter plnting in pots nd weekly therefter, 2 ml of modified Long Ashton nutrient solution ontining 25% of the P onentrtion (Hewitt, 1966) ws dded to prevent myorrhizl inhiition s result of exess of phosphorous. Plnts were hrvested 5 d fter inoultion, New (211) 19: Ó 211 The Authors New Ó 211 New Trust

3 Reserh 195 nd the root system ws wshed nd rinsed severl times with sterilized distilled wter. The root system ws weighed nd used for the different mesurements ording to the nture of the experiments. In eh experiment, five independent plnts were nlysed per tretment. Sitiens plnts were spryed dily with wter to prevent wilting. Estimtion of root oloniztion The nonvitl trypn lue histohemil stining proedure ws used ording to the Phillips & Hymn (197) method. Stined roots were oserved with light mirosope, nd the intensity of root ortex oloniztion y AM fungus ws determined s desried y Trouvelot et l. (1986) using the MYCOCALC softwre ( inr.fr/myhinte/myol-prg/downlod.html). The prmeters mesured were frequeny of oloniztion (%F), intensity of oloniztion (%M) nd rusulr undne (%A) in the whole root s well s intensity of oloniztion (%m) nd rusulr undne (%) in myorrhizl root frgments. At lest five mirosope slides were nlysed per iologil replite, nd eh slide ontins 3 root piees of 1 m. reverse trnsription, 8 ll of MilliQ wter ws dded to otin finl volume of 1 ll for eh DNA solution. Quntittive RT-PCR (qrt-pcr) ws rried out to mesure the trnsript undnes of elongtion ftor 1 (GinEF) nd glutmine synthse (GinGS) G. intrrdies genes nd tomto ethylene reeptor genes (LeETR3, LeETR4, LeETR6) in myorrhizl roots. All primer nmes nd orresponding sequenes re listed in Tle 1. qrt-pcr ws rried out with n icyler pprtus (Bio-Rd). Eh 2 ll PCR retion ontined 1 ll of diluted DNA (1 : 1), 1 ll 2 SYBR Green Supermix (Bio-Rd), nd 2 nm of eh primer using 96-well plte. The PCR progrmme onsisted of 3 min inution t 95 C, followed y 35 yles of 3 s t 95 C, 3 s t C, nd 3 s t 72 C. The speifiity of the PCR mplifition proedure ws heked with melting urve fter the finl yle of the PCR (7 steps of 3 s from 6 to 95 C with heting rte of.5 C). qpcr experiments were rried out from three iologil replites nd the threshold yle (C T ) ws determined in triplite. The reltive levels of trnsription were lulted y using the 2 )DDCt method (Livk & Shmittgen, 21). C T vlues of ll genes were normlized to the C T vlue of the LeEF-1 (X14449) housekeeping gene. RNA extrtions nd gene expression For the reverse trnsription polymerse hin retion (RT- PCR) experiments, totl RNA ws isolted from 1 g of pooled mteril (iologil replite) tht ontined representtive portions of roots from t lest five different plnts. Totl RNA ws isolted from the roots stored t )8 C using the RNesy Plnt Mini Kit (Qigen, Vleni, Cliforni) following the mnufturer s instrutions. DNAs were otined from 1 lg of totl DNse-treted RNA in 2 ll retion volume ontining 2 U of vin myelolstosis virus (AMV) reverse trnsriptse (Rohe, Mnnheim, Germny), 4 ng of rndom hexmer primers, 1 mm eh deoxynuleoside triphosphte (dntp), 5 U of RNse inhiitor nd 1 reverse trnsription uffer. After Chemil tretments The tomto plnts were treted in soil with ABA (Sigm), sodium tungstte (N 2 WO 4 ) (Pnre, Brelon, Spin), nd minoethoxyvinyl glyine hydrohloride (AVG) (Sigm-Aldrih, Steinheim, Germny). Sodium tungstte is potent inhiitor of molydo-enzymes in plnts suh s ABA ldehyde oxidse (Milorrow, 21) nd AVG strongly inhiits the onversion of methionine to ACC in the ethylene iosyntheti pthwy (Adms & Yng, 1979). The solutions were prepred y dilution from stok. Twenty millilitres of the orresponding diluted solution were pplied twie week to eh 2 ml pot ontining one tomto plnt. The first pplition strted 1 wk fter AM fungl inoultion. Stok solutions ontined 1 mm ABA in Tle 1 Primers used in this study for quntittive reverse trnsription polymerse hin retion (qrt-pcr) experiments Primer nme Orgnism Trget gene Primer sequene (5 3 ) Referene GinGS-F Glomus intrrdies GinGS (5 -CCTCAAGGTCCCTATTATTGTTCTG-3 ) Gomez et l.(29) GinGS-R (5 -ACGATAATGAGCTTCCACAACGT-3 ) GinEF-F G. intrrdies GinEF (5 -GCTATTTTGATCATTGCCGCC-3 ) Bendellh et l. (29) GinEF-R (5 -TCATTAAAACGTTCTTCCGACC-3 ) EF-1F Solnum lyopersium LeEF-1 (5 -GGTGGCGAGCATGATTTTGA-3 ) Grí-Grrido et l. (21) EF-1R (5 -CGAGCCAACCATGGAAAACAA-3 ) ETR3F S. lyopersium LeETR3 (NR) (5 -AGGGAACCACTGTCACGTTTG-3 ) Kevny et l. (28) ETR3R (5 -CTCTGGGAGGCATAGGTAGCA-3 ) ETR4F S. lyopersium LeETR4 (5 -GGTAATCCCAAATCCAGAAGGTTT-3 ) Kevny et l. (28) ETR4R (5 -CAATTGATGGCCGCAGTTG-3 ) ETR4F S. lyopersium LeETR6 (5 -CAATTGATGGCCGCAGTTG-3 ) Kevny et l. (28) ETR4R (5 -GGATGTGGATATGTGGGATTAGAAG-3 ) Ó 211 The Authors New Ó 211 New Trust New (211) 19:

4 196 Reserh New 1% ethnol, 25 lm AVG nd 1 mm N 2 WO 4 in wter. Control tretments used.1% ethnol solution. The finl ABA, AVG nd sodium tungstte onentrtions used were in the rnge of onentrtions used in previous studies (Hnsen & Grossmnn, 2; Audenert et l., 22; Mrtín-Rodríguez et l., 21). Ethylene quntifition The ethylene ontent in roots ws mesured y pling exised root systems in 16 ml test tue seled with ruer stopper nd inuted for 1 h t room temperture. The umultion of ethylene in eh tue ws determined from three different smples of 1 ml tken from the tue using syringe. Mesurements were rried out in gs hromtogrph (Hewlett Pkrd 589, Plo Alto, Cliforni) fitted with flme ioniztion detetor, using ommeril ethylene s stndrd for identifition nd quntifition purposes. The ethylene onentrtion ws expressed s g 1 root FW. ABA quntifition Plnt hrvest nd root proessing for ABA quntifition in tomto roots were rried out ording to Mrtín- Rodríguez et l. (21). ABA quntifition ws repeted three times with different pooled root mteril. Freeze-dried roots (equivlent to minimum 1 mg FW of root mteril) were extrted with mixture of isopropnol nd eti id (95 : 5, v v). To eh smple ws dded 1 ng (d 6 )-ABA (Plnt Biotehnology Institute, Ntionl Reserh Counil of Cnd, Ssktoon, Cnd). Smple preprtion ws performed ording to Meixner et l. (25); the smples were inuted, ontinuously shken for 2 h t 4 C, nd entrifuged for 1 min t 1 g; the superntnt ws removed nd evported to dryness under strem of N 2. The residue ws resuspended in methnol, entrifuged gin for 1 min t 1 g, nd the superntnt ws removed nd pled in glss vil. The methnol ws evported under strem of N 2 nd the smple ws resuspended in 1 ll ethyl ette. Methyltion ws rried out ording to the method desried y Cohen (1984) using freshly prepred dizomethne. Gs hromtogrphy mss spetrometry nlysis ws rried out using Vrin Sturn 21 ion-trp mss spetrometer with eletron impt ioniztion t 7 ev onneted to Vrin CP-39 gs hromtogrph equipped with CP- 84 utosmpler (Vrin, Wlnut Creek, CA, USA). For the nlysis, 2 ll of the methylted smple dissolved in 3 ll ethyl ette ws injeted in splitless mode (splitter opening 1 : 1 fter 1 min) onto Phenomenex (Ashffenurg, Germny) ZB-5 olumn (3 m.25 mm.25 lm) using He rrier gs t 1 ml min )1. Injetor temperture ws 25 C, nd the temperture progrmme ws 6 C for 1 min followed y n inrese of 25 C min )1 to 18 C, 5 C min )1 to 25 C, 25 C min )1 to 28 C, nd then 5 min isothermilly t 28 C. The methyl ester of ABA eluted t 13.5 min under these onditions. For greter sensitivity, the lsis mode (Vrin Mnul; Wells & Huston, 1995) ws used. The endogenous ABA onentrtion ws lulted on the sis of isotope dilution priniples using the ions from the methylted sustne t m z= (ions derived from endogenous nd d6-aba onentrtions; Wlker- Simmons et l., 2). Sttistil nlysis The dt were sujeted to one- or two-wy ANOVA. The men vlues for five replite smples were ompred using Dunn s multiple rnge test (P =.5). Results Plnt ethylene sensitivity medites the degree of fungl oloniztion fter ABA iosynthesis inhiition Previous results hve shown tht sodium tungstte pplition to roots redued myorrhiztion nd hd positive effet on ethylene prodution nd negtive impt on ABA iosynthesis in tomto plnts (Mrtín-Rodríguez et l., 21). In order to determine whether the negtive effet of tungstte pplitions on myorrhiztion is lwys used y ethylene pthwy tivtion, we studied the impt of sodium tungstte pplitions on different mutnt tomto plnts with vrying degrees of ethylene sensitivity. These experiments show tht ll the myorrhiztion prmeters mesured, exept oloniztion frequeny, were negtively ffeted in wild-type Flor-Dde plnt roots y the pplition of sodium tungstte (Fig. 1). This effet ws limited in wild-type plnts to etween 16% in %M nd 4% in %A, nd no negtive effet on myorrhizl prmeters ws oserved when the pplition of ABA ws omined with tungstte. Plnts with redued ethylene pereption, nd thus lower sensitivity to ethylene (NRO), were unffeted y tungstte pplitions in terms of myorrhizl frequeny nd oloniztion intensity, nd, s with wild-type plnts, 35% redution ws deteted in the % prmeter (Fig. 1). As the tungstte pplition did not ffet the %M, the redution in %A ws less pronouned (. 2%) in NRO plnts. Nevertheless, in the presene of tungstte, the plnts with inresed sensitivity to ethylene (ETR6-AS) reorded shrply redued myorrhiztion prmeters (Fig. 1). In ETR6-AS plnts, %M nd %m deresed y 6%, while % prmeters fell y. 4%. The ltter redution lso ourred in wild-type nd NRO plnts. The shrp derese in %M nd % prmeters with respet to ETR6-AS tungstte-treted plnts redued their %A y 75% (Fig. 1). When ABA ws pplied together with tungstte to ETR6-AS plnts, %M nd %m prmeters rehed only 8% of their former vlues in ontrol tretments (Fig. 1). New (211) 19: Ó 211 The Authors New Ó 211 New Trust

5 Reserh 197 Our results revel tht the redution in root oloniztion y tungstte pplition (%M) ws highly dependent on the plnt genotype (two-wy ANOVA, F 2,45 = 27.44, P <.1). By ontrst, the redution in rusulr undne () Myorrhiztion prmeters (Flor-Dde wild-type plnts) % () 12 % () 1 % (d) 2 M vlue d %F %M %m % %A Myorrhiztion prmeters (NRO plnts), %F %M %m % %A Myorrhiztion prmeters (ETR6-AS plnts) %F %M %m % %A f,g Flor- Dde GiEF e,f h NRO e ETR6- AS d,d Flor- Dde GiGS i d NRO d,e ETR6- AS in the myorrhizl root zones (%) ws minly the result of tungstte pplition (two-wy ANOVA, F 2,27 = 174.7, P <.1), regrdless of the plnt s ethylene sensitivity. Nevertheless, there is signifint intertion etween plnt genotype nd tungstte ABA pplition ftors for the two myorrhizl prmeters, %M (two-wy ANOVA, F 4,45 = 4.21, P =.5) nd % (two-wy ANOVA, F 4,27 = 6.13, P =.1), demonstrting tht these ftors intert. Fungl oloniztion ws lso