Differential Regulation of the Nodulation Zone by Silver Ions, L-α-(2-Amino-Ethoxyvinyl)-Glycine, and the skl Mutation in Medicago truncatula

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1 HAYATI Journl of Biosciences Mrch 2010 Vol. 17 No. 1, p EISSN: Aville online t: DOI: /hj Differentil Regultion of the Nodultion Zone y Silver Ions, L-α-(2-Amino-Ethoxyvinyl)-Glycine, nd the skl Muttion in Medicgo trunctul JOKO PRAYITNO 1, ULRIKE MATHESIUS 2 1 Institute for Environmentl Technology, BPP Teknologi, Gd. 412 Puspiptek Serpong, Tngerng 15314, Indonesi 2 Reserch School of Biology, Austrlin Reserch Council Centre of Excellence for Integrtive Legume Reserch, Austrlin Ntionl University, Cnerr ACT 0200, Austrli Received Decemer 2, 2009/Accepted Ferury 19, 2010 Nodule formtion in Rhizoium-legume symiosis is negtively regulted y ethylene. Ethylene inhiitors such s L-α-(2-mino-ethoxyvinyl)-glycine (AVG) nd silver ions (Ag + ), the ethylene-insensitive sickle mutnt, nd trnsgenic plnts were used to study ethylene-medited responses in nodultion. The mode of ction of ethylene inhiitors AVG nd Ag +, nd the skl muttion occur t different steps in ethylene iosynthesis nd perception. Their effects on root growth nd nodultion phenotypes, in prticulr nodule distriution long the primry root, were compred in this study. Ag + nd AVG tretments showed similr root growth responses to skl mutnt. However, nodule distriution in the hypernodulting skl mutnt is different from tht of wild-type plnts grown on either AVG or Ag +. AVG incresed nodule numers nd widened the nodultion zone, while the skl mutnt hd n incresed numer of nodules within the susceptile zone of nodultion. Ag + reduced nodule numers, restricted the nodultion zone, nd restored the nodultion phenotype of skl to tht of the wild type. Key words: ethylene, nodultion, Rhizoium-legume symiosis, Medicgo trnctul INTRODUCTION Phytohormones re known to control nodule initition nd development in Rhizoium-legume symiosis. Ethylene, gsous phytohormone, negtively ffects the process of nodule development (Hirsch & Fng 1994; Guinel & Geil 2002; Fergusson & Mthesius 2003). The involvement of ethylene in nodultion hs een suggested for more thn three decdes (Hirsch & Fng 1994; Guinel & Geil 2002; Ferguson & Mthesius 2003). Lter, the involvement of ethylene in nodultion ws demonstrted using ethylene inhiitors such s AVG nd Ag +, using n ethylene-insensitive mutnt, nd trnsgenic plnts. The inhiitory effect of ethylene on nodultion could e overcome y the tretment with AVG (Peters & Crist- Estes 1989; Ligero et l. 1991; Lee & LRue 1992), or Ag + (C et l. 1998; Nukui et l. 2000). AVG inhiits ethylene iosynthesis y inctivtion of ACC synthse, n enzyme tht converts S-denosyl methionine to ACC (Lin et l. 2009). Ag + is thought to lock ethylene perception y replcing the copper cofctor present in the ethyleneinding site of the receptor. Receptors contining Ag + ind ethylene ut fil to trnsduce the signls from the receptors to their downstrem signlling cscde (Rodriguez et l. 1999). Also, the use of n ethylene-insensitive mutnt of Medicgo trunctul (the skl mutnt), which hs n Corresponding uthor. Phone: , Fx: , E-mil: joko2812@yhoo.co.id incresed numer of nodules when inoculted with S. meliloti (Penmets & Cook 1997), confirms the negtive role of ethylene in the nodule developmentl progrm. The skl mutnt hs muttion in component of ethylene signlling, most likely similr to AtEIN2 (Penmets & Cook 1997). Another support for the role of ethylene in nodultion is shown in trnsgenic L. jponicus plnts crrying the melon ethylene receptor gene Cm-ERS1/ H70A tht re defective in ethylene inding s result of point muttion t the 70th mino cid (Nukui et l. 2004). In this trnsgenic plnt, nodule primordi nd infection thred numers re incresed ecuse of its ethylene insensitivity. The mode of ction of ethylene inhiitors AVG nd Ag +, nd the skl muttion occur t different steps in ethylene iosynthesis nd perception. However, none of previous studies directly compred their effects on root growth nd nodultion phenotypes, in prticulr nodule distriution long the primry root following rhizoiol inocultion. This nodultion phenotype is n importnt feture to study the utoregultion of nodultion which controls the formtion of nodule numers in younger root tissues (Cetno-Anollés & Gresshoff 1991; vn Noorden et l. 2006). In this report, the effect of the ethylene inhiitors Ag + nd AVG on nodule distriution long the primry root ws investigted nd compred to tht of the skl mutnt. In ddition, the ethylene inhiitor Ag + nd AVG were used to test whether Ag + - or AVG-treted wildtype plnts would phenocopy the nodultion nd root phenotypes of skl upon inocultion with rhizoi such s Copyright 2010 Institut Pertnin Bogor. Production nd hosting y Elsevier B.V. This is n open ccess rticle under the CC BY-NC-ND license (

2 16 PRAYITNO AND MATHESIUS HAYATI J Biosci the incresed nodule numers, nd reduction of root growth. Likewise, AVG ws used to test whether it induced similr nodule nd root growth responses s Ag + in the skl mutnt. MATERIALS AND METHODS Plnt nd Bcteril Growth Conditions. Seeds of skl mutnt were otined from Prof. Dougls R. Cook (Penmets & Cook 1997). Seeds of cv Jemlong A17 were used s the wild type. Seeds were scrified nd surfce sterilized with 6.25% (v/v) sodium hypochlorite for 15 min. After severl wshing, seeds were incuted on nitrogenfree Fåhreus gr medium (Fåhreus 1957) in drk-cold condition (4 o C) for 2 dys to rek their dormncy. A drop of sterile wter ws pplied on ech seed to prevent the seeds from drying. Seeds were then germinted y incuting in the drk t 28 o C overnight, Seedlings with similr root length were selected nd trnsferred to 15 cm Petri dishes contining Fåhreus gr medium. The seedlings were incuted verticlly in the growth chmer with photon flux density of 90 µmol m -2 s -1, nd 16 h of light per dy t 20 o C for 2 dys. To reduce light intensity round roots, pieces of drk-thick pper were plced t the lower hlf etween the pltes. After 2 dys incution, the seedlings were trnsferred to fresh Fåhreus pltes contining AVG nd/or Ag +, nd incuted in the sme growth chmer. The seedlings were flood-inoculted t the root tips with 5 µl of diluted Sinorhizoium suspension 24 h lter. The positions of the root tips t the time of inocultion ( ) were mrked on the pltes to provide n initil point of mesurement for root growth nd nodule position. Sinorhizoium meliloti strin 1021 ws grown in liquid Bergensen s modified medium (Rolfe et l. 1980) t 28 o C overnight, nd diluted with sterile wter to n opticl density (OD600) of 0.1 or pproximtely 10 7 cells/ml. As controls, roots were inoculted with n equivlent mount of diluted Bergensen s modified medium. RESULTS Ag + Restores the Wild-Type Nodule Phenotype in skl. After trnsferring seedlings to medium contining Ag +, newly grown wild-type roots were thinner with smller root dimeter (Figure 1). These morphologicl chnges were not oserved on the wild-type plnts trnsferred to control pltes devoid of Ag +. The plnts continued forming thinner roots fter inocultion with S. meliloti. Fresh root sections of the region 15 to 20 mm elow showed tht the reduced root dimeter ws result of decrese in cell expnsion, consistent with the role of ethylene on rdil cell expnsion (Figure 1 compred to 1c). The inoculted skl plnts, regrdless of Ag + tretments, lso hd thinner roots compred to the control wild-type plnts (Tle 1). In inoculted wild-type plnts, the increse of Ag + concentrtion to 0.1 µm grdully enhnced the primry root growth (Figure 1d). In inoculted skl plnts, 0.01 µm Ag + hd no effect on primry root growth, ut tretment of 0.1 µm Ag + shrply incresed the primry root growth to lmost doule tht of control tretment (197%) (Figure 1d). In contrst to its primry root growth response, nodule numers of wild-type plnts were not ffected y 0.01 nd 0.1 µm Ag +, yet t higher Ag + concentrtion (1 µm), nodule numers were significntly reduced (LSD test, á=0.05) (Figure 1e). Similrly, in skl plnts, nodule numers were not ffected y low concentrtion of Ag + (0.01 µm). Higher Ag + concentrtions (0.1 nd 1 µm), however, shrply reduced nodule numers in skl plnts to the level of the corresponding wild-type plnts (Figure 1e). Nodule distriution in skl plnts grown on medium contining Ag + were then nlysed nd compred with control wild-type nd skl plnts (devoid of Ag + ). Nodules were formed on wild-type roots within the region close to (Figure 1f). In skl plnts, n incresed numer of nodules were formed in the susceptile zone of nodultion, producing cluster of nodules with reduced size (Figure 1g). The sme nodultion phenotype ws lso oserved in skl plnts grown on medium contining 0.01 μm Ag + (Figure 1h). In contrst, skl plnts grown on 0.1 nd 1 μm Ag + hd significntly reduced nodule numers (Figure 1e), with nodules formed only in the nrrow zone close to (Figure 1i). In ddition, the nodule size ws lso comprle to tht of the wild-type plnts (Figure 1f nd i). Therefore, Ag + concentrtions of 0.1 nd 1 μm restored the wild-type nodule phenotype in skl plnts. The events of Ag + -enhncement of primry root growth nd Ag + -inhiition of nodultion in skl plnts occurred t the sme Ag + concentrtion (0.1 μm) (Figure 1d nd e). A significnt correltion etween primry root growth nd nodultion ws oserved in Ag + -treted skl plnts (R = ; P < 0.001), ut not in Ag + -treted wildtype plnts (R = ; P = 0.133). Tken together, the hypernodultion phenotype of skl cused root growth inhiition, nd when the nodultion phenotype ws restored to the wild-type level, the inocultion-reduced root growth ws not present. AVG Increses Nodultion y Incresing the Susceptile Zone of Nodultion. As shown in Figure 2, AVG concentrtion determined the response of primry root growth. In inoculted wild-type plnts, for exmple, 0.1 µm AVG incresed primry root growth, while 10 µm AVG ppered toxic to the plnt nd severely reduced primry root growth. Similrly, 10 µm AVG inhiited primry root growth in inoculted skl plnts. AVG lso reduced the root dimeter of inoculted wild-type plnts in similr fshion to Ag + tretment (s shown in Figure 1-c). AVG incresed nodule numers of wild-type roots in dose dependent mnner, with 0.1 nd 1 µm AVG giving the highest result (Figure 2). These AVG tretments induced pproximtely three times the numer of nodules developed in the untreted control wild-type roots, ut with smller size. Interestingly, 0.1 nd 1 µm AVG induced nodule formtion further down towrd the root tips (Figure 2c), indicting tht AVG increses nodule numers in wildtype roots y expnding the nodultion zone. Nodules were spred long the primry root to t lest 70 mm from. In terms of nodule distriution long the root, AVGtreted wild-type plnts hd distinct nodule phenotype

3 Vol. 17, 2010 Ag +, AVG, skl on M. Trunctul 17 d c e f g h i Figure 1. Root nd nodultion responses of A17 (wild type) nd skl mutnt to Ag + tretments upon inocultion with S. meliloti strin () morphologicl chnges of wild-type root fter eing exposed to Ag + (indicted y white rrow hed). (-c) comprison of root dimeter of untreted (), or Ag + -treted (c) wild-type roots in the region mm elow. (d) primry root growth t 9 dpi. Vlues re the men ± SE of 15 plnts. (e) nodule numers of wild-type nd skl plnts t vrious Ag + concentrtions scored t 21 dpi. Vlues re the men ± SE of 15 plnts. (f-i) nodule phenotype of (f) wild type, (g) skl, (h) skl treted with 0.01 µm Ag +, nd (i) 0.1 µm Ag +. White rrowheds indicte the position of. White rs = 2 mm (, f-i), nd 100 µm (-c). Strs indicte significnt differences to their corresponding control tretment (LSD test, á=0.05). : WT, : skl. Tle 1. Effect of Rhizoium inocultion nd Ag + on corticl cell length nd root dimeter of wild-type nd skl plnts Tretment A17 (wild type) A17 + Sm1021 A17 + Sm Ag + skl skl + Sm1021 skl + Sm Ag + Corticl cell length Root dimeter # (μm) (μm) c c c c c c Corticl cell length ws determined from twenty cells selected rndomly from the second nd third corticl lyers of the root in the region mm elow. Vlues re men + SD of ten plnts. Vlues followed y the sme letter do not differ significntly ccording to the Lest Significnt Difference (LSD) test, with the significnce level (á)=0.05. # Root dimeter ws determined from cross-sections of fresh roots in the region mm elow. Vlues re men + SD of five plnts. Vlues followed y the sme letter in the column do not differ significntly ccording to LSD test (á = 0.05). from the skl mutnt, since the nodules did not form cluster (Figure 1g compred to Figure 2c). In ddition, incresed nodule numers in AVG-treted wild-type plnts did not reduce primry root growth s oserved in skl plnts (Figure 2-). In contrst to its effect on wild type, 0.1 µm AVG hd no effect on nodule numers nd nodultion zone of skl (Figure 2). A higher AVG concentrtion (10 µm) decresed nodule numers of skl plnts, nd the cluster of nodules in the susceptile zone of nodultion ws mintined. Thus, when compred to Ag +, AVG tretments hd distinct effects on the nodultion response in oth skl nd wild-type plnts. The Negtive nd Positive Role of Ag + nd AVG, Respectively, in the Regultion of the Nodultion Zone. As 0.1 µm Ag + restored the nodultion phenotype of the skl mutnt to tht of wild-type plnts (Figure 1e nd i), the sme concentrtion of Ag + ws used to test if it ws le to restore the nodultion phenotype of AVG-treted wild type (0.1 µm) to untreted wild type. Ag + supplement hd no detectle effect on nodule numers in AVG-treted wild-type roots. For exmple, nodule numers in AVGtreted nd Ag + -AVG-treted wild-type roots were 9.5 nd 11.6, respectively (LSD test, P 0.05 = 0.184; Figure 3). However, Ag + supplement nrrowed the nodultion zone of AVG-treted wild-type plnts, which ws close to the

4 18 PRAYITNO AND MATHESIUS HAYATI J Biosci c (Figure 3). In skl plnts, Ag + supplement significntly reduced nodule numers in AVG-treted plnts from 34.5 to 13.9 (Figure 3), in which nodule distriution ws lso close to the (Figure 3c). Nodule numers of Ag + -AVGtreted skl plnts (13.9), however, were significntly higher thn tht of Ag + -treted skl plnts (5.5), ut were comprle to tht in Ag + -AVG-treted wild-type plnts (Figure 3). In ddition, the nodultion zone of Ag + -AVGtreted skl plnts ws similr to tht in Ag + -AVG-treted wild type plnts (Figure 3-c) d Figure 2. Root nd nodultion responses of A17 (wild-type) nd skl plnts to AVG upon inocultion with S. meliloti. () Primry root growth mesured t 9 dpi. () nodule numers scored t 21 dpi. Vlues re men + SE of 15 plnts. (c) nodule distriution in the wild-type root treted with 0.1 µm AVG. White rrowheds indicte the position of. Br = 2 mm. Strs indicte significnt vlues to their corresponding control tretment (LSD test, á = 0.01). : A17, : skl. c c Figure 3. Effect of dul tretment of AVG nd Ag + on nodule numers. () nodule numers of A17 (wild type) nd skl in response to 0.1 μm AVG nd 0.1 μm Ag +. Tretments hving the sme lowercse letters re not significntly different ccording to the Lest Significnt Difference test (á = 0.05) (+ SE; n=15). () nodule phenotype of AVG-Ag + -treted A17 plnts. (c) nodule phenotype of AVG-Ag + -treted skl plnts. White rrows indicte. Brs = 2 mm. : A17, : skl. AVG (μm) c DISCUSSION AVG nd Ag + Effect on Non-Symiotic nd Symiotic Properties of M. trunctul. In this study, the root growth nd nodultion responses of inoculted wild-type plnts to the ethylene inhiitors AVG nd Ag + hve een exmined nd compred to tht of the skl mutnt. The results showed tht in inoculted wild-type plnts, the ppliction of AVG stimulted root growth nd reduced root dimeter in similr fshion to tht of Ag + tretment or tht of skl plnts. This indictes tht AVG nd Ag + tretments, nd the skl muttion give similr effects on non-symiotic properties of M. trunctul upon inocultion with Rhizoium, which cn e ttriuted to defect or reduction in ethylene ction on cell expnsion (Figure 1). This is consistent with the role of ethylene in the root growth of Aridopsis (Ecker 1995; Smlle & vn der Streten 1997). In contrst to their effect on non-symiotic properties, Ag +, AVG nd the skl muttion cused sustntilly different symiotic properties upon inocultion, which re summrised in Figure 4. Ag + reduced nodule numers in wild-type plnts nd restored the nodultion phenotype of the skl mutnts to tht of the wild-type plnts (Figure 1e, i). In this study, Ag + concentrtions etween 0.01 nd 0.1 µm were criticl for nodule development in skl, ecuse shrp reduction of nodule numer occurred etween these two concentrtions (Figure 1e). Previous reports hve demonstrted the positive effect of Ag + tretment on nodultion in pe, lflf nd L. jponicus (Lee & LRue 1992; C et l. 1998; Nukui et l. 2000). In the present study, high Ag + concentrtion, up to 1 µm, did not induce ny visile negtive effect on other spects of plnt growth, thus excluding the possiilities tht the toxic level of Ag + is the cuse for the restortion of wildtype nodultion in skl plnts. Fern nd LRue (1991) reported tht 0.1 µm Ag + decresed colony size of Rhizoium, suggesting tht this concentrtion ws inhiitory to cteril growth. Results in this chpter showed tht 0.1 µm Ag + did not decrese nodule numers of wild-type plnts. Therefore, the cteri lone re not likely cuse for the reduction of nodule numers in skl. Ag + is thought to inhiit ethylene perception y inding to ethylene receptors nd then lock the trnsmission of ethylene signls (Rodriguez et l. 1999). Ag + lso reduced the expression of ethylene receptor genes in rice such s OsERS1, OsERS2, nd OsETR1 (Yu et l. 2004). However, the restortion of the nodule phenotype in skl y Ag + suggests tht Ag + my not specificlly lock the ethylene

5 Vol. 17, 2010 Ag +, AVG, skl on M. Trunctul 19 perception, ecuse ethylene perception is ltered in the skl mutnt (Penmets & Cook 1997). Ag + hs long een known s n inhiitor of ATPses (Knee 1992), enzymes involved in ion trnsport such s N + /K +, C 2+ nd hevy metl ions (Axelsen & Plmgren 2001). Since the inhiition of ATPses would prevent norml cellulr functions, Ag + might inhiit the specific nd non-lethl ATPse function in nodultion. AVG enhnced nodule numers nd widened the nodultion zone in wild-type plnts (Figure 2-c, 4). This is the first time tht AVG hs een shown to increse the nodultion zone in legumes, nd rises the possiility tht AVG cn lock the utoregultion mechnism in M. trunctul. An erlier report, however, showed tht AVG incresed nodule numers in lflf (M. stiv) without the increse of susceptile nodultion zone (Peters & Crist-Estes 1989). Similrly, ddition of AVG to Vici stiv roots in split root system using Jensen s liquid medium did not olish utoregultion (vn Brussel et l. 2002). This pprent contrdiction might reflect differences in the species context or the experimentl conditions. Becuse AVG is n ethylene inhiitor, it would e expected then, tht AVG tretment would give essentilly the sme result s the ltertion in ethylene signlling, such s found in skl. The nodultion phenotype of AVGtreted wild-type plnts, however, is different from tht of skl mutnts, s enhnced nodule numers in skl plnts re restricted to the susceptile zone of nodultion (Penmets & Cook 1997; Figure 1g). Thus, these conflicting results could not e reconciled s inhiition of ethylene signlling. In Aridopsis, studies using AVG Ag A17 A17 A17 skl A17 skl skl skl ethylene inhiitors nd the ein2 mutnt to determine the interction of ethylene with scisic cid (ABA) in root growth gve similr prdox (Ghssemin et l. 2000). Similr results were lso found in studies to determine the role of ethylene inhiitors on germintion nd seedling growth of rley (Locke et l. 2000). One possile explntion for this prdox is tht AVG inhiits nother pthwy distinct from ethylene iosynthesis, which could e involved in the utoregultion mechnism. AVG inhiits ethylene iosynthesis y inctivtion of ACC synthses through inding to their cofctor, pyridoxl 5 -phosphte (PLP) (Cpitni et l. 1999). ACC synthses elong to the fmily of PLP-dependent enzymes (Alexnder et l. 1994). Other PLP-dependent enzymes include ornithine decroxylse (ODC) nd rginine decroxylse (ADC), the enzymes involved in the synthesis of polymines such s putrescine, spermine nd spermidine. AVG is lso known s n inhiitor of cystthionine β lyse, PLPdependent enzyme involved in methionine iosynthesis (Rvenel et l. 1998). It is possile tht the incresed re of nodule distriution y AVG is result of the inctivtion of PLP-dependent enzyme other thn ACC synthses. Further experiment would e necessry to test this hypothesis. Nevertheless, the nodule phenotype of skl exposed 0.1 μm AVG, the concentrtion tht stimulted nodule numers nd widened the nodultion zone in wild type, ws similr to untreted skl (Figure 1g), indicting tht AVG ction in widening the nodultion zone my require the SKL gene product. This could lso e prt of the pleiotrophic effects of the skl muttion in nodultion. Consistent with single ppliction of the ethylene inhiitor, Ag + supplement reduced the nodultion zone without ffecting nodule numers in AVG-treted wild type (Figure 3-), indicting the mintennce of nodultion homeostsis under these tretments. Conversely, AVG supplement incresed nodule numers nd the nodultion zone of Ag + -treted skl (Figure 3, c). Together, results presented here suggest tht AVG nd Ag + hve positive nd negtive role, respectively, in the regultion of nodule numers nd nodultion zone in M. trunctul. They lso suggest tht inhiitor studies should e treted with cution ecuse of unknown side effects of the inhiitors. The involvement of AVG nd Ag + s positive nd negtive inducer in determining the nodultion zone s presented here my provide dditionl tools to study the regultion of nodule development in legumes, in prticulr in reltion to utoregultion of nodultion. REFERENCES Figure 4. Summry of nodultion phenotypes in A17 (wild type) nd skl treted with AVG nd/or Ag +. The tle indictes comintion tretments (mrked with +) of AVG nd Ag + in wild type nd skl. The nodultion phenotype of ech comintion is shown elow the column. Blck dots represent nodules. The horizontl line represents. Alexnder FW, Sndmeier E, Meht PK, Christen P Evolutionry reltionships mong pyridoxl-5'-phosphtedependent enzymes. Regio-specific lph, et nd gmm fmilies. Eur J Biochem 219: Axelsen KB, Plmgren MG Inventory of the superfmily of P-type ion pumps in Aridopsis. Plnt Physiol 126: C JM, Reclde L, Ligero F Nitrte-induced ethylene iosynthesis nd the control of nodultion in lflf. Plnt Cell Environ 21:87-93.

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