Root and Nodulation Phenotypes of the Ethylene-Insensitive Sickle Mutant of Medicago truncatula

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1 HAYATI Journl of Biosienes Septemer 2010 Vol. 17 No. 3, p EISSN: Aville online t: DOI: /hj Root nd Nodultion Phenotypes of the Ethylene-Insensitive Sikle Mutnt of Medigo truntul JOKO PRAYITNO Institute for Environmentl Tehnology, The Ageny for the Assessment nd Applition of Tehnology Gd. 412 Puspiptek Serpong, Tngerng 15314, Indonesi Phone: , Fx: , E-mil: joko2812@yhoo.o.id Reeived Deemer 2, 2009/Aepted August 18, 2010 The sikle (skl) mutnt of the model legume Medigo truntul is n ethylene-sensitive mutnt tht hve ten-fold inrese in nodule numers. The nodultion nd root phenotypes of the skl mutnt were investigted nd further hrterised. The skl mutnt hd longer roots thn the wild type, ut when inoulted with Sinorhizoium, its root length ws redued to the level of wild type. Furthermore, lterl root numers in uninoulted skl were similr to those in uninoulted wild type. However, when the root tips were depitted, fewer lterl roots formed in skl thn in wild type. Nodule numers of the skl mutnt were signifintly redued y low nitrte onentrtion (2.5 mm). These results suggest tht skl mutnt hs ltertions in oth root nd nodule development. Key words: sikle mutnt, ethylene insensitive, Medigo truntul INTRODUCTION Nodule development is tightly ontrolled y the host plnt. Nodules re formed mostly in the root region etween the root tip nd elongtion zone t the time of inoultion, whih is known s the suseptile zone of nodultion (Bhuvneswri et l. 1980, 1981). Autoregultion of nodultion (AON) is one of the mehnisms ontrolling the formtion of nodule numers in younger root tissues (Kinkem et l. 2006). Mutnts defetive in utoregultion llow mny new nodules to grow in newly developing roots, resulting in n enhned nodultion or so-lled supernodultion phenotype (Croll et l. 1985; Sgn & Du 1996; Wopereis et l. 2000; Penmets et l. 2003). The gene regulting utoregultion hs een identified in severl legumes nd enodes leuine-rih repet reeptor-like kinse (Krusell et l. 2002; Serle et l. 2003; Shnel et l. 2005). The utoregultion mutnts re le to nodulte in the presene of high nitrte onentrtions (Croll et l. 1985,; Wopereis et l. 2000; Kwguhi et l. 2002). This muttion lso hs pleiotropi effets on other spets of plnt development s shown in the root phenotype of the hr1-1 mutnt of Lotus jponius nd the sym29 mutnt of Pisum stivum nd the sunn (super numerry nodules) mutnt of M. truntul (Sgn & Du 1996; Wopereis et l. 2000; vn Noorden et l. 2006). These mutnts hve short root systems nd ltered numers of lterl roots. Grfting studies on these mutnts determined tht utoregultion is shoot ontrolled (Delves et l. 1986; Jing & Gresshoff 2002; Krusell et l. 2002; Penmets et l. 2003). Other endogenous signls tht ontrol nodule development re phytohormones. Ethylene hs negtive role in the nodultion of severl legumes nd is involved t vrious stges of nodule development (Guinel & Geil 2002; Ferguson & Mthesius 2003; Mulder et l. 2005). Consistent with the negtive role of ethylene in nodultion, the ethylene-insensitive mutnt of Medigo truntul, sikle (skl), hs hypernodultion phenotype nd n inresed numer of sustined infetion threds (Penmets & Cook 1997). In ontrst to utoregultion mutnts, the hypernodultion phenotype of skl is root ontrolled (Pryitno et l. 2006). It is not known whether the skl mutnt hs pleiotrophi effets other thn its insensitivity to ethylene, suh s found in utoregultion mutnts. Beuse the skl mutnt is defetive in ethylene signlling pthwy ortholog of Aridopsis EIN2 (Penments et l. 2008), it is likely tht the skl muttion hs n effet on some spets of ethylene-medited plnt growth, suh s dventitious nd lterl roots formtion. It hs een known tht ethylene medites wound-stress response in plnts. Depittion of the root tip indues ethylene-medited stress responses from wounding (O Donell et l. 1996) nd indues lterl root formtion (Aloni & Plotkin 1985). Conversely, the formtion of dventitious roots in uttings of the ethyleneinsensitive Never-ripe (NR) tomto is signifintly redued ompred to tht of the wild type (Wilkinson et l. 1997). This pper reports the effets of skl muttion on the spets of root growth nd nodultion other thn those lredy desried y Penmets nd Cook (1997). These spets inlude primry root growth nd lterl root formtion, nodule numers nd nodule distriution long the root, nd the nodultion response to nitrte. Results presented here showed tht tht the skl mutnt displys pleiotrophi effets on root growth nd nodultion.