quntified y qrt-pcr t the moleulr level to verify the mirosopi mesurement of myorrhiztion in tungstte, tungstte plus ABA-treted nd nontreted plnts with vrying degrees of ethylene sensitivity. We quntified the umultion of mrna for the G. intrrdies elongtion ftor 1-lph gene (GinEF) (GenBnk ession no. DQ ) in tomto roots in order to mesure the rte of fungl oloniztion. The presene of rusules in the whole myorrhizl root ws quntified t the moleulr level through trnsript detetion in the G. intrrdies glutmine synthse gene (GinGS), whih hs een desried elsewhere s rusuleexpressed (Gomez et l., 29). The dt in Fig. 1(d) show the M vlues for gene indution. M vlue represents the log2 rtio of gene expression in treted plnts with respet to gene expression for nontreted plnts. A more signifint hnge in GinEF expression ws oserved in reltion to tungstte-treted ETR6-AS plnts, where M vlues were found to e )2 (four times down-regulted) (Fig. 1d). The M vlues for GinEF were ).9 (lmost twie down-regulted) nd ).4 (< 1.5 times down-regulted) in tungstte wild-type nd NRO-treted plnts, respetively. GinGS expression deresed threefold in wild-type nd nerly eightfold in ETR6-AS myorrhizl roots treted with tungstte (M vlues of nerly )1.5 nd )2.7, respetively) (Fig. 1d). The pplition of tungstte ffeted GinGS gene expression in NRO plnts to lesser degree thn in the other Fig. 1 Effet of sodium tungstte pplition on myorrhiztion prmeters (,, ) nd expression of Glomus intrrdies GinGS nd GinEF genes (d) in plnts with different ethylene sensitivities. After 1 wk of trnsplnting nd inoultion with G. intrrdies, wild-type Flor-Dde, ethylene-insensitive NRO nd ethylene-hypersensitive ETR6-AS plnts were treted with sodium tungstte lone (1.5 mm, lk rs, ) nd in omintion with ABA (75 lm, grey rs, ). White rs, ontrol ( ). In (d) white rs, treted with sodium tungstte; lk rs, sodium tungstte + ABA. Solutions were pplied to soil twie per wk nd myorrhiztion ws mesured 5 d fter inoultion. Myorrhiztion prmeters of frequeny (%F), intensity (%M) nd rusulr undne in the whole root (%A), nd intensity (%m) nd rusulr undne (%) in myorrhizl zones of the root were determined using MYCOCALC softwre. Quntittive reverse trnsription polymerse hin retion dt represent the M vlue (log 2 rtio). The vlue of M is if there is no hnge, nd +1 or )1 if there is twofold indution or redution with respet to the expression of eh gene under nontretment onditions. Vlues re mens ± SE. For eh myorrhiztion prmeter, rs with similr letters re not signifintly different (P =.5) ording to Dunn s multiple rnge test. Ó 211 The Authors New Ó 211 New Trust New (211) 19:

6 198 Reserh New M vlue LeETR6 plnt vrieties, while the pplition of ABA in omintion with tungstte resued oth GinEF nd GinGS expression in ll plnts (Fig. 1d). These results therefore show tht geneti mnipultion in the ethylene reeptors ffets the myorrhiztion proess fter tungstte pplition differently ording to ethylene sensitivity, nd lerly illustrte tht the effet of the redution in myorrhizl root oloniztion s result of tungstte pplition is medited y the ethylene pthwy. We lso performed LeETR3 (NR) nd LeETR6 gene expression nlysis in order to onfirm the tivtion of the ethylene pthwy during AM formtion. qrt-pcr experiments with speifi primers on LeETR3 nd LeETR6 gene fmilies were rried out during myorrhiztion on wild-type nd ABA-defiient sitiens tomto plnt roots, whih re inherently ple of inresing ethylene. An indution of oth ethylene reeptor fmily genes during myorrhiztion of wildtype plnt roots ws lerly oserved (Fig. 2). In sitiens plnts, oth myorrhizl nd nonmyorrhizl root plnts showed upregultion of ETR6 nd ETR3 tomto ethylene reeptor genes ompring with wild-type nonmyorrhizl plnts (Fig. 2). Disrete ethylene nd ABA roles during AM formtion To determine the ethylene-independent effet of ABAregulted AM formtion in tomto plnts, we studied the effet of sodium tungstte pplition on ABA onentrtions, ethylene prodution nd myorrhizl formtion in ACCD-expressing trnsgeni tomto plnts. This trnsgeni line expresses teril ACC deminse (Klee et l., 1991), whih is involved in irreversile degrdtion of the ethylene preursor ACC. Consequently, these plnts exhiited redution in ethylene ontent s result of ethylene iosynthesis impirment. This trnsgeni line hs ABA onentrtions similr to those of the wild-type, while the derese in ABA ontent s result of tungstte pplition ws similr in oth wild-type nd ACCD-expressing plnts (Fig. 3). There ws no signifint intertion etween genotype nd sodium tungstte ABA pplition ftors using two-wy ANOVA (F 2,12 =1.77,P =.212), inditing tht the ABA ontent in roots is only dependent on the tungstte ABA pplition ftor. The onentrtion of ethylene in ACCDexpressing plnt roots ws lwys elow tht for wild-type plnts (Fig. 3). In the se of ethylene ontent, the intertion etween genotype nd sodium tungstte ABA pplition ftors ws signifint, demonstrting tht the effet on ethylene ontent of tungstte pplition ws dependent on plnt ethylene synthesis pity (two-wy ANOVA, F 2,12 = 18, P =.2) (Fig. 3). This indites tht this trnsgeni tomto line is n idel tool for seprtely nlysing the effets of ABA nd ethylene. The pplition of sodium tungstte to wild-type UC82B plnts negtively ffeted ll the prmeters of myorrhiztion mesured in the experiment, suh s myorrhizl frequeny nd intensity nd rusulr undne (Fig. 3). Nevertheless, in ACCD-expressing plnts, only rusulr undne (%) ws negtively ffeted following tungstte pplition (Fig. 3d). In the se of %M there ws signifint intertion etween genotype nd sodium tungstte ABA pplition ftors (two-wy ANOVA, F 2,18 = 3.88, P =.5), demonstrting tht the pplition of tungstte seletively ltered the %M prmeter ording to the plnt phenotype studied. The pplition of ABA omined with sodium tungstte resued ll the myorrhiztion prmeters in oth wild-type UC82B nd ACCD-expressing plnts (Fig. 3,d). Even the vlues for myorrhizl intensity (%M nd %m) were signifintly higher in ACCD-expressing plnts treted with tungstte plus ABA. After tungstte pplition, the myorrhiztion prmeters for wild-type UC82B plnts were lower, while ABA ontent deresed nd ethylene prodution in roots inresed (Fig. 3,). The derese of lmost 4% in ABA ontent following tungstte pplition ws similr for oth wild-type UC82B nd ACCD-expressing plnts (Fig. 3). The onentrtion of ethylene in ACCD-expressing plnts remined elow tht reorded for UC82B nontreted plnts s well s those treted with sodium tungstte (Fig. 3). For the % prmeter there ws no signifint intertion etween genotype nd sodium tungstte ABA pplition ftors (two-wy ANOVA, F 2,18 =.18, P =.836), inditing tht the redution in % oserved in these plnts ws minly used y the redution in ABA iosynthesis used y tungstte pplition (two-wy ANOVA, F 2,18 = 16.32, P =.1). The derese in %M nd % for tungstte-treted UC82B plnts redued %A in these plnts lmost 2.2-fold. In ACCD-expressing plnts, the pplition of tungstte only ffeted the perentge of rusules (%) in the myo, LeETR3 Fig. 2 Expression of LeETR6 nd LeETR3 ethylene reeptor genes in roots of wild-type Rheinlnds Ruhm (Rhe) nd ABA-defiient sitiens (Sit, white rs) tomto plnts olonized (I, lk rs) nd nonolonized (NI, grey rs) y Glomus intrrdies. Gene expression ws mesured y quntittive reverse trnsription polymerse hin retion (qrt-pcr) 5 d fter inoultion. qrt- PCR dt represent the M vlue (log 2 rtio). The vlue of M is if there is no hnge, nd +1 or )1 if there is twofold indution or redution with respet to the expression of eh gene in wild-type plnts under nonolonized onditions. Vlues re mens ± SE. Brs with similr letters re not signifintly different (P =.5) ording to Dunn s multiple rnge test. New (211) 19: Ó 211 The Authors New Ó 211 New Trust

7 Reserh 199 () ABA (ng g 1 ) Control ABA ontent Sodium tungstte Tretment Sodium tungstte + ABA ().35.3 ET (ng g 1 h 1 ) Control Ethylene ontent d Sodium tungstte Tretment d, Sodium tungstte + ABA () 12 % Myorrhiztion prmeters (UC82B wild-type plnts), %F %M %m % %A (d) , Myorrhiztion prmeters (ACCD-expressing plnts),, %F %M %m % %A Fig. 3 Effet of sodium tungstte pplition on ABA ontent (UC82B, open rs; 1-minoylopropne-1-roxylte deminse (ACCD)- expressing, lk rs, () nd ethylene umultion (ET) in roots (UC82B, open rs; ACCD-expressing, lk rs, () nd myorrhiztion prmeters (ontrol, open rs; sodium tungstte, lk rs; sodium tungstte + ABA, grey rs, (, d) of plnts with different ethylene synthesis pities. After 1 wk of trnsplnting nd inoultion with Glomus intrrdies, wild-type UC82B nd the ethylene trnsgeni tomto lines expressing ACCD, tomto plnts were treted with sodium tungstte lone (1.5 mm) nd in omintion with ABA (75 lm). Solutions were pplied to soil twie per wk nd the myorrhiztion prmeters, ABA ontent nd ethylene umultion were mesured 5 d fter inoultion. Myorrhiztion prmeters of frequeny (%F), intensity (%M) nd rusulr undne in the whole root (%A), nd intensity (%m) nd rusulr undne (%) in myorrhizl zones of the root were determined using MYCOCALC softwre. Vlues re mens ± SE. For eh myorrhiztion prmeter, rs with similr letters re not signifintly different (P =.5) ording to Dunn s multiple rnge test. % rrhizl zones of the root nd did not ffet myorrhizl intensity (%M). Consequently, the %A prmeter for tungstte-treted ACCD-expressing plnts (lulted from % nd %M dt) fell only 1.4-fold (Fig. 3d). Fungl oloniztion ws lso quntified y qrt-pcr t the moleulr level to verify the mirosopi mesurements of myorrhiztion in tungstte, tungstte plus ABA-treted nd nontreted wild-type UC82B nd ACCD-expressing trnsgeni plnts. The dt shown in Fig. 4 represent the M vlue for gene indution. M vlue represents the log 2 rtio of gene expression in treted plnts with respet to gene expression for nontreted plnts in eh genotype. No signifint hnge in GinEF expression ws oserved for ACCDexpressing plnts, s the M vlues for tungstte nd tungstte plus ABA tretments were found to e etween nd )1 (less thn twie down-regultion). Nevertheless, GinEF expression deresed in wild-type UC82B myorrhizl roots treted with tungstte (M vlue of nerly )4, or 16 times down-regultion), nd the pplition of ABA in omintion with tungstte resued GinEF expression in these plnts (the M vlue ws up from )4 to)1). The dt for GinGS gene expression, refleting the presene of fungl tivity in rusule ells, show tht tungstte tretment negtively ffets GinGS gene expression in oth types of plnts. The pplition of tungstte diminished GinGS expression lmost eight- nd fourfold in wild-type nd ACCD-expressing trnsgeni plnts, respetively (M vlues of )2.7 nd )1.8 for wild-type nd ACCD-expressing plnts, respetively). There ws no negtive impt on GinGS gene expression when the pplition of ABA ws omined with tungstte (Fig. 4). LeETR4 nd LeETR6 gene expression nlysis ws rried out to determine the differing responses t the ethylene signlling pthwy tivtion level in UC82B wild-type nd ACCD-expressing plnts fter tunsgstte pplitions. LeETR4 nd LeETR6 ethylene reeptor trnsript umultion ws lerly indued in wild-type plnt roots when tungstte ws pplied on its own, while the pplition of tungstte omined with ABA only hd slightly positive effet on trnsript umultion of LeETR4 (Fig. 5). In ACCD-expressing plnts, the LeETR4-6 ethylene reeptor trnsript umultion ws unffeted y tungstte pplied either lone or in omintion with ABA (Fig. 5). LeETR4 expression levels were greter thn nd equl to levels found for LeETR6 in wild-type nd ACCD-expressing plnts, respetively (Fig. 5). The dul ABA defiieny ethylene enhnement mehnism using myorrhiz impirment in ABAdefiient mutnt sitiens plnts To onfirm tht the redued perentge of rusules reorded in myorrhizl sitiens plnts nd wild-type myorrhizl roots Ó 211 The Authors New Ó 211 New Trust New (211) 19:

8 2 Reserh New M vlue GinEF,d UC82B wildtype ACCDexpressing e d,e GinGS UC82B wildtypexpressing ACCD- e Fig. 4 Quntittive reverse trnsription polymerse hin retion (qrt-pcr) nlysis of the expression of Glomus intrrdies GinGS nd GinEF genes in root plnts with different ethylene synthesis pities during rusulr myorrhiz (AM) formtion. After 1 wk of trnsplnting nd inoultion with G. intrrdies,wild-type UC82B nd the ethylene trnsgeni tomto lines expressing 1-minoylopropne-1-roxylte deminse (ACCD), tomto plnts were treted with sodium tungstte lone (1.5 mm, open rs) nd in omintion with ABA (75 lm, losed rs). Solutions were pplied to soil twie per wk nd dt for qrt-pcr were mesured in 5-d-old roots fter inoultion. qrt-pcr dt represent the M vlue (log 2 rtio). The vlue of M is if there is no hnge, nd +1 or )1 if there is twofold indution or redution with respet to the expression of eh gene under nontretment onditions. Vlues re mens ± SE. Brs with similr letters re not signifintly different (P =.5) ording to Dunn s multiple rnge test. M vlue LeETR4 e d, UC82B wildtype ACCDexpressing LeETR6 d,,, UC82B wildtype ACCDexpressing Fig. 5 Quntittive reverse trnsription polymerse hin retion (qrt-pcr) nlysis of the expression of LeETR4 nd LeETR6 ethylene reeptor genes in root plnts with different ethylene synthesis pities during rusulr myorrhiz (AM) formtion. After 1 wk of trnsplnting nd inoultion with Glomus intrrdies, wild-type UC82B nd the ethylene trnsgeni tomto lines expressing 1-minoylopropne-1-roxylte deminse (ACCD), tomto plnts were treted with sodium tungstte lone (1,5 mm, open rs) nd in omintion with ABA (75 lm, losed rs). Solutions were pplied to soil twie per wk nd dt for qrt-pcr were otined from 5-d-old roots fter inoultion. qrt-pcr dt represent the M vlue (log 2 rtio). The vlue of M is if there is no hnge, nd +1 or )1 if there is twofold indution or redution with respet to the expression of eh gene under nontretment onditions. Vlues re mens ± SE. Brs with similr letters re not signifintly different (P =.5) ording to Dunn s multiple rnge test. e % Myorrhiztion prmeters treted with tungstte ws speifilly used y ABA suppression rther thn ethylene enhnement, we performed experiments where ethylene synthesis ws loked in sitiens plnts y the pplition of AVG. AVG nd ABA were pplied lone nd together to sitiens-plnted soil, nd dt on frequeny, intensity nd rusule undne in myorrhizl roots were olleted. No signifint vritions in the vlues for myorrhiztion frequeny were oserved in sitiens plnts fter ABA tretment s ompred with nontreted sitiens plnts (Fig. 6). A slight inrese in myorrhiztion frequeny ws oserved fter AVG nd AVG + ABA tretments. This inrese in frequeny ws suffiient to reh the vlues reorded for the wild-type plnts (Fig. 6). Nevertheless, the intensity of myorrhiz development notiely inresed in the plnts treted with AVG (lone or in omintion with ABA) (Fig. 6). The perentge of rusule undne (%) in the olonized prts of the roots ws resued to the vlues reorded y wild-type Rheinlnds Ruhm plnts only when ABA ws present (Fig. 6). The single pplition of ABA or AVG did not resue the perentge of rusules in whole sitiens roots, while only the omined pplition of oth ompounds hd positive effet on the reovery of this prmeter in myorrhizl sitiens roots (Fig. 6). Aminoethoxyvinyl glyine hydrohloride, pplied lone or in omintion with ABA, redued ethylene prodution in sitiens plnts to the mounts reorded y wild-type plnts, while the pplition of ABA lone ws not effetive in signifintly deresing ethylene onentrtions in sitiens plnts (Fig. 7). Rhe Sit Sit AVG Sit ABA Sit AVG + ABA %F %M % %A Fig. 6 Effet of ABA nd minoethoxyvinyl glyine hydrohloride (AVG) pplition on myorrhiztion prmeters of ABA-defiient sitiens tomto plnts. After 1 wk of trnsplnting nd inoultion with Glomus intrrdies, wild-type (Rhe ) nd set of sitiens (Sit ) tomto plnts were treted with.1% ethnol solution, nd three sets of sitiens plnts were treted with AVG lone nd in omintion with ABA (Sit AVG; Sit ABA; Sit AVG + ABA). AVG (5 lm) nd ABA (75 lm) solutions were pplied to soil twie per wk. Myorrhiztion prmeters of frequeny (%F), intensity (%M), rusulr undne in myorrhizl zones of the root (%), nd rusulr undne in whole root (%A) were determined 5 d fter inoultion using MYCOCALC softwre. Vlues re mens ± SE. For eh myorrhiztion prmeter rs with similr letters do not signifintly differ (P =.5) ording to Dunn s multiple rnge test. New (211) 19: Ó 211 The Authors New Ó 211 New Trust

9 Reserh 21 ng g 1 h Disussion Ethylene ontent Rhe Sit Sit AVG Sit ABA Sit AVG + ABA Tretments Fig. 7 Effet of ABA nd minoethoxyvinyl glyine hydrohloride (AVG) pplition on ethylene ontent in roots of ABA-defiient sitiens tomto plnts olonized y Glomus intrrdies. After 1 wk of trnsplnting nd inoultion with G. intrrdies, wild-type (Rhe ) nd set of sitiens (Sit ) tomto plnts were treted with.1% ethnol solution nd three sets of sitiens plnts were treted with AVG lone nd in omintion with ABA (Sit AVG; Sit ABA; Sit AVG + ABA). AVG (5 lm) nd ABA (75 lm) solutions were pplied to soil twie per wk, nd ethylene ontent ws determined 5 d fter inoultion. Vlues re mens ± SE. Brs with similr letters do not signifintly differ (P =.5) ording to Dunn s multiple rnge test. As in previous studies on plnt growth nd development, ABA-defiient mutnts were used in our study to investigte the role of ABA during AM formtion. Reent reserh hs shown tht ABA-defiient sitiens plnts re less suseptile to G. intrrdies infetion thn wild-type plnts nd tht ABA is involved in rusule formtion nd its funtionlity to promote sustinle oloniztion of the plnt root during the estlishment of AM (Herrer-Medin et l., 27). An dditionl role for ABA in the estlishment of AM symiosis nnot therefore e ruled out, s the redution in AM oloniztion oserved in sitiens my lso e used y redution in strigoltone prodution y this mutnt (López-Ráez et l., 21). Interestingly, the exogenous pplition of ABA to sitiens did not ffet strigoltone prodution (López-Ráez et l., 21). The filure to resue sitiens strigoltone phenotype with exogenous ABA pplitions is in line with the reported filure to omplement sitiens AM-oloniztion phenotype y exogenous ABA pplition (Aro et l., 28). The pplition of ABA to sitiens plnts normlized growth nd led to n lmost omplete reovery in the frequeny nd intensity of lkline phosphtse tivity. Nevertheless, exogenous ABA pplitions were unle to restore the frequeny nd intensity of myorrhiztion (Herrer-Medin et l., 27). There ws some evidene tht the role plyed y ABA in AM formtion ould, t lest in prt, e ttriutle to ntgonisti intertion with ethylene (Herrer-Medin et l., 27; Mrtín-Rodríguez et l., 21). We therefore investigted the reltionship etween ABA nd ethylene with respet to the regultion of the formtion of AM,,, symiosis in tomto plnts nd ttempted to define the speifi roles of these different phytohormones during the myorrhiztion proess. Previous studies hve shown orreltion etween ABA root ontent nd limittions in AM fungl oloniztion with respet to the ABA-defiient notilis nd sitiens mutnts in whih the root ethylene ontent inresed ompred with their respetive wild-type plnts (Mrtín-Rodríguez et l., 21). This suggests tht ABA plys role in symiosis funtionlity regrdless of the ethylene ontent in roots. In ddition, sodium tungstte pplition to roots used redution in myorrhiztion nd hd positive effet on ethylene prodution nd negtive impt on ABA iosynthesis in tomto plnts (Mrtín-Rodríguez et l., 21). It is therefore neessry to determine whether the prmeters inditing the negtive impt of myorrhiztion used y ABA defiieny were ffeted y the derese in ABA or y the ethylene umultion ssoited with ABA defiieny. To nswer this question, we used two lterntive strtegies: nlysis of the effets on the pttern of myorrhiztion s result of the inhiition of ABA iosynthesis in trnsgeni tomto plnts with n ltered ethylene pthwy; nd omprison of the effets on myorrhiztion etween sitiens plnts treted with the AVG ethylene iosynthesis inhiitor lone nd those omined with ABA pplitions. We used two different kinds of trnsgeni tomto lines with ltered ethylene pthwys. The NRO nd ETR6-AS lines orrespond to plnts whose ethylene pereption ws ffeted y ltertions in the expression of ethylene reeptors (Cirdi et l., 2; Kevny et l., 27). The ACCD-expressing trnsgeni plnt line ws mde up of plnts with ethylene synthesis impirment (Klee et l., 1991). Our results revel tht the redution in root oloniztion y tungstte pplition orreltes with the inresed sensitivity of plnts to ethylene. The NRO plnts with higher insensitivity to ethylene s result of overexpression of the NR reeptor (Cirdi et l., 2) were unffeted y tungstte pplition with respet to the perentge of myorrhizl intensity of oloniztion. This result ws well orrelted with the finding tht the expression of GinEF gene ws unhnged in NRO myorrhizl root fter tungstte pplition. NRO trnsgeni plnts overexpress the NR tomto ethylene reeptor, whih redues ethylene sensitivity in seedlings nd mture plnts, inditing tht this reeptor is negtive regultor of ethylene response (Cirdi et l., 2). Conversely, ETR6-AS plnts, with inresed sensitivity to ethylene s result of ETR6 mrna intivtion y ntisense overexpression (Kevny et l., 27), were more ffeted in the prmeters of intensity of myorrhiztion (%M, %m), nd derese in GinEF expression when tungstte ws pplied either lone or in omintion with ABA. The redution in LeETR6 mrna levels in ETR6-AS lines produed erly-ripening phenotypes, whih re onsistent with onstitutive ethylene response (Kevny et l., 27). Ó 211 The Authors New Ó 211 New Trust New (211) 19:

10 22 Reserh New Our results lerly show tht the impt of this redution on the intensity of myorrhizl root oloniztion used y tungstte pplition is medited y the ethylene pthwy. This result is in line with previous dt where epinsti (epi) nd Never ripe (Nr) mutnts, ethylene overproduer nd low sensitivity, respetively, hve the intrrdil oloniztion y Glomus lrum highly inhiited, s ompred with the ontrol Miro-Tom (Zsögön et l., 28). Furthermore, qrt- PCR nlysis of LeETR6 nd LeETR3 ethylene reeptor genes indites tht their expression ws enhned in wildtype Rheinlnds Ruhm s result of myorrhiztion, suggesting the role of ethylene during AM formtion. In sitiens roots, LeETR6 nd LeETR3 ethylene reeptor gene expression ws onstitutively inresed regrdless of myorrhiztion, s these plnts re inherently ple of inresing ethylene. By ontrst, the presene of tungstte redued rusulr undne in the myorrhizl root zones (%) regrdless of the plnt s ethylene sensitivity. In this sense, redution in GinGS gene expression, mrker for the presene of rusules in roots, ws oserved in ll plnt vrieties treted with tungstte. The dt of GinGS expression were etter orrelted with the prmeter of undne of rusules in whole myorrhizl root (%A) sine oth represent vlues from the whole root system. Unlike the proess oserved for the intensity of oloniztion in roots, these results lerly show tht the impt of this redution on the rusule undne in the myorrhizl zones of the roots (%) s result of tungstte pplition is not medited y the ethylene pthwy. 1-Aminoylopropne-1-roxylte deminse-expressing trnsgeni plnts (ACCD plnts) re sujet to ethylene synthesis impirment s result of the tolism of its immedite preursor, ACC, y the tion of the teril ACC deminse loned nd introdued into tomto plnts (Klee et l., 1991). We used this trnsgeni tomto plnt line to seprtely nlyse the effet of oth ABA nd ethylene. ACCD-expressing plnts, whih hve less ethylene ontent in their roots, showed no ltertion in myorrhiztion pity. These plnts lso responded to tungstte pplition in similr wy to the UC82B wild-type, reording redution of nerly 4% in ABA ontent in roots. The derese in ethylene synthesis in trnsgeni ACCD-expressing plnts did not use ny pprent vegettive phenotypi normlities, nd only some delys in fruit ripening hve een oserved (Klee et l., 1991). Sutle differenes in response to the environmentl effets on trnsgeni plnts nnot e ruled out (Klee et l., 1991). In our experiments, the pplition of tungstte seletively ltered the prmeters of myorrhiztion ording to the plnt phenotype studied. In ACCD-expressing plnts, only the perentge of rusules (%) in myorrhizl roots ws ffeted, while in wild-type UC82B plnts, the pplition of tungstte ffeted ll the myorrhiztion prmeters. The histohemil dt orrelted losely with the trnsript umultion results for GinGS nd GinEF. The pttern of trnsript umultion for GinGS, fungl gene expressed in rusulr ells (Gomez et l., 29) prlleled the dt on rusule undne (%, %A) in myorrhizl roots. As with these prmeters, GinGS gene expression ws downregulted in oth wild-type nd ACCD-expressing plnts fter tungstte pplition. On the other hnd, the expression pttern of GinEF ws losely orrelted with the dt of myorrhizl intensity (%M) nd its expression ws downregulted only in wild-type plnts fter tungstte pplition. The redution in myorrhiztion prmeters for UC82B plnts ws ssoited with the effets of lower ABA ontent nd higher ethylene prodution in roots, wheres the redution in % oserved in ACCD-expressing plnts ws minly used y the derese in ABA iosynthesis. In ontrst to wild-type UC82B, no hnges in the expression of the ETR4 or ETR6 tomto ethylene reeptors were reported following tungstte pplition in ACCD-expressing plnts. These genes re ethylene-induile (Kevny et l., 27), nd the sene of up-regultion onfirms tht the ethylene response in ACCD-expressing plnts fter tungstte pplition ws ineffetive. These results indite tht ABA positively ffets rusule formtion (mesured s %), while ethylene minly regulted the rtio of oloniztion (mesured s myorrhizl intensity). To onfirm this hypothesis, we determined the effet of ABA nd n ethylene synthesis inhiitor on the resue of myorrhiz prmeters in sitiens plnts. We lerly demonstrted here tht myorrhiz resue in sitiens plnts vried depending on the ompound pplied. The dt show signifint inrese in the intensity of myorrhiz development ssoited with the inhiition of ethylene iosynthesis resulting from AVG tretment (lone or in omintion with ABA) s well s resue of rusule undne in sitiens myorrhizl roots when ABA is present. In previous work, the higher rte of reovery for fungl lkline phosphtse tivity in the sitiens mutnt ourred fter ABA pplitions ut not with silver thiosulphte pplitions, whih loked ethylene pereption, suggesting tht ABA my hve n dditionl role in rusulr funtionlity prt from its funtion s n inhiitor of ethylene prodution (Herrer-Medin et l., 27).The results shown in this study re in line with previous dt, whih onfirmed tht ABA pplitions restored fungl lkline phosphtse tivity (mostly ssoited with rusules) in sitiens roots, though not fungl spred, s mesured y trypn lue stining (Herrer-Medin et l., 27). In previous study, the reovery rte rehed 25% in %F, while only slight inrese in % ws oserved in sitiens myorrhizl roots fter ABA pplition (Herrer-Medin et l., 27). However, in our experiments, we found tht the exogenous ABA pplitions were unle to restore myorrhiztion frequeny or fully resue rusule undne. Although similr plnt nd fungl lines were used, this disrepny ould e used y differing New (211) 19: Ó 211 The Authors New Ó 211 New Trust

11 Reserh 23 Strigoltones? sitiens plnts Hyphl rnhing nd presori formtion oloniztion dynmis in oth experiments, s, ompred with previous studies, the %F nd % vlues reorded in our study for nontreted sitiens plnts were > three times higher for plnts with pproximtely the sme hrvest time. In myorrhiztion experiment rried out in prllel with tht shown in Fig. 6, we used G. mossee s fungl inoulum, nd oserved similr inrese in the intensity of myorrhiz development ssoited with the inhiition of ethylene iosynthesis used y AVG tretment. We lso noted tht rusulr undne in sitiens myorrhizl roots ws resued when ABA ws present (Supporting Informtion, Fig. S1). It is therefore ler tht, in zones with sitiens myorrhizl roots olonized y the fungus, the exogenously pplied ABA restored rusulr formtion nd funtioning pity, s mesured y fungl lkline phosphtse tivity (Herrer-Medin et l., 27) or in terms of rusulr undne resue in sitiens myorrhizl roots s shown in the urrent study. The vlues for ethylene prodution in roots demonstrte tht AVG, pplied lone nd in omintion with ABA, redued ethylene prodution in sitiens plnts to wild-type plnt mounts, wheres the pplition of ABA lone ws not suffiiently effetive in signifintly deresing the mount of ethylene in sitiens plnts. Interestingly, when ABA ws exogenously pplied to sitiens plnts, omplete resue of rusulr undne ws oserved, while the pplition of AVG did not produe totl reovery in myorrhiztion intensity. The oserved differentil response to similr ethylene onentrtions in wild-type nd sitiens plnts ould e result of their different ethylene sensitivities. The effet of the redution in ethylene onentrtions s result of exogenous AVG pplition to the mutnt plnt my not hve een suffiient to restore ll physiologil defiienies used y the muttion ffeting myorrhizl oloniztion. This study shows tht dul ethylene-dependent ethylene-independent mehnism is ssoited with ABA regultion of the AM formtion (Fig. 8). ABA is neessry for rusule formtion, inditing tht ABA defiieny hs diret negtive effet on the perentge of rusules in myorrhizl roots. ABA defiieny enhnes ethylene ontent, whih funtions s negtive regultor of myorrhizl intensity. This is in line with previous dt, where the exogenous pplition of ethylene restrited the spred of the AM fungus long the length of the root s xis, lthough its movement towrds the inner ortex, where rusules re formed, did not pper to e impeded (Geil et l., 21). Furthermore, ethylene hs een suggested s negtive regultor in erly infetion events suh s infetion thred formtion or elongtion nd ortil ell tivtion during nodultion in legumes (Sugwr et l., 26), nd erly phses of the symioti intertion of Medigo truntul with myorrhizl fungi (Penmets et l., 28). In this study, the pplition of ABA to myorrhizl sitiens roots led to reovery in rusule formtion pity. Further reserh is required to lrify the role of ABA in rusule formtion nd to identify puttive rosstlk etween plnt hormones in this proess. Aknowledgements ABA Arusule formtion nd funtioning We wish to thnk the Tomto Genetis Resoure Centre (TGRC) of the University of Cliforni nd Dr H. Klee t the University of Florid for providing tomto seeds. We would lso like to thnk Isel Tmyo nd Nuri Molinero for their help with the plnt experiments. Finnil support for this study ws provided y grnts from the Comisión Interministeril de Cieni y Tenologí (CICYT) nd Fondos Europeos de Desrrollo Regionl (FEDER) through the Ministerio de Cieni e Innovión, Spin (AGL25-? Ethylene Ethylene Fungl spred in the root Sodium tungstte AVG Fig. 8 Proposed model of the regultion of rusulr myorrhiztion in tomto roots y ABA ethylene intertion. A dul ethylene-dependent ethylene-independent mehnism is suggested for ABA regultion of rusulr myorrhiz (AM) formtion. ABA is neessry for the proess of rusule formtion nd funtioning, nd n dditionl role plyed y ABA in the estlishment of AM symiosis medited y strigoltone prodution nnot e ruled out (dshed rrow). In ddition, ABA interts ntgonistilly with ethylene, whih negtively regultes the rtio of fungl spred in the root. ABA defiieny s result of sodium tungstte pplition or muttion in sitiens plnts redues rusulr undne nd inreses ethylene onentrtions, using derese in the rte of oloniztion. Inhiition of ethylene synthesis y minoethoxyvinyl glyine hydrohloride (AVG) in ABA-defiient sitiens plnts speifilly inreses the intensity of myorrhiz development. Ó 211 The Authors New Ó 211 New Trust New (211) 19: