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1 CSIRO PUBLISHING Funtionl Plnt Biology, 23, 3, Aumultion of solule rohydrtes, trehlse nd surose synthse in effetive (Fix + ) nd ineffetive (Fix ) nodules of soyen ultivrs tht differentilly nodulte with Brdyrhizoium jponium Zhi-Ping Xie A,C, Christin Stehelin A,D, Willim J. Broughton B, Andres Wiemken A, Thoms Boller A nd Johim Müller A,E A Botnishes Institut der Universität Bsel, Heelstrsse 1, CH-456 Bsel, Switzerlnd. B Lortoire de Biologie Moléulire des Plntes Supérieures, Université de Genève, 1, Chemin de l Impértrie, CH-1292 Chmésy/Genève, Switzerlnd. C Present ddress: Euhinus Ltd, Rheinshnze 8, CH-456 Bsel, Switzerlnd. D Present ddress: Lortoire de Biologie Moléulire des Plntes Supérieures, Université de Genève, 1, Chemin de l Impértrie, CH-1292 Chmésy/Genève, Switzerlnd. E Present ddress: Johim Müller InnoLife, Shreinrstrsse 51, CH-84, Zürih, Switzerlnd. Corresponding uthor; emil: info@jminnolife.om Astrt. Roots of soyens hve the ility to form symioses with nitrogen-fixing rhizoil teri to form nitrogen-fixing (Fix + ) nodules, thus llowing the plnt to grow in the sene of minerl nitrogen. Severl soyen ultivrs from Chin nodulted normlly with Brdyrhizoium jponium 11 sp4, ut developed only few nodules with, nother B. jponium strin. When soyens were infeted with Rhizoium sp. NGR234, ineffetive (Fix ) nodules tht do not fix nitrogen were formed. Plnts infeted with NGRΩnodD2, mutnt strin overproduing lipo-hitooligoshridi nodultion signls (Nod ftors), showed signifintly higher numers of ineffetive nodules. Nodules from the different plnt mirosymiont omintions were hrterized with respet to their umultion of solule rohydrtes nd their indution of trehlse nd surose synthse. These two plnt enzymes re known to e nodule-stimulted proteins. Pool sizes of solule rohydrtes in nodules showed strin-speifi ltertions in surose nd trehlose, wheres myo-inositol nd pinitol were ffeted in more ultivr-speifi wy. Immunolots with nodulin-speifi ntiserum indited tht surose synthse is indued in Fix + nodules, ut undetetle in Fix nodules, inditing strin-speifi indution profile. Trehlse tivity in nodules showed similr strin-speifi indution profile. High enzyme tivity ws mesured for nodules hroring the Brdyrhizoium strins, wheres ineffetive nodules ontining NGR234 exhiited tivities in the rnge of uninfeted. Nodules indued y NGRΩnodD2 showed inresed trehlse tivity. A similr indution of trehlse ws oserved when uninfeted were treted with Nod ftors purified from NGR234. The dt otined re disussed in the ontext of rohydrte llotion in nodules nd the question of how rhizoil teri influene the rohydrte metolism of their host plnt is ddressed. Introdution On of legumes, rhizoi my indue formtion of nodules, highly speilized root orgns, in whih tmospheri dinitrogen is redued to mmoni (effetive nodules with nitrogen-fixing teroids, Fix + ). It is well estlished tht the nodule symiosis depends on genotype genotype reltionships etween the plnt nd the mirosymiont. The first steps of the intertion re determined y rhizoil signls, nmely the lipo-hitooligomeri Nod ftors nd vrious exopolyshrides (Perret et l. 2). Reent findings lso indite role for effetor moleules sereted y the teril type III seretion-system, whih hs een identified in ertin rhizoil strins (Mrie et l. 21). Nodultion of soyen, the most importnt legume rop worldwide, depends on vriety of geneti determinnts of oth the host nd the mirosymiont (Qin et l. 1996). Reent results indite, for instne, tht Sinorhizoium fredii strins with defetive inositol dehydrogense hve ompetitive disdvntges ompred with their respetive wild-types (Jing et l. 21). Similr oservtions were mde for Rhizoium leguminosrum v. viie mutnts tht nnot use myo-inositol s ron soure (Fry et l. 21). Arevitions used: Fix +, effetive nodules; Fix, ineffetive nodules; fw, fresh weight; nkt, nnoktl (nnomole sustrte trnsformed per seond);, norml nodultion potentil; pkt, pioktl;, restrited nodultion potentil. CSIRO /FP /3/9965

2 966 Funtionl Plnt Biology Z.-P. Xie et l. This is interesting s myo-inositol is one of the more undnt, non-struturl rohydrtes in soyen nodules. Conversely in uninfeted, pinitol, methylted derivtive of inositol, is more undnt thn myo-inositol (see Müller et l. 1994). A similr shift in the metoli profile from myo-inositol to pinitol n e oserved in nodules of poorly nodulting soyen mutnts (Müller et l. 1995) nd in ineffetive (Fix ) soyen nodules olonized y rdyrhizoi unle to fix nitrogen (Müller et l. 1994). A mjor ftor in the effetiveness of nodules is the orret llotion of surose to the nodule (for review see Vne nd Heihel 1991) nd its tolism y surose synthse (EC ). This key enzyme is indued in nodules (Morell nd Copelnd 1985; Thummler nd Verm 1987; Gordon et l. 1992) nd down-regulted in response to stresses tht inhiit nitrogen fixtion (e.g. Gonzlez et l. 1995; Müller et l. 1996). The resulting tolites, frutose nd UDPgluose, re used to fuel the energy-onsuming proess of nitrogen fixtion nd provide sustrtes for the iosynthesis of other rohydrtes. Of prtiulr interest in this perspetive re trehlose, non-reduing dishride umulted y teroids of some rhizoil strins, nd the indution of the trehlose-leving plnt enzyme trehlse (EC ) in nodules (Müller et l. 1995; Fris- Rodriguez et l. 1998; Aeshher et l. 1999). In previous studies (Xie et l. 1996, 1999) Chinese soyen ultivrs hve een desried s forming nodules upon inoultion with Brdyrhizoium jponium. Certin soyen ultivrs were poorly nodulted y this strin, wheres they retined the pity to estlish symiosis with B. jponium 11 sp4. This strin nodultes soyen very effetively (Rvuri nd Hume 1992) nd umultes more thn five times less trehlose thn strin in nodules, thus, drwing less ssimilte from the plnt host (Müller et l. 1994, 1998). In ontrst to the rdyrhizoil strins, soyen nodules indued y the rod host-rnge Rhizoium sp. NGR234 re ineffetive (Pueppke nd Broughton 1999). Here we investigted the umultion of solule rohydrtes nd the indution profile of surose synthse nd trehlse in Fix + nd Fix nodules of Chinese soyen ultivrs with different symioti potentil. Plnts were inoulted with 61-A11, 11 sp4, NGR234 s well s with strin NGRΩnodD2, derivtive of NGR234, whih overprodues Nod ftors (Felly et l. 1998). We present generl nodultion prmeters nd show tht rohydrte pools, surose synthse nd trehlse depend on oth the plnt genotype nd the rhizoil strins hrored y the plnt. Mterils nd methods Biologil mterils Seeds of soyen ultivrs [Glyine mx (L.) Merr.] were kindly provided y the Chinese Ademy of Agriulturl Siene (Beijing, People s Repuli of Chin). For this study the following ultivrs were seleted: D Hei Qi, D Zi Hu, Dong D Li, Du Lu Hung, An Tu Bi Hu Lu D Dou nd Gong Jio Glyine mx v. Mple Arrow ws otined from Semenes UFA (Busigny, Switzerlnd). All seeds were surfe-sterilized with 3% H 2 2 for 2 min, followed y wshing with sterile tp-wter nd inution t 27 C on 1% wter gr pltes for germintion (Stehelin et l. 1992). Brdyrhizoium jponium (Stripf nd Werner 1978) nd 11 sp4 (Regensurger nd Henneke 1983) were grown to sttionry phse in 2E-medium (Werner et l. 1975) t 27 C on rotry shker t 14 rpm. Rhizoium sp. NGR234 (Stnley nd Cervntes 1991; Pueppke nd Broughton 1999) nd its mutnt strin NGRΩnodD2 (Felly et l. 1998) were rised s desried y Felly et l. (1998). Estlishment of the symioti intertions Soyen seedlings were trnsferred to sterilized Leonrd jrs filled with perlite nd vermiulite (1:1) nd nutrient solution supplemented with 1 mm KNO 3 s desried (Stripf nd Werner 1978). In previous studies, 1 mm KNO 3 ws suffiient to sustin growth of Fix nodules without impiring nodultion y Fix + strins (Stehelin et l. 1992; Müller et l. 1994). After one week, plntlets were inoulted with 5 ml teril ultures (see Müller et l. 1996) or left uninoulted. Plnts were ultivted in phytotron (14-h dy t photon flux of 3 µmol m 2 s 1 nd 26 C, 1-h night t 2 C) nd hrvested fter four weeks of o-ultivtion. Anlysis of non-struturl rohydrtes Pool sizes of solule rohydrtes were mesured y gs hromtogrphy s desried previously (Müller et l. 1994, 1996). Extrtion of solule proteins, trehlse ssy nd immunolotting Frozen or nodule smples were ground in morpholinoethnesulfoni id (K + ) uffer (ph 6.3,.1 M) ontining EDTA nd phenylmethylsulfonyl fluoride (2 mm eh). The uffer:smple rtio ws 1 ml g 1 fresh weight (fw) for or 2 3 ml g 1 fw for nodules. The rude extrts were entrifuged t 2 g for 1 min. Superntnts (.1 ml) were diluted with.9 ml of sturted mmonium sulfte nd kept on ie for t lest 3 h. Proteins were preipitted y entrifugtion (1 g, 1 min) followed y resuspension in morpholinoethnesulfoni id (K + ) uffer (ph 6.3, 5 mm; totl volume.1 ml). Trehlse ws ssyed ording to Müller et l. (1995) y inuting liquots of resuspended proteins in morpholinoethnesulfoni id (K + ) uffer (ph 6.3, 5 mm) ontining 1 mm trehlose t 37 C for 15 6 min. Retions were terminted y oiling nd the gluose generted ws determined with gluose oxidse peroxidse (Boehringer Mnnheim GmH, Mnnheim, Germny). Solule proteins were ssyed ording to Brdford (1976). Solule nodule proteins were seprted on SDS PAGE gels in whih the lower seprting gel ontined 1% (w/v) rylmide in.375 M Tris HCl, ph 8.8,.1% (w/v) SDS, nd the upper stking gel ontined 3% rylmide in.125 M Tris HCl, ph 6.8 nd.1% SDS. Immunolotting (western lotting) ws performed s desried y Müller et l. (1994) using ommeril soyen milk insted of skim milk for loking. The protein onentrtion of the soyen milk ws djusted to 3% w/v y dilution in Tris-uffered sline (Tris ph 7.5, 5 mm, NCl 15 mm). The ntiserum ws rised s desried nd reognizes leghemogloin nd nodule-speifi surose synthse (Müller et l. 1994). Tretment with Nod ftors Roots of soyens (v. Mple Arrow ) were treted with etylted nd sulfted Nod ftors purified from strin NGR234 (Prie et l. 1992) s desried erlier (Xie et l. 1995, 1998). These Nod ftors re iologilly tive on soyen (Shmid et l. 1994; Xie et l. 1995, 1999).

3 Crohydrtes, trehlse nd surose synthse in soyen nodules Funtionl Plnt Biology 967 Results We hve previously reported tht totl of 92 soyen vrieties ommonly grown in the People s Repuli of Chin were tested for nodultion defiieny fter infetion with B. jponium. Severl ultivrs formed only few nodules with this strin, inditing restrited nodule potentil ( ultivrs), ut nodulted normlly with B. jponium 11 sp4 (Xie et l. 1999). For this study, we used four of these ultivrs ( D Hei Qi, D Zi Hu, Dong D Li, nd Du Lu Hung ) nd ompred them with three ultivrs with norml nodultion potentil (), whih inluded An Tu Bi-Hu Lu D Dou, Gong Jio nd the Cndin ultivr Mple Arrow. Plnts of eh ultivr were inoulted with, 11 sp4, Rhizoium sp. NGR234 nd with the mutnt strin NGRΩnodD2, whih overprodues Nod ftors. Nodule numer nd iomss were similr within nd ultivrs, ut differed etween these groups in strinspeifi mnner. Therefore, these results were grouped ording to nd ultivrs insted of listing eh ultivr seprtely. The ultivrs were strongly nodulted y oth tested Brdyrhizoium strins (> 5 nodules per plnt). Strin-speifi differenes were found for the ultivrs, whih formed less thn five nodules with. Strin 11 sp4 restored nodultion on ll ultivrs tested. Strin NGR234 indued pproximtely 55 ineffetive nodules per plnt on ultivrs, while ultivrs formed less thn 3 nodules with this strin. When the Nod-ftor-overproduing mutnt strin NGRΩnodD2 ws used s n inoulum, ultivrs formed signifintly more nodules thn with the wild-type, on verge, 136 nodules per plnt. ultivrs lso formed slightly higher numers of nodules, pproximtely 5 (ANOVA followed y LSD test; Fig. 1A). Nodule iomss ws higher thn.4 g per plnt in ultivrs olonized y or 11 sp4. In ultivrs infeted with 11 sp4, nodule iomss (verge.39 g) ws signifintly lower thn in ultivrs (verge.55 g). Nodules of ultivrs olonized y hd n verge iomss of.11 g per plnt. This vlue ws signifintly lower thn nodule iomsses of ultivrs olonized y eh B. jponium nd signifintly lower thn the iomss of nodules of ultivrs olonized y 11 sp4. ultivrs inoulted with the NGR234 formed out.2 g of Fix nodules per plnt. This vlue ws nerly 1 times lower in ultivrs. When the Nod-ftor-overproduing mutnt NGRΩnodD2 ws used, iomss did not signifintly differ etween nd ultivrs (ANOVA followed y LSD test; Fig. 1B). In most ses, nodules indued y nd 11 sp4 were effetive, s determined y the etylene redution method. In nodules of ll seleted ultivrs olonized y 11 sp4, nitrogense tivity did not show pronouned differenes. In ontrst, nitrogense tivity of nodules olonized y vried strongly within ultivrs, from less thn.1 nkt g 1 fw ( D Zi Hu ) to 2 nkt g 1 fw ( Du Lu Hung ). The NGR strins only osionlly indued teri-ontining nodules nd these were hrterized y sent or very low etylenereduing tivity in ll ultivrs (dt not shown). Upon mirosopi investigtion, most of these ineffetive nodules hroring NGR234 or NGRΩnodD2 looked like empty nodules free of teri or pseudonodules, whih sometimes rehed the size of the teri-ontining nodules. Aumultion of solule rohydrtes in nodules Uninfeted of nd ultivrs ontined 1% surose on dry weight sis (% dw). In oth types of ultivrs, effetive nodules olonized y ontined % dw surose. These vlues were not signifintly Nodule numer Nodule fw (mg) A B d 11sp4 e NGRwt NGRΩnodD2 d Sterile Fig. 1. Numer (A) nd iomss (B) of nodules. Soyen ultivrs hd norml nodultion potentil () or redued nodultion potentil () with respet to B. jponium. Plnts were infeted with strin, B. jponium 11 sp4, Rhizoium sp. NGR234 or with its mutnt NGRΩnodD2. Men vlues ± s.e. re given for three ultivrs nd four ultivrs respetively. Vlues topped y the sme letters re not signifintly different (P<.5; ANOVA followed y Student Newmn Keuls test).

4 968 Funtionl Plnt Biology Z.-P. Xie et l. inresed ompred with. High onentrtions of surose (up to 3% dw) were found, however, in extrts from nodules of oth types of ultivrs olonized y 11 sp4. In ontrst to effetive nodules, surose ontents of ineffetive nodules olonized y NGR234 were, in generl, lower thn in uninfeted nd showed signifint differenes etween nd ultivrs. In nodules from ultivrs, surose onentrtion ws twie s high s in nodules from ultivrs. Nodules from ultivrs olonized y NGRΩnodD2 exhiited surose pool sizes in the sme rnge s uninfeted (Fig. 2A; ANOVA followed y Student Newmn Keuls test). The teril dishride trehlose ws found in nodules in strin-dependent mnner. In nodules of ll tested ultivrs olonized y, trehlose ontents were in the sme rnge s surose ontents ( 1% dw), thus, onfirming erlier results otined with v. Mple Arrow (Müller et l. 1998). Trehlose ontents of nodules olonized y 11 sp4 were signifintly lower (.2% dw). Trehlose ws ompletely sent in NGR234- or NGRΩnodD2-hroring ineffetive nodules of ll seven ultivrs. Where present, trehlose ontents were not signifintly different etween nd ultivrs (Fig. 2B; ANOVA followed y Student Newmn Keuls test). Nodule ontents of pinitol nd myo-inositol, diret preursor of pinitol iosynthesis, re shown in Figs 3A nd B, respetively. Pinitol ws lwys mjor rohydrte in uninfeted ( 1% dw) nd in the pseudonodules of NGR234. Conversely, uninfeted ontined very smll pools of myo-inositol. The ontents of pinitol nd myoinositol in nodules were found to e dependent on oth ultivrs nd teril strins. Nodules from ultivrs hroring hd more thn three times more pinitol thn nodules from ultivrs olonized y this strin, while myo-inositol pool sizes were reiprolly ffeted. In nodules olonized y 11 sp4, pinitol nd myo- Surose (mg g 1 dw) A e d de Pinitol (mg g 1 dw) A Trehlose (mg g 1 dw) B 11sp4 <.5 mg g 1 dw NGRwt NGRΩnodD2 Sterile Fig. 2. Surose (A) nd trehlose (B) pool sizes of nodules. Soyen ultivrs hd norml nodultion potentil () or redued nodultion potentil () with respet to strin. Plnts were left uninfeted (sterile ) or infeted with, 11 sp4, NGR234 nd its mutnt NGRΩnodD2. Crohydrtes were nlysed y gs hromtogrphy. Men vlues ± s.e. re given for three ultivrs nd four ultivrs respetively. Vlues topped y the sme letters re not signifintly different (P<.5; ANOVA followed y Student Newmn Keuls test). Inositol (mg g 1 dw) B 11sp4 NGRwt NGRΩnodD2 Sterile Fig. 3. Content of ylitols in nodules. Soyen ultivrs hd norml nodultion potentil () or redued nodultion potentil () with respet to strin. Plnts were left uninfeted (sterile ) or infeted with, 11 sp4, NGR234 nd its mutnt NGRΩnodD2. Cylitol (pinitol nd myo-inositol) ontents were nlysed y gs hromtogrphy. Men vlues ± s.e. re given for three ultivrs nd four ultivrs respetively. Vlues topped y the sme letters re not signifintly different (P<.5; ANOVA followed y Student Newmn Keuls test). (A) Pinitol; (B) myo-inositol.

5 Crohydrtes, trehlse nd surose synthse in soyen nodules Funtionl Plnt Biology 969 inositol pool sizes did not signifintly differ etween nd ultivrs. Differenes etween nd ultivrs were lso oserved upon nlyses of ineffetive nodules indued y NGR234. Here, pinitol ontents were twie s high in nodules from ultivrs s in nodules from ultivrs, thus rehing levels found in uninfeted (1% dw). Indution of surose synthse An ntiserum ginst leghemogloin (14 kd) nd nodulespeifi surose synthse (mjor nd t 9 kd) ws used to exmine the indution of the plnt enzyme surose synthse in the different nodule types. In ll effetive nodules indued y the Brdyrhizoium strins, strong signl orresponding to leghemogloin ws deteted. Surose synthse ws indued in nodules of ll ultivrs olonized y nd in nodules of ultivrs hroring 11 sp4. Interestingly, ultivrs like Dong D Li hd weker surose synthse signl when inoulted with strin 11 sp4 in omprison with strin. Surose synthse ws lmost or ompletely undetetle in nodules indued y NGR234 or NGRΩnodD2 (Fig. 4). Similr findings were otined with proteins from other nd ultivrs (lots not shown). Indution of trehlse tivity The different nodule types were further studied y determining their hydrolyti tivities of the plnt enzyme trehlse. As shown in Fig. 5, strin-speifi indution ws oserved. Trehlse ws strongly indued in Fix + nodules ontining the Brdyrhizoium strins (12 14 nkt g fw 1 for oth strins nd ll ultivrs) ompred with uninfeted (less thn.1 nkt g fw 1 ). Fix nodules indued y the NGR strins hd trehlse tivities in the rnge of uninfeted. Interestingly, in nodules indued y the Nod-ftor-overproduing NGRΩnodD2, trehlse tivity ws more thn ten times higher (Fig. 5). To demonstrte tht Nod ftors ould, indeed, e responsile for the high stimultion of trehlse tivity, 9 kd 14 kd Fig. 4. Indution of nodule-speifi surose synthse. Nodules of n ultivr ( Dong D Li ) were indued y (lne 1), 11 sp4 (lne 2), NGR234 (lne 3) nd NGRΩnodD2 (lne 4). After SDS gel eletrophoresis, the proteins (15 µg per lne) were lotted onto nitroellulose memrne nd treted with the ntiserum (surose synthse, doule nd t 9 kd; leghemogloin, 14 kd). Tle 1. Trehlse tivity in soyen treted with Nod ftors Soyens (v. Mple Arrow ) were xenilly grown in Leonrd jrs supplemented with 5 mm KNO 3 nd treted with.1 µm Nod ftors purified from strin NGR234 or left untreted. Plnts were hrvested fter four weeks nd trehlse tivity ws mesured s desried. Men vlues ± s.e. re given for n independent plnts. Vlues followed y the sme supersript letters re not signifintly different (P s indited; ANOVA followed y Student Newmn Keuls test) Trehlse tivity Tretment (nkt g 1 protein) (pkt g 1 fw) Control (n = 6) 51 ± 6 39 ± 6 Aetylted Nod ftor (n = 6) 181 ± ± 8 Sulfted Nod ftor (n = 3) 212 ± ± 21 P <.5 <.5 uninfeted from soyen (ultivr Mple Arrow ) were treted with Nod ftors (.1 µm) purified from NGR234 nd plnts hrvested fter four weeks. Upon tretment with oth sulfted nd etylted Nod ftors, 4-fold stimultion of root trehlse tivity oth with protein nd with iomss s referene ould e oserved. This stimultion ws highly signifint (ANOVA followed y Student Newmn Keuls test; Tle 1). Disussion Aumultion of rohydrtes in soyen nodules depended on oth the host plnt genotype nd the mirosymiont. Strin-speifi ltertions were found for the pool sizes of surose nd trehlose, wheres the ontents of ylitols were ffeted in more ultivr-speifi mnner. ultivrs seem to umulte more pinitol thn Trehlse (nkt g 1 fw) sp4 NGRwt NGRΩnodD2 Sterile Fig. 5. Trehlse tivity of nodules. Soyen ultivrs hd norml nodultion potentil () or redued nodultion potentil () with respet to strin. Three ultivrs nd four ultivrs were left uninfeted (sterile ) or infeted with, 11 sp4, NGR234 nd NGRΩnodD2. Men vlues ± s.e. re given for three ultivrs nd four ultivrs respetively. Vlues topped y the sme letters re not signifintly different (P<.5; ANOVA followed y Student Newmn Keuls test).

6 97 Funtionl Plnt Biology Z.-P. Xie et l. ultivrs in effetive nodules infeted with 61-A11 nd in ineffetive nodules hroring NGR234, ut not in effetive nodules indued y 11 sp4. Similrly, levels of myo-inositol differed etween nd ultivrs when plnts were infeted with strin or NGR234. Hene, the umultion of ylitols in soyen nodules is signifintly influened y the plnt genotype, inditing more root-like rohydrte profile. Surose synthse ws indued in nodules in strinspeifi mnner. Reently pulished studies hve shown tht externlly pplied trehlose indued surose synthse tivity in uninfeted soyen (Müller et l. 1998). Thus, trehlose-produing teroids ould, indeed, ffet ssimilte sink properties in the surrounding tissue. This is onsistent with our oservtion tht the nodule-speifi surose synthse, whih is responsile for surose rekdown, is higher in nodules indued y in omprison with 11 sp4 (Fig. 4; see lso Müller et l. 1998). Consequently, the nodules indued y generlly showed low rtio of surose to trehlose, when ompred with those of strin 11 sp4. By ompring more thn 9 different soyen nodule types, signifint, negtive orreltion etween surose nd trehlose pool sizes hs een oserved (Fig. 2 nd dt not shown). It seems likely therefore, tht the strin-speifi umultion of trehlose redues the surose pool of the host, inditing ertin ompetition for rohydrtes etween the teroids nd the host. In this perspetive, the indution of trehlse ould e possile wy for the plnt to limit these sink-induing effets of trehlose in nodules. The trehlose-free pseudonodules indued y NGR234 (Fig. 5) exhiited low trehlse tivity, suggesting orreltion etween trehlose nd trehlse in nodules. It ws postulted tht trehlose, possily relesed from the teroids, indued trehlse tivity in the host ytoplsm (Mellor 1992). Experiments hve shown, however, tht trehlse tivity is not indued diretly y trehlose, ut regulted y uxins (Müller et l. 1995). The present work suggests tht Nod ftors re positive regultors of trehlse tivity. Nod ftors hve een shown to inhiit uxin trnsport (Mthesius et l. 1998) nd ffet rohydrte prtitioning (Xie et l. 1998). Hene, the stimulting effet of Nod ftors on trehlse tivity in soyen ould e relted to hnges in uxin levels. Further studies re required to understnd the mehnism y whih phytohormones, Nod ftors nd unknown metolites of rhizoil origin ontrol rohydrte metolism during nodule symiosis. Aknowledgments We re indeted to Prof. D Werner (University of Mrurg, Germny) nd to Prof. H Henneke (ETH Zürih, Switzerlnd) for providing us teril strins. This work ws supported y the Swiss Ntionl Foundtion nd grnt from the Rohe Foundtion to JM. Referenes Aeshher RA, Müller J, Boller T, Wiemken A (1999) Purifition of the trehlse GMTRE1 from soyen nodules nd loning of its DNA. GMTRE1 is expressed t low level in multiple tissues. Plnt Physiology 119, Brdford M (1976) A rpid nd sensitive method for the quntittion of mirogrm quntities of protein utilizing the priniple of proteindye inding. Anlytil Biohemistry 72, Fris-Rodriguez R, Mellor RB, Aris C, Pen-Criles JJ (1998) The umultion of trehlose in nodules of severl ultivrs of ommon en (Phseolus vulgris) nd its orreltion with resistne to drought stress. Physiologi Plntrum 12, Felly R, Hnin M, Montorzi G, Frey J, Freierg C, Golinowski W, Stehelin C, Broughton WJ, Jouri S (1998) nodd2 of Rhizoium sp. NGR234 is involved in the repression of the nodabc operon. Moleulr Miroiology 27, Fry J, Wood M, Poole PS (21) Investigtion of myo-inositol tolism in Rhizoium leguminosrum v. viie nd its effet on nodultion ompetitiveness. Moleulr Plnt Miroe Intertions 14, Gonzlez EM, Gordon AJ, Jmes CL, Arrese-Igor C (1995) The role of surose synthse in the response of soyen nodules to drought. 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