2 132 PRAYITNO HAYATI J Biosi MATERIALS AND METHODS Plnt nd Bteril Growth Conditions. Seeds of skl mutnt were otined from Prof. Dougls R. Cook (Penmets & Cook 1997). Seeds of v Jemlong A17 were used s the wild type. Seeds were srified nd surfe sterilized with 6.25% (v/v) sodium hypohlorite for 15 min. After severl wshes, seeds were inuted on nitrogenfree Fåhreus gr medium (Fåhreus 1957) in the drk t 4 o C for 2 dys to rek their dormny. A drop of sterile wter ws pplied to eh seed to prevent the seeds from drying. Seeds were then germinted y inuting in the drk t 28 o C overnight. Seedlings with similr root length were seleted nd trnsferred to 15 m Petri dishes ontining Fåhreus gr medium. The seedlings were inuted vertilly in the growth hmer 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. After 2 dys inution, the seedlings were trnsferred to fresh Fåhreus pltes, nd inuted in the sme growth hmer. The seedlings were floodinoulted t the root tips with 5 µl of diluted Sinorhizoium suspension 24 h lter. The positions of the root tips t the time of inoultion (RT 0 ) were mrked on the pltes to provide n initil point of mesurement for root growth nd nodule position. Root growth ws mesured from RT 0 to the root tip using ruler t ertin time points. 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 optil density (OD600) of 0.1 or pproximtely 10 7 ells/ml. As ontrols, roots were inoulted with n equivlent mount of diluted Bergensen s modified medium. Depitted Root Experiments. Roots of skl nd wildtype plnts were depitted 5 mm from the root tip, nd grown on Fåhreus gr medium. Lterl roots (LRs) emerging from the depitted primry root were reorded t 14 dys post depittion. To exmine the effet of IAA on lterl root formtion in skl, n gr lok (5 x 10 x 5 mm) ontining 10 µm IAA ws pled on the hypootyls immeditely fter root tip depittion. For ontrol tretments, n gr lok without IAA ws pled on nother set of hypootyls. type. This inoultion-redued PRG ws oserved until the experiment ended t 21 dys post inoultion (DPI). Altered Lterl Root Formtion in Depitted Roots of the skl Mutnt. Altertion of lterl root (LR) phenotypes is ommonly found in hypernodultion mutnts. Therefore, the effet of skl muttion on LR formtion ws exmined. Under non-symioti onditions, LR formtion in skl ws not signifintly different to tht in wild-type roots t 21 dy of growing, with the verge numer of 4.9 nd 3.2 lterl roots per plnt, respetively (Student s t-test, P = 0.074, n = 20). It hs een estlished tht ethylene medites woundstress response in plnts. Sine intt seedlings of the skl mutnt showed no ltertion in LR formtion, the effet of skl muttion on the numer of LR in depitted seedlings (wound stress) of M. truntul ws investigted. Depittion of the root tip indued the formtion of LRs in wild-type nd skl plnts mostly t the utting site. The numer of LR formed in skl t 7 dys fter depittion (DAD) ws only 37% of the wild-type plnts, nd this proportion ws slightly redued t 14 dys (35%). LR numers of the wild-type nd skl plnts t 14 DAD were 3.7 nd 1.3, respetively (Figure 2). Initilly, skl plnts hd signifintly shorter LR length thn wildtype plnts (35% of the wild-type) t 7 DAD (Figure 2). Lter t 14 DAD, its LR length ws not signifintly different from tht found in the wild-type plnts. These results suggest tht defet in ethylene signlling suh s found in the skl mutnt, uses redution in LR formtion fter wounding nd dely in LR elongtion during erly stge of LR development (7 DAD). Auxin is known to stimulte lterl root formtion in intt plnts nd uttings. Sine the numer of LR ws redued in the depitted roots of skl, the skl mutnt my hve n ltered uxin response for the indution of LR formtion fter wounding. To test this, roots of skl nd wild-type plnts were depitted 5 mm from the root tip, nd grown on Fåhreus medium in the sene or RESULTS The Inoulted skl Mutnt hs Redued Root Growth. Uninoulted skl plnts hd longer primry root growth (PRG) thn uninoulted wild-type plnts (Figure 1). This differene ws oserved s erly s 3 dys fter initil mesurement (Student s t-test, P < 0.01). At 9 dys fter initil mesurement, the PRG of uninoulted skl ws doule tht of uninoulted wild type. Inoulted skl roots were then nlysed to see whether their growth ws pertured s result of the hypernodultion phenotype. Inoultion hd no effet on the PRG of wild type (Figure 1). In ontrst, inoultion redued the PRG of skl to length omprle to wild dpi Figure 1. Root growth of A17 (wild type) nd skl mutnt. dpi = dys post inoultion. Vlues re the men + SE of 15 plnts. A17, A17 + Sm1021, skl, skl + Sm1021.

3 Vol. 17, 2010 Nodule nd Root Phenotypes of skl Mutnt 133 presene of IAA (Figure 3). LRs emerging from the depitted primry root were reorded t 14 DAD. IAA indued LR formtion in the depitted roots of skl nd wild type to similr extent (1.7 nd 1.6 fold inrese, respetively; Figure 3). To determine LR distriution in the depitted primry roots, the distne of LRs from the utting site ws mesured nd ompred etween wild type nd skl. As shown in Figure 3, in the sene of Figure 2. Lterl root formtion fter root tip depittion. () lterl root numers per plnt. () lterl root length. Vlues re men + SE of the verge lterl root length from plnts. Different lower se letters indite signifint differene etween tretments ording to the non-prmetri Mnn-Whitney test t P < WT, skl. d IAA, the depittion of root tips indued LR formtion in wild type nd skl lose to the utting site. The enhnement of LR numer following IAA tretments in oth genotypes ws minly due to the inrese of LR numer towrd the hypootyl (sipetl). This ws more pronouned in wild type, where the mrked inrese of LRs ws found in the zone of 1-15 mm from the utting site (Figure 3). Although there ws n inrese in sipetl LR numer in skl, the distriution pttern of LR in the depitted primry roots ws similr regrdless of IAA tretment, with the highest numer of LR t the utting site (Figure 3). These results demonstrte tht when the root tip is depitted, the skl mutnt hs n ltered response to IAA-stimulted LR formtion in the primry root. Nodule Numers nd Nodule Distriution in the skl Mutnt. Nodule numers nd nodule distriution in skl were exmined t 21 DPI. In wild type, the verge numer of nodules per plnt t 21 DPI ws 3.2. These nodules were formed in the suseptile zone of nodultion, whih ws pproximtely 10 mm ove RT 0 nd 20 mm elow RT 0 (Figure 4,). In skl, higher numer of nodules ws lso oserved in the suseptile zone with the verge numers per plnt eing In this region, nodules formed luster with redued size of eh nodule (Figure 4,d). In smll perentge of plnts (two in fifteen plnts), two lusters were present long the primry root of skl, one of whih ws found eyond the suseptile zone, or pproximtely in the root region etween 25 to 35 mm elow RT 0 (Figure 4e). The Nodultion of the skl Mutnt in the Presene of High Nitrte Conentrtion. Nitrte tolerne is n importnt hrteristi of supernodultion mutnts defetive in utoregultion of nodultion. To investigte the nodultion response of skl to nitrte, vrious levels of nitrte were tested. Nitrte onentrtion s low s 2.5 mm shrply redued nodule numers in wild type to 12% of the ontrol tretment (Figure 5). The sme nitrte onentrtion lso deresed nodule numers in skl, ut not s severely s in the wild type, i.e. to 56% of the Figure 3. Lterl root formtion t 14 dys fter root-tip depittion. () shemti digrm of the experiment. The root ws depitted 5 mm from the root tip (X), nd n gr lok (5 x 10 x 5 mm) ontining 10 µm IAA ws pled on the hypootyl. The plnts were inuted on gr medium. () effet of 10 µm IAA on lterl root numers. Brs with different lowerse letters re signifintly different ording to the non-prmetri Mnn-Whitney test, P < 0.05 (n = 15-17, men + SE). () distriution of lterl roots on the depitted primry roots. The depitted primry roots were divided into five zones, t the utting site (0 mm), 1-5, 6-10, nd >15 mm ove the utting site (Men + SE, n = 15-17).

4 134 PRAYITNO HAYATI J Biosi Figure 4. Nodule formtion nd distriution t 21 DPI. () nodule formtion in wild type. () nodultion region oxed in () is highlighted. () nodule formtion in skl. (d) luster of nodules oxed in () is highlighted. Blk or white rrows indite RT 0. Brs = 1 mm. (e) two lusters of nodules formed in skl in the region etween white strs nd white dots. White rrow indites RT 0. Br = 2.5 mm. Figure 5. Nodule numers in response to nitrte t 21 DPI. Vlues re normlised to untreted ontrol plnts (men + SE of 20 plnts). The verge nodule numers of wild-type nd skl plnts t 0 mm nitrte re 4.7 nd 19.6, respetively. WT, skl. ontrol plnts. Higher nitrte onentrtions from 5 to 20 mm further redued nodule numers in skl to 22%, while nodule numers in wild type were redued to 5% of the untreted ontrol (Figure 5). These results indite tht nitrte inhiits nodultion in the skl mutnt; however the skl mutnt is more tolernt to nitrte thn the wild type. DISCUSSION Root Growth Inhiition of skl Plnts y Sinorhizoium. Results reported here showed tht the skl muttion hd n effet on root growth. In the sene of the symioti prtner, skl roots were longer thn wild-type roots (Figure 1). Other fetures of uninoulted skl roots hve een desried in previous reports inluding n inrese of ell elongtion nd redution of root dimeter (Pryitno & Mthesius 2010) nd root hir elongtion (Oldroyd et l. 2001). Together, these root phenotypes of the skl mutnt showed hrteristis of ethylene insensitivity. In ontrst, the primry root growth in skl mutnt ws redued in the presene of rhizoi. In wild type, inoultion did not redue the primry root growth. Beuse Sinorhizoium inoultion indued the hypernodultion phenotype in skl, these results suggest tht the root growth inhiition of skl upon inoultion is orrelted to its hypernodultion response. Ethylene insensitivity is usl to hypernodultion phenotype of skl, nd the hypernodultion response then redues the primry root growth. Redued plnt growth following inoultion hs een previously reported in supernodulting mutnts hr1-1 of L. jponius nd nts382 of soyen (Croll et l. 1985; Wopereis et l. 2000). However, the root retrdtion in hr1-1 is more extreme thn in skl, nd is oserved oth in the sene nd presene of rhizoi. In ddition, the root growth redution in nts382 ours t lter stge, when the roots hve eome extensively nodulted. Therefore, it ppers tht root growth inhiition in supernodulting mutnts following inoultion is ommon phenomenon in legumes. This inhiition of root growth in supernodulting mutnts ould e result of n inrese in photosynthte llotion into the nodultion region t the expense of the root growth (Wopereis et l. 2000), or hnges in hormonl response or trnsport suh s uxin (vn Noorden et l. 2006). Lterl Root Formtion in the Ethylene-Insensitive Mutnts fter Root Tip Depittion. In trnsgeni plnts onstitutively expressing the Aridopsis etr1-1, the dventitious root formtion of the uttings ws severely inhiited s result of its redued sensitivity to ethylene (Wilkinson et l. 1997; Shiuy et l. 2004), suggesting tht the endogenous uxin, whih is thought to e the primry induer of lterl root formtion, requires ethylene signlling to stimulte the formtion of dventitious roots in uttings. Results presented here demonstrted tht the ethylene-insensitive skl mutnt hs lso n ltered lterl root formtion fter root-tip depittion. The redued pity of the skl mutnt to form lterl roots fter roottip depittion is similr to tht found in the tomto mutnt Never-ripe, n ethylene insensitive mutnt with muttion in the ETR1 reeptor, nd to tht found in the trnsgeni petuni line 44,568 plnts onstitutively expressing the etr1-1 gene (Clrk et l. 1997; Wilkinson et l. 1997; Shiuy et l. 2004). However, lterl root formtion in skl ws more like in the Never-ripe tomto plnts. In skl nd Never-ripe plnts, rooting ws not totlly inhiited (pproximtely 50% of the wild-type plnts), while in the trnsgeni petuni line 44,568 rooting ws lmost totlly inhiited (less thn 1% of the wild type). The dventitious roots of the Never-ripe mutnt nd trnsgeni line 44,568 were shorter thn the wild-type roots t three weeks fter utting. During erly growth of lterl roots, skl plnts lso hd shorter lterl roots thn wildtype plnts, lthough lter, their lterl root elongtion ws similr to tht of wild type. These differenes ould e due to the different muttion onferring ethylene sensitivity in different speies. Following root-tip depittion, LR numers formed on the skl primry roots were signifintly lower thn those on wild-type roots (Figure 2). In ontrst, intt skl plnts hd omprle numers of lterl roots to wild-type

5 Vol. 17, 2010 Nodule nd Root Phenotypes of skl Mutnt 135 plnts. These results suggest tht the redution of LR formtion in skl fter root-tip depittion ws orrelted with its insensitivity to ethylene. Beuse uxin trnsport from the shoot to the root is required for the formtion of lterl roots (Csimiro et l. 2001), nd uxin tretment t the hypootyl indued sipetl LR formtion in wild type (Figure 3), it is likely tht the skl mutnt hs n ltered uxin trnsport or response following root-tip depittion. A model for the role of ethylene nd uxin trnsport in lterl root formtion hs een proposed (Aloni et l. 2006). In this model, ethylene is produed lolly following wounding, flooding or externl ethylene pplition. Lol ethylene umultion lolly inhiits uxin trnsport in the periyle. Immeditely ove this inhiition site, newly rriving IAA is umulted nd then stimultes ell division in the periyle (Aloni et l. 2006). Similrly, the effet of the skl muttion on uxin trnsport nd uxin response during nodultion hs een reported (Pryitno et l. 2006). In their report, ethylene signling negtively ffets uxin uptke into the nodultion zone, prtilly y ffeting PIN1 nd PIN2 gene expression (Pryitno et l. 2006). Nodultion nd Nitrte Responses of the skl Mutnt. Nodule development is restrited to the suseptile zone of nodultion in roots, whih is ontrolled y the host plnt through long distne signlling or known s AON (Kinkem et l. 2006). Altertion of this regultion suh s found in the AON mutnt nts 382 of soyen resulted in n inrese of nodule numers within the wider zone of nodultion (Croll et l. 1985). In the ethylene-insensitive skl mutnt, the inresed nodultion ours within the suseptile zone of nodultion (Figure 4,d), without n inrese in nodultion zone. These results suggest tht the hypernodultion phenotype in skl is regulted y ethylene tht is different from AON. Nitrte inhiition of nodultion is ommon phenomenon mong legume speies. A supernodultion of soyen, nts382, whih hs defet in AON, ws otined from sreens of plnts for nitrte tolerne (Croll et l. 1985,). Nodule numers in this mutnt were inresed in the presene of 5 mm nitrte ompred to wild type (Croll et l. 1985). Another AON mutnt, the hr1-1 mutnt of L. jponius, is lso insensitive to high onentrtions of nitrte (5-15 mm nitrte), with nodules developed t 6 weeks fter inoultion (Wopereis et l. 2000). The wild type of L. jponius only forms few umps in the presene of 15 mm KNO 3. Unlike AON mutnts, nodule numers in skl were redued in the presene of low nitrte onentrtion (2.5 mm), lthough the nodultion in skl ws less sensitive to the inhiitory effet of nitrte thn tht in wild type (Figure 5). Previous reports hve suggested tht ethylene might medite the suppression of nodultion y nitrte, sine nitrte inresed ethylene prodution in lflf roots, nd L-α- (2-minoethoxyvinyl)-glyine (AVG) ould overome the inhiitory effet of nitrte y the inhiition of ethylene iosynthesis (Ligero et l. 1986; Ligero et l. 1991). In ontrst, the nodultion in skl, mutnt hving defet in ethylene pereption, ws suppressed y low nitrte onentrtion (Figure 5). In ddition, two lines of evidene in pe hve suggested tht ethylene might not e involved in nitrte inhiition of nodultion (Lee & LRue 1992). Lee nd LRue (1992) showed tht ddition of Ag +, n ethylene pereption inhiitor, were not le to reverse the inhiitory effet of nitrte on nodultion, nd tht the lok of nodultion y nitrte ourred t n erlier stge thn tht y ethylene. Overll, this study hs shown tht the hypernodulting skl mutnt shows ltertions in oth root nd nodule development. Sine the skl mutnt is defetive in ethylene signling, this mutnt n e used to further nlysis the role of ethylene in stges of nodule development t the tissue, ellulr nd moleulr level to gin etter understnding of plnt development. ACKNOWLEDGEMENT This work ws supported in prt y Austrlin Reserh Counil Centre of Exellene for Integrtive Legume Reserh, Austrlin Ntionl University, Cnerr, Austrli. A grteful to Brry Rolfe nd Ulrike Mthesius for helpful disussion nd supervision in this work. REFERENCES Aloni R, Aloni E, Lnghns M, Ullrih CI Role of ytokinin nd uxin in shping root rhiteture: regulting vsulr differentition, lterl root initition, root pil dominne nd root grvitropism. Ann Bot 97: Aloni R, Plotkin T Wound indued nd nturlly ourring regenertive differentition of xylem in Ze mys L. Plnt 163: Bhuvneswri TV, Bhgwt AA, Buer WD Trnsient suseptiility of root ells in four ommon legumes to nodultion y rhizoi. Plnt Physiol 68: Bhuvneswri TV, Turgeon BG, Buer WD Erly events in the infetion of soyen (Glyine mx (L.) Merr.) y Rhizoium jponium. I. Loliztion of infetile root ells. Plnt Physiol 66: Crroll BJ, MNeil DL, Gresshoff PM Isoltion nd properties of soyen Glyine mx (L.) Merr. mutnts tht nodulte in the presene of high nitrte onentrtions. Pro Ntl Ad Si USA 82: Crroll BJ, MNeil DL, Gresshoff PM A supernodultion nd nitrte tolernt symioti (nts) soyen mutnt. Plnt Physiol 78: Csimiro I, Mrhnt A, Bhlero RP, Beekmn T, Dhooge S, Swrup R, Grhm N, Inze D, Snderg G, Csero PJ, Bennett M Auxin trnsport promotes Aridopsis lterl root initition. Plnt Cell 13: Clrk DG, Gurium EK, Brrett JE, Nell TA, Klee HJ Root formtion in ethylene-insensitive plnts. Plnt Physiol 121: Delves AC, Mthews A, Dy DA, Crter AS, Gresshoff PM Regultion of the soyen-rhizoium symiosis y shoot nd root ftors. Plnt Physiol 82: Fåhreus G The infetion of lover root hirs y nodule teri studied y simple glss tehnique. J Gen Miroiol 16: Ferguson BJ, Mthesius U Signling intertions during nodule development. J Plnt Growth Regul 22: Guinel FC, Geil RD A model for the development of the rhizoil nd rusulr myorrhizl symioes in legumes nd its use to understnd the roles of ethylene in the estlishment of these two symioses. Cn J Bot 80:

6 136 PRAYITNO HAYATI J Biosi Jing Q, Gresshoff PM Shoot ontrol of hypernodultion nd ernt root formtion in the hr1-1 mutntt of Lotus jponius. Fun Plnt Biol 29: Kwguhi M, Imizumi-Anrku H, Koiw H, Niw S, Ikut A, Syono K, Ako S Root, root hir, nd symioti mutnts of the model legume Lotus jponius. Mole Plnt- Miroe Intert 15: Kinkem M, Sott PT, Gresshoff PM Legume nodultion: suessful symiosis through short- nd long-distne signlling. Fun Plnt Biol 33: Krusell L, Mdsen LH, Sto S, Auert G, Genu A, Szzyglowski K, Du G, Kneko T, Tt S, de Bruijn FJ, Pjuelo E, Sndl N, Stougrd J Shoot ontrol of root development nd nodultion is medited y reeptor-like kinse. Siene 420: Lee KH, LRue TA Exogenous ethylene inhiits nodultion of Pisum stivum L. v. Sprkle. Plnt Physiol 100: Ligero F, C JM, Lluh C, Olivres J Nitrte inhiition of nodultion n e overome in the presene of the ethylene inhiitor minoethoxyvinylglyine. Plnt Physiol 97: Ligero F, Luh C, Olivres J Evolution of ethylene from roots of Medigo stiv plnts inoulted with Rhizoium meliloti. J Plnt Physiol 125: Mulder L, Hogg B, Bersoult A, Cullimore J Integrtion of signling pthwys in the estlishment of the legume-rhizoi symiosis. Physiol Plnt 123: O Donnell PJ, Clvert C,Atzorn R, Wsternk C, Leyser HMO, Bowles DJ Ethylene s signl mediting the wound response of tomto plnts. Siene 274: Oldroyd GED, Engstrom EM, Long SR Ethylene inhiits the nod ftor signl trnsdution pthwy of Medigo truntul. Plnt Cell 13: Penmets RV, Cook DR A legume ethylene-insensitive mutnt hyperinfeted y its rhizoil symiont. Siene 275: Penmets RV, Frugoli JA, Smith LS, Long SR, Cook DR Dul geneti pthwys ontrolling nodule numer in Medigo truntul. Plnt Physiol 131: Penmets RV, Urie P, Anderson J, Lihtenzveig J, Gish JC, Nm YW, Engstrom E, Xu K, Skisel G, Pereir M, Bek JM, Lopez- Meyer M, Long SR, Hrrison MJ, Singh KB, Kiss GB, Cook DR The Medigo truntul ortholog of Aridopsis EIN2, sikle, is negtive regultor of symioti nd pthogeni miroil ssoitions. Plnt J 55: Pryitno J, Mthesius U Differentil regultion of the nodultion zone y silver ions, L-α-(2-minoethoxyvinyl)- glyine, nd the skl muttion in Medigo truntul. Hyti J Biosi 17: Pryitno J, Rolfe BG, Mthesius U The ethylene insensitive sikle mutnt of Medigo truntul shows ltered uxin trnsport regultion during nodultion. Plnt Physiol 142: Rolfe BG, Gresshoff PM, Shine J Rpid sreening for symioti mutnts of Rhizoium nd white lover. Plnt Si Let 19: Sgn M, Du G Sym28 nd Sym29, two new genes involved in regultion of nodultion in pe (Pisum stivum L.). Symiosis 20: Shnel E, Journet E, de Crvlho-Nieel F, Du G, Frugoli J The Medigo truntul SUNN gene enodes CLV1- like leuine-rih repet reeptor kinse tht regultes nodule numer nd root length. Plnt Mol Biol 58: Serle IR, Men AE, Lniy TS, Buzs DM, Iture-Ormetxe I, Crroll BJ, Gresshoff PM Long-distne signling in nodultion direted y CLAVATA1-like reeptor kinse. Siene 299: Shiuy K, Brry KG, Cirdi JA, Lous HM, Underwood BA, Nourizdeh S, Eker JR, Klee HJ, Clrk DG The entrl role of PhEIN2 in ethylene responses throughout plnt development in petuni. Plnt Physiol 136: vn Noorden GE, Ross JJ, Reid JB, Rolfe BG, Mthesius U Defetive long distne uxin trnsport regultion in the Medigo truntul sunn mutnt. Plnt Physiol 140: Wilkinson JQ, Lnhn MB, Yen HC, Giovnnoni JJ, Klee HJ A dominnt mutnt reeptor from Aridopsis onfers ethylene insensitivity in heterologous plnts. Nt Biotehnol 15: Wopereis J, Pjuelo E, Dzzo FB, Jing Q, Gresshoff PM, de Bruijn FJ, Stougrd J, Szzyglowski K Short root mutnt of Lotus jponius with drmtilly ltered symioti phenotype. Plnt J 23: