Regular Paper. Introduction

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1 Regulr Pper Serotonin, Tryptophn-Derive Signl Conserve in Plnts n Animls, Regultes Root System Arhiteture Proly Ating s Nturl Auxin Inhiitor in Ariopsis thlin Rmón Pelgio-Flores, Rny Ortíz-Cstro, Alfonso Ménez-rvo, Loures Mís-Roríguez n Jose López-uio* Instituto e Investigiones Químio-iológis, Universi Mihon e Sn Niolás e Hilgo, Eifiio 3, Ciu Universitri. CP 83 Moreli, Mihoán, Méxio *Corresponing uthor: E-mil, juio@zeus.umih.mx; Fx, (Reeive Septemer 2, 21; Aepte Jnury 11, 211) Serotonin (-hyroxytryptmine) is well-known neurotrnsmitter in mmmls n is wiely istriute in plnts. This ompoun is synthesize from tryptophn n shres struturl similrity with IAA. To te, little is known out the morphologil, physiologil n moleulr responses of plnts to serotonin. In this stuy, we hrterize the effets of serotonin on growth n evelopment in Ariopsis thlin seelings. Gs hromtogrphy mss spetrometry (GC-MS) nlysis showe tht plnts re le to tke up serotonin from the growth meium, whih oinie with gretly stimulte lterl root evelopment t onentrtions from 1 to 16 mm. In ontrst, higher oses of serotonin represse lterl root growth, primry root growth n root hir evelopment, ut stimulte ventitious root formtion. To investigte the role of serotonin in moulting uxin responses, we performe experiments using trnsgeni Ariopsis lines expressing the uxin-responsive mrker onstruts DR:uiA, A3:uiA n HS::AXR3NT-GUS, s well s vriety of Ariopsis mutnts efetive t the AUX1, AXR1, AXR2 n AXR4 uxin-relte loi. We foun tht serotonin strongly inhiite oth DR:uiA n A3:uiA gene expression in primry n ventitious roots n in lterl root primori. This ompoun lso olishe the effets of IAA or nphthleneeti i on uxin-regulte evelopmentl n geneti responses, initing n nti-uxin tivity in the plnt. Mutnt nlysis further showe tht lterl root inution eliite y serotonin ws inepenent of the AUX1 n AXR4 loi ut require AXR1 n AXR2. Our results show tht serotonin regultes root evelopment proly y ting s nturl uxin inhiitor. Keywors: Ariopsis Auxin signling Root rhiteture Serotonin. Arevitions: ARF, uxin response ftor; ER, enoplsmi retiulum; GC-MS, gs hromtogrphy mss spetrometry; GUS, -gluuronise; MS, Murshige n Skoog; NAA, nphthleneeti i; NPA, 1-nphthylphthlmi i; LR, lterl root; LRP, lterl root primorium; PAT, polr uxin trnsport; PCI, p-hlorophenoxyisoutyri i; RNAi, RNA interferene; TDC, tryptophn eroxylse; TIA, triiooenzoi i; WT, wil-type; YFP, yellow fluoresent protein. Introution Plnts synthesize n use vriety of signls to just growth n evelopment throughout their life yle. Auxins, inluing IAA, omprise group of tryptophn-erive signls, whih re involve in most spets of plnt evelopment (Woowr n rtel 2). Extensive stuies over the pst ee hve investigte the ftors involve in the regultion of plnt morphogenesis y uxins. These ompouns exert strong iologil tivity t very low onentrtions in oth in vivo n in vitro systems n re essentil for mintenne of physiologil n morphogeneti proesses inluing grvity n light responses, root hir evelopment, n lterl root (LR), ventitious root n shoot system evelopment (Woowr n rtel 2). Optiml plnt growth requires tight ontrol of IAA tivity, whih is omplishe y iverse mehnisms tht inlue IAA iosynthesis, its trnsport mong tissues, yling etween tive n intive forms of uxin, n signl pereption through fmily of IAA reeptors (Ljung et l. 22, Leyser 26, Mokitis n Estelle 28). Although IAA is mong the most highly hrterize metolites of tryptophn, reltively high levels of IAA-relte ompouns hve een reporte in plnts suh s the mmmlin neurotrnsmitter serotonin (-hyroxytryptmine). This ompoun is uiquitous signl, whih plys multiple roles in neurotrnsmission, hormone n mitogeni funtions s well s ting in immunomoultory n nti-inflmmtory proesses in niml ells (Frzer n Hensler 1999). In plnts, serotonin hs een foun in roots, leves, fruits n sees from Downloe from y guest on 4 Septemer 218 Plnt Cell Physiol. 2(3): 49 8 (211) oi:1.193/pp/pr6, ville online t The Author 211. Pulishe y Oxfor University Press on ehlf of Jpnese Soiety of Plnt Physiologists. All rights reserve. For permissions, plese emil: journls.permissions@oup.om 49 Plnt Cell Physiol. 2(3): 49 8 (211) oi:1.193/pp/pr6! The Author 211.

2 Role of serotonin in root evelopment t lest 42 speies (Grose 1982, Engstrom et l. 1992, Roshhin 21). Serotonin hs een implite in importnt physiologil n evelopmentl funtions inluing flowering, senesene, shoot formtion n efense responses (Ojkov n Hjiivnov 1997, Murh et l. 21, Roshhin 21, Ishihr et l. 28, Kng et l. 29). Serotonin iosynthesis ours vi two enzymti steps. Tryptophn eroxylse (TDC) tlyzes the onversion of tryptophn into tryptmine, followe y tryptmine -hyroxylse (TH), whih hyroxyltes the C- position of tryptmine to form serotonin (Kng et l. 27, Kng et l. 27, Kng et l. 28, Kng et l, 29). In leves n sees of rie (Oryz stiv) plnts, TDC expression is very low or negligile. In leves, the serotonin onentrtion ws reporte to e roun.3 mgg 1 FW. However, levels gretly inrese in response to senesene signls suh s nutrient eprivtion or lef ethment, n serotonin synthesis is losely ouple with trnsriptionl n enzymti inution of tryptophn iosyntheti genes s well s TDC in senesing leves (Kng et l. 29). Trnsgeni rie plnts overexpressing TDC proue 2-fol higher serotonin levels thn wil-type (WT) plnts n showe elye lef senesene, wheres lines in whih expression of TDC ws suppresse through n RNA interferene (RNAi) system proue less serotonin n senese fster thn the WT line (Kng et l. 29). Serotonin umultion ws lso reporte to our in rie leves infete y the fungl pthogen ipolris oryze (Ishihr et l. 28). Serotonin umultion ws preee y trnsient inrese in tryptmine ontent n y mrke tivtion of TDC. Serotonin tretment suppresse the growth of fungl hyphe, initing tht the tivtion of the tryptophn pthwy is involve in the estlishment of effetive efenses ginst the pthogen through serotonin proution in rie plnts. Colletively, this informtion inites tht serotonin levels in plnt tissues my inrese y emn, uner prtiulr evelopmentl trnsitions or when hllenge y pthogens. Although serotonin is nturlly present in wie vriety of plnts, little is known out the moleulr mehnisms involve in plnt evelopmentl responses to this ompoun. The Ariopsis root system is n exellent moel to hrterize the effets of ompouns with novel n interesting tivities in plnts (López-uio et l. 26, Contrers-Cornejo et l. 29). Roots perform the essentil tivities of proviing wter, nutrients n physil support to the plnt. The primry root origintes in the emryo n proues mny LRs uring the lifetime of plnt, n eh of these will proue more LRs. The quntity n plement of these strutures etermine the rhiteture of the root system, n this in turn plys mjor role in etermining whether plnt will survive in prtiulr limte or environment (Mlmy n enfey 1997, Csimiro et l. 23, López-uio et l. 2). During the post-emryoni evelopment of plnts, new xes of growth emerge from shoot tissues through ventitious orgnogenesis. This is prtiulrly importnt in rops suh s mize, in whih ventitious root formtion provies flexile wy for plnts to lter their form n resoure llotion in response to environmentl hnges or fter injury. While LRs typilly form from lterl root primori (LRPs) initite on the primry root periyle, ventitious roots form nturlly from stem tissue. LR n ventitious root formtion is omplex proess ffete y multiple enogenous ftors, inluing phytohormones suh s uxin, n environmentl ftors suh s light n nutrient eprivtion (Csimiro et l. 23, López-uio et l. 23, Péret et l. 29). The ontrol of post-emryoni root growth n LR formtion is tightly regulte y uxin (IAA). IAA moves throughout the plnt in the phloem or y more ontrolle polr trnsport system [polr uxin trnsport (PAT)]. PAT is proess regulte y AUXIN RESISTANT 1/LIKE AUX1 (AUX1/LAX) uptke proteins, PIN-FORMED (PIN) efflux rriers n P-GLYCOPROTEIN (MDR/PGP/AC) efflux/onitionl trnsporters (Swrup et l. 24; Mrve et l. 28). There re severl Ariopsis mutnts efetive in the proution of uxin trnsport proteins or in the orret lotion of these proteins tht show uxin-relte phenotypes, inluing ux1-7 n xr4-1. The ux1-7 mutnt is efetive t the AUX1 lous enoing n uxin influx trnsporter (Swrup et l. 24), while the xr4-1 mutnt is efetive in n essory protein of the enoplsmi retiulum (ER) tht regultes loliztion of AUX1 proteins. Loss of AXR4 results in norml umultion of AUX1 in the ER of epierml ells, initing tht the xr4 grvitropi phenotype is use y efetive AUX1 trffiking in the root epiermis (Dhrmsiri et l. 26). Auxin is pereive y the TRANSPORT INHIITOR RESPONSE1/AUXIN SIGNALING F-OX PROTEIN 1-3 (TIR1/ AF1-3) reeptor fmily. TIR1 is prt of the uiquitin ligse omplex SCF TIR1/AF tht tlyzes the uiquitintion n estrution of AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA) proteins (Gry et l. 21, Dhrmsiri et l. 2, Kepinski et l. 2). These proteins, uner low uxin onentrtions, form imers with the uxin response trnsription ftors (ARFs), therey loking their tivity. The Ariopsis uxin-resistnt 1 (xr1) mutnts were initilly isolte in sreen for uxin non-responsive seelings (Linoln et l. 199). Susequent nlysis emonstrte tht AXR1 is suunit in the relte to uiquitin (RU)-tivting enzyme, the first enzyme in the pthwy tht onjugtes the uiquitin-relte protein RU to memers of the uiquitin protein ligses (el Pozo et l. 22). One free from the AUX/IAAs, ARFs regulte the expression of uxin-responsive genes (Lu et l. 28, Kieffer et l. 21). Gin-of-funtion muttions in IAA3/SHY2, IAA7/AXR2, IAA12/ DL, IAA14/SLR, IAA18/CRANE, IAA19/MSG2 n IAA28 genes le to plnts with ltere root evelopment (Tin n Ree 1999, Ngpl et l. 2, Rogg et l. 21, Fukki et l. 22, Ttemtsu et l. 24, Uehr et l. 28). The slr n i28 gin-of-funtion mutnts show strong reution in LR formtion (Rogg et l. 21, Fukki et l. 22). These oservtions inite tht the AUX/IAA proteins funtion s negtive regultors of LR evelopment, lthough etile iret Downloe from y guest on 4 Septemer 218 Plnt Cell Physiol. 2(3): 49 8 (211) oi:1.193/pp/pr6! The Author

3 R. Pelgio-Flores et l. omprison of the LR phenotypes of these mutnts hs not een performe. se on its struturl similrity to IAA, we hypothesize tht serotonin might t through nonil uxin signling pthwy to moulte evelopmentl responses y either tivting or repressing uxin responses. We therefore hrterize the effets of serotonin on root system rhiteture n uxin-meite responses in Ariopsis thlin. Interestingly, our results show tht the supply of serotonin hs ul effet on LR formtion, stimulting it t low (1 16 mm) onentrtions, ut with repressing effets t higher (1 6 mm) onentrtions. Although t high onentrtions serotonin lso inue ventitious root formtion, it represse typil uxin responses suh s primry root growth n root hir formtion. Moreover, n nlysis of root rhiteture responses in the ux1-7, xr1-3, xr2-1 n xr4-1 Ariopsis uxin-relte mutnts n uxin-inuile gene expression tests revele tht serotonin my t s nturl uxin inhiitor in plnts. Results Serotonin ffets Ariopsis thlin root system rhiteture Serotonin n IAA re tryptophn-erive ompouns, with similr hemil strutures (Fig. 1). To evlute the effets of serotonin on plnt growth n evelopment, we use A. thlin s moel system. Ariopsis thlin (Col-) seelings were grown in Petri pltes ontining soli.2 Murshige n Skoog (MS) meium supplemente with the solvent or with inresing onentrtions of serotonin from 1 to 16 mm. Twelve ys fter germintion, the primry root length, LR numer n LR ensity were etermine for 3 seelings. We foun tht serotonin strongly promote LR evelopment, without ffeting primry root growth. This les to plnts with inrese LR numer n ensity (LR m 1 )(Fig. 2A D). An importnt evelopmentl trit wiely use to monitor uxin responses is primry root growth (Woowr n rtel 2). To etermine whether serotonin tretments of H N CH 2 COOH Inole-3-eti i Tryptophn H N CH 2 C NH 2 H COOH HO H N Serotonin CH 2 CH 2 NH 2 Fig. 1 Comprtive hemil strutures of serotonin (N-etyl-- hyroxytryptmine), IAA n their ommon preursor tryptophn. >16 mm oul ffet primry root growth n other root rhiteturl prmeters, Ariopsis seelings were supplie with 1 6 mm serotonin. It oul e seen tht oth LR numer n ensity inrese t serotonin onentrtion of 1 mm ut erese t greter onentrtions of this ompoun (Fig. 3, C). Supplementry Fig. S1 illustrtes the root rhiteturl responses of Ariopsis seelings to high serotonin onentrtions; it n e seen tht this ompoun rmtilly inhiits primry root growth while promoting root rnhing use y prolifertion of ventitious roots. Our results show tht serotonin hs ul effet in moulting root system rhiteture, promoting LR evelopment t low onentrtions (1 16 mm) ut inhiiting primry root growth n LR evelopment t higher onentrtions. Serotonin ffets ell ivision n ell growth in Ariopsis roots The post-emryoni root evelopmentl effets of high serotonin onentrtions in Ariopsis seelings suggeste tht this ompoun oul ply n importnt role in ell ivision n/or ell elongtion. To stuy the effets of this ompoun on ell ivision n elongtion, we mesure the length of oth fully evelope ortil ells from the ifferentition region n the primry root meristem from 7 ol WT Ariopsis (Col-) seelings. In ition, we nlyze the expression of pprz1:uia, whih mrks only tive meristems (Sieerer et l. 23), n CyC1:uiA, whih is expresse only in ells in the G 2 /M phse of the ell yle n is mrker of mitoti tivity (Colón-Crmon et l. 1999). Strong primry root growth inhiition uner onentrtions of serotonin 3 mm orrelte with oth erese ell size of ortil ells n the loss of -gluuronise (GUS) expression in the primry root meristem of pprz1:uia n CyC1:uiA trnsgeni seelings (Fig. 4A C). In ition to these effets, meristem length signifintly erese from 3 mm in solvent-trete seelings to 22 mm tonen- trtion of 6 mm serotonin. These results inite tht serotonin inhiits primry root growth y ffeting oth ell ivision n elongtion. Serotonin inues lterl root growth ut not lterl root primori initition To etermine whether serotonin promotes LR evelopment y stimulting LRP growth or inuing e novo formtion of LRPs, or moulting oth of these proesses, we investigte the stges of LRP evelopment ffete y serotonin. LRPs were quntifie 7 fter germintion in plnts trete with the solvent or with 1 mm serotonin, whih inreses LR numer n ensity without ffeting primry root growth (Fig. 2). Seeling roots were first lere to enle LRPs t erly stges of evelopment to e visulize n ounte. Eh LRP ws lssifie oring to its stge of evelopment s reporte y Mlmy n enfey (1997). We foun tht the stge istriution of LRPs ws ffete y tretment with serotonin. In prtiulr, LRP stge I, whih esries LRPs t the erliest stge of Downloe from y guest on 4 Septemer Plnt Cell Physiol. 2(3): 49 8 (211) oi:1.193/pp/pr6! The Author 211.

4 Role of serotonin in root evelopment A Primry root length (m) D evelopment, ws signifintly erese in serotonin-trete seelings (Fig. A). In mrke ontrst, LRP stge VII, overing the most evelope LRPs giving rise to emerge LRs, ws inue 3-fol y serotonin (Fig. A). The totl numer of LRPs per seeling i not hnge in response to serotonin tretments (Fig. ). These t suggest tht serotonin i not inue e novo LRP initition n proly inreses root rnhing in Ariopsis y inuing the mturtion of pre-forme LRPs from periyle ells. Serotonin promotes ventitious root evelopment To etermine whether serotonin is involve in regultion of prolifertive events in the shoot system, we ssesse its regenertive properties y ultivting stem explnts from etiolte Ariopsis seelings uner inresing onentrtions of serotonin n monitoring ventitious root formtion s reporte y Cmpos-Cuevs et l. (28). Ariopsis explnts trete Lterl root numer 8 C C C C Solvent mm Ser Fig. 2 Effets of low serotonin onentrtions on Ariopsis root system rhiteture. Ariopsis Col- seelings were germinte n grown for 12 uner inresing serotonin onentrtions. (A) Primry root length. () Lterl root numer. (C) Lterl root ensity. Vlues shown represent the mens of 3 seelings ± SD. Different letters represent mens sttistilly ifferent t the. level. (D) Representtive photogrphs of Ariopsis seelings grown in the inite serotonin tretments. The experiment ws repete twie with similr results. Sle r = 1 m. Lterl root ensity with 1 6 mm serotonin showe roughly 2-fol inrese in ventitious root numer ompre with solvent-trete explnts (Fig. 6A). Fig. 6 n C shows representtive photogrphs of the effets of serotonin on ventitious root formtion. This result illustrtes tht serotonin is ompoun with strong effet on Ariopsis ventitious root orgnogenesis. Serotonin inhiits root hir evelopment n expnsin gene expression The serotonin effets of inhiiting primry root growth n promoting ventitious root formtion re reminisent of those use y treting plnts with uxins (Woowr n rtel 2). Auxins hve lso een foun to inue root hir evelopment in severl plnt speies (Prker et l. 2). To etermine whether serotonin oul ffet root hir evelopment, we performe experiments in whih Ariopsis seelings were germinte n grown in Petri pltes ontining.2 gr MS meium supplemente with inrese Downloe from y guest on 4 Septemer 218 Plnt Cell Physiol. 2(3): 49 8 (211) oi:1.193/pp/pr6! The Author

5 R. Pelgio-Flores et l. A Primry root length (m) Lterl root numer C Lterl root ensity onentrtions of the ompoun, n fter germintion root hirs from the ifferentition n mturtion zones of the primry root were nlyze. In mrke ontrst to ventitious root evelopment, serotonin tretments rmtilly inhiite root hir evelopment oth in the ifferentition zone n in the mturtion zone (Fig. 7A). Next, we etermine whether the ompoun ltere root hir initition, root hir elongtion or oth, y mirosopilly ounting n mesuring triholst ells present in the mturtion zone of the primry root. It ws foun tht 1 mm serotonin signifintly inhiite root hir growth, while inrese onentrtions rmtilly loke hir growth (Fig. 7). The root hir ensity nlysis showe tht serotonin lso inhiite root hir formtion in ose-epenent wy (Fig. 7C). To etermine whether serotonin ffets root hir formtion t n erly step in evelopment, we use the patexp7:uia trnsgeni line, whih expresses GUS in triholst ells n is mrker of root hir initition (Cho n Cosgrove 22). Serotonin proue efiient ell ifferentition progrm in root epierml ells, eviene y loss of GUS expression in triholst ells in serotonin-trete seelings (Fig. 7D). GC-MS nlysis of serotonin levels in A. thlin seelings To etermine whether serotonin is nturlly proue in A. thlin n if the effets oserve on root system rhiteture orrelte with serotonin umultion in plnt tissues, we performe experiments to quntify serotonin from root n shoot tissues from solvent- or serotonin-trete WT (Col-) seelings y using gs hromtogrphy mss spetrometry (GC-MS) nlysis. Smll yet etetle mounts of serotonin were ientifie in root n shoot tissue of solventtrete Ariopsis seelings; however, serotonin levels in plnt tissues rmtilly inrese in seelings trete with this ompoun (Fig. 8A). Serotonin is etylte in the erivtiztion proess y eti nhyrie, prouing N-etylserotonin (Fig. 8). Fig. 8C n D shows mss spetr of the N-etylserotonin stnr n the extrte smple, respetively. Representtive hromtogrms of root n shoot smples from solvent- or serotonin-trete seelings re shown in Fig. 8E H. These finings provie the first eviene tht serotonin is proue nturlly in Ariopsis, n tht plnts re le to tke up serotonin from the growth meium. Downloe from y guest on 4 Septemer 218 C Fig. 3 Effets of high serotonin onentrtions on Ariopsis root system rhiteture. Ariopsis Col- seelings were germinte n grown for 1 uner inresing serotonin onentrtions. (A) Primry root length. () Lterl root numer. (C) Lterl root ensity. Vlues shown represent the mens of 3 seelings ± SD. Different letters represent mens sttistilly ifferent t the. level. The experiment ws repete twie with similr results. Serotonin inhiits uxin-inuile gene expression We next investigte whether serotonin ts in n uxinrelte signling pthwy y nlyzing the expression of the uxin-inuile DR:uiA n A3:uiA gene mrkers. Sine low serotonin onentrtions tivte LR evelopment y inuing LRP outgrowth (Figs. 2, ), we first etermine histohemil GUS expression uring LRP evelopment in 7 trnsgeni DR:uiA Ariopsis seelings, in response to the solvent or 1 mm serotonin. Interestingly, serotonin lerly 494 Plnt Cell Physiol. 2(3): 49 8 (211) oi:1.193/pp/pr6! The Author 211.

6 Role of serotonin in root evelopment A Cell size (mm) C PRZ1:uiA Cy1:uiA C 1 3 inhiite DR:uiA expression in LRPs from ll teste evelopmentl stges (Fig. 9A). We lso teste the response of the mrkers to high serotonin onentrtion y nlyzing histohemil stining of trnsgeni DR:uiA n A3:uiA Ariopsis seelings tht were grown for 7 in.2 gr MS meium n then trnsferre to liqui.2 MS meium supplemente with the solvent, mm IAA or 4 mm serotonin, n inute for 9 h t 22 C. As previously reporte (Ulmsov et l. 1997), in solvent-trete DR:uiA seelings, GUS expression ws sent from otyleons n leves n ws expresse primrily in the root tip region (Fig. 9). DR:uiA seelings grown uner onentrtion of mm IAA showe strong GUS Solvent Meristem length (mm) C tivity throughout the plnt (Fig. 9). The pttern of GUS expression in DR:uiA seelings trete with 4 mm serotonin further erese when ompre with solvent-trete plnts (Fig. 9), initing the lk of uxin tivity for this ompoun. Untrete A3:uiA seelings i not show etetle levels of GUS tivity (Fig. 9C), wheres, when trete with mm IAA, they showe GUS expression minly in the petioles of the otyleons (Fig. 9C) n in the root elongtion zone (Fig. 9C). GUS expression in seelings trete with serotonin ws unetetle (Fig. 9C), initing tht this ompoun file to tivte A3:uiA expression. We lso nlyze DR:uiA expression in eveloping ventitious roots from mm Ser Fig. 4 Effet of serotonin on ell ivision n elongtion. WT (Col-), pprz:uia n Cy1:uiA A. thlin seelings were grown for 7 on.2 MS meium supplemente with the inite onentrtions of serotonin. (A) Men epierml ell length. () Meristem length. Dt points represent the men ± SD (n = 3). (C) Plnts were stine for GUS tivity n lere to show gene expression. Photogrphs show representtive iniviuls from t lest 1 stine plnts. The experiment ws replite twie with similr results. Different letters inite sttistil ifferenes t P <.. Sle rs in pprz:uia imges = mm, n in Cy1:uiA imges = mm. Downloe from y guest on 4 Septemer 218 Plnt Cell Physiol. 2(3): 49 8 (211) oi:1.193/pp/pr6! The Author

7 R. Pelgio-Flores et l. A Lterl root primori numer Totl lterl root primori * * C 1 mm Ser l ll lll lv V Vl Lterl root primori stge Solvent 1 mm Serotonin Fig. Effets of serotonin on lterl root primori evelopment. Ariopsis Col- seelings were grown for 7 on gr pltes supplemente with the solvent or with 1 mm serotonin. Dt re presente for LRP evelopmentl stges (A) n totl LRPs per seeling (). LRP stges were reore oring to Mlmy n enfey (1997). Vlues shown represent the men of 1 seelings ± SD. Different letters re use to inite mens tht iffer signifintly (P <.). The experiment ws repete twie with similr results. Ariopsis seelings trete with 1 6 mm serotonin (Supplementry Fig. S2). A ose response inhiitory effet on GUS expression ws lerly oserve, initing tht serotonin i not stimulte ut rther represse uxin-inuile gene expression. To etermine in more etil the possile mehnism of tion of serotonin, we performe ompetene ssys y using the uxin-inuile DR:uiA n A3:uiA gene mrkers. Trnsgeni Ariopsis seelings expressing eh of these mrkers were grown for 7 in.2 gr MS meium n then trnsferre to liqui.2 MS meium supplemente * Vll * with the solvent, 1 mm IAA or nphthleneeti i (NAA), 4 mm serotonin or eh uxin in omintion with serotonin. When serotonin n IAA were supplie together, mrke reution of uxin-inue DR:uiA n A3:uiA expression ws evient (Fig. 1A). Serotonin lso ntgonize the effets of NAA on uxin-inuile gene expression when supplie t 4 mm (Fig. 1) or uner lower onentrtion, nmely 1 mm (Supplementry Fig. S3). These results suggest tht serotonin my t s ompetitive inhiitor of uxin-regulte gene expression in Ariopsis. Serotonin i not ntgonize uxin-meite Aux/IAA protein egrtion Auxin promotes the egrtion of Aux/IAA repressor proteins vi the uiquitin protesome pthwy n therey inues primry uxin-responsive gene expression (Gry et l. 21). To ress the effet of serotonin on uxin-meite egrtion of Aux/IAA proteins, we exmine the effet of IAA n serotonin on Aux/IAA stility using the Ariopsis HS::AXR3NT-GUS line, in whih trnsltionl fusion etween omins I n II of AXR3 n the GUS reporter protein is expresse uner the ontrol of het shok promoter (Gry et l. 21). Seelings expressing the HS::AXR3NT-GUS onstrut were het shoke t 37 C for 2 h n further trete with mm IAA, 4 mm serotonin or mm IAA plus 4 mm serotonin for 1, 3 n 6 min. Tretment with IAA showe enhne egrtion of the fusion protein in otyleons n in the primry root, ut serotonin file to inue egrtion of the fusion protein even fter 6 min of tretment (Fig. 11A L). Moreover, this ompoun stilize the expression of this mrker in otyleons (ompre Fig. 11A C with I K), initing uxin ntgonist tivity. When oth ompouns were supplie together, the effet of IAA preomintes, giving rise to GUS expression pttern similr to tht oserve in Ariopsis seelings trete with IAA lone (Fig. 11M P). Our t inite tht serotonin ts inepenently of uxin or ownstrem of uxin reeptors, whih moulte the egrtion of the AXR3 protein. Effets of serotonin on uxin-inue lterl root formtion euse serotonin strongly inhiite the expression of the uxin-inuile DR:uiA n A3:uiA gene mrkers, it ws possile tht the repressing effets of this ompoun on root hir n LR evelopment oul e ue to serotonin ting s n uxin inhiitor. We next evlute the LR responses of WT (Col-) seelings to serotonin n NAA y growing Ariopsis WT (Col-) seelings on Petri pltes ontining.2 gr MS meium supplemente with ifferent onentrtions of serotonin, NAA or NAA plus serotonin. Seven ys fter germintion, primry root length n LR numer were quntifie. In these experiments, serotonin onentrtions of 1 mm i not ffet primry root growth (Fig. 12A), ut signifintly inrese LR numer (Fig. 12). NAA tretment or NAA plus Downloe from y guest on 4 Septemer Plnt Cell Physiol. 2(3): 49 8 (211) oi:1.193/pp/pr6! The Author 211.

8 Role of serotonin in root evelopment A 6 Aventitious root numer mm serotonin showe strong effet of inue root rnhing. Interestingly, serotonin speifilly inue LR growth when supplie together with NAA, initing tht it llevites the growth-repressing effets of NAA on LR elongtion (Fig. 12C, D). Supply of 3 n 4 mm serotonin inhiite oth primry root growth n LR formtion. In ontrst, onentrtions of.1 n.2 mm NAA, whih inhiit primry root growth, strongly stimulte LR formtion (Supplementry Fig. S4). Although tretment of seelings with oth serotonin n NAA showe n itive effet on primry root growth inhiition, we foun tht serotonin h n ntgonisti effet on LR response to NAA y eresing LR formtion (Supplementry Fig. S4). These results imply tht lthough the effets of serotonin n NAA on primry root growth re similr, they t in n opposite fshion to regulte LR evelopment. Effet of serotonin on root rhiteturl trits in uxin-relte Ariopsis mutnts To evlute t the geneti level the role plye y selete uxin-relte loi in serotonin responses, we ompre the primry root growth n ventitious root formtion of WT (Col-) seelings n the xr2-1, xr4-1, ux1-7 n xr1-3 Ariopsis mutnts in response to 1 mm serotonin tretment. Serotonin signifintly inue oth LR numer n ensity in WT n in xr2-1, xr4-1 n ux1-7 seelings ut not in xr1-3 mutnts (Fig. 13A C). Surprisingly, the xr2-1 mutnts showe inrese LR numers when grown in meium without serotonin, initing tht this mutnt is C C inherently potentite in LR formtion (Fig. 13, C). We lso teste the effets of high serotonin onentrtion on ventitious root evelopment in intt Ariopsis seelings. Supply of 4 mm serotonin use n 8% inhiition in primry root growth in WT seelings ompre with solventtrete ontrol seelings. All four uxin-resistnt mutnts ux1-7, xr1-3, xr2-1 n xr4-1 showe similrly inhiite primry root growth to WT plnts (Supplementry Fig. S). When grown in meium without serotonin, WT n mutnt seelings showe n sene of ventitious roots, wheres when trete with the ompoun the formtion of 3 6 ventitious roots ws oserve. This effet ws similr in WT seelings n in xr4-1 n ux1-7 mutnts (Supplementry Fig.S). In ontrst, xr2-1 mutnts showe exerte responses to the ompoun while xr1-3 mutnts showe erese ventitious root numers (Supplementry Fig. S). Supplementry Fig. S6 illustrtes the root rhiteturl responses of Ariopsis seelings to IAA tretments; the primry root growth resistne of the lines use in this stuy with n lmost norml ventitious root inution exept in xr1-3 n e seen. Our results inite tht LR n ventitious root inution y serotonin re inepenent of the xr2-1, xr4-1 n ux1-7 loi ut require n intt xr1-3 lous. Disussion Solvent Serotonin Fig. 6 Effets of serotonin on ventitious root evelopment from Ariopsis shoot explnts. Ariopsis seelings were germinte n grown in rkness for on the surfe of gr pltes ontining.2 MS meium n hypootyl explnts were otine. Hypootyl explnts were trnsferre to.2 MS meium ontining the inite onentrtions of the ompoun n ultivte for further 9 perio to quntify ventitious root formtion. (A) Aventitious root numer in response to serotonin tretments. () Representtive photogrph of solvent-trete Ariopsis (Col-) explnts. (C) Photogrph of explnts supplie with 4 mm serotonin. Vlues shown in (A) represent the men ± SD (n = 2). Different letters inite sttistil ifferenes t P <.. The experiment ws repete twie with similr results. Sle r = 1 m. Serotonin is highly onserve inoli ompoun ourring in evolutionrily istint orgnisms from humns to plnts. The results of reserh with serotonin hve unovere severl fts: Downloe from y guest on 4 Septemer 218 Plnt Cell Physiol. 2(3): 49 8 (211) oi:1.193/pp/pr6! The Author

9 R. Pelgio-Flores et l. A Solvent Root hir length (mm) D C C C Solvent Root hir numer 1 1 Downloe from y guest on 4 Septemer 218 Fig. 7 Effets of serotonin on root hir evelopment. Ariopsis thlin seelings were grown for on.2 MS meium supplemente with the inite onentrtions of serotonin. (A) Representtive photogrphs of root hirs forme t the ifferentition n mturtion regions of Continue 498 Plnt Cell Physiol. 2(3): 49 8 (211) oi:1.193/pp/pr6! The Author 211.

10 Role of serotonin in root evelopment (i) it is present in wie numer of plnt speies; (ii) it is proue from tryptophn; n (iii) its onentrtion my vry in plnt tissues or in response to environmentl onitions, suggesting importnt evelopmentl n ptive funtions (Ishihr et l. 28, Kng et l. 29). However, little is known out its signling role in the plnt. In previous stuy y Kng et l. (29), it ws foun tht serotonin is gretly umulte in rie (O. stiv) leves unergoing senesene inue y either nutrient eprivtion or ethment, n its synthesis is losely ouple with trnsriptionl n enzymti inution of the tryptophn iosyntheti genes s well s TDC. Trnsgeni rie plnts tht overexpresse TDC umulte higher levels of serotonin thn the WT n showe elye senesene of rie leves. In ontrst, trnsgeni rie plnts, in whih expression of TDC ws suppresse through n RNAi system, proue less serotonin n senese fster thn the WT, suggesting tht serotonin is involve in ttenuting lef senesene. Sine the role of serotonin in plnt evelopment is not well unerstoo, in this work we teste the hypothesis tht it oul t s plnt growth regultor y proly ting s n uxin or interfering with uxin tion. Phrmologil tools tht hve inrese our unerstning of uxin signling re uxin response inhiitors, whih elong to two min lsses: those tht lter uxin trnsport n those tht pertur uxin signling. Most stuies hve employe syntheti inhiitors, suh s 1-nphthylphthlmi i (NPA), p-hlorophenoxyisoutyri i (PCI) n triiooenzoi i (TIA), or smll moleule ntgonists of the TIR1 reeptor funtion proue y introuing ifferent lkyl hins to the -position of IAA (Fujit n Syono 1996, Csimiro et l. 21, Oono et l. 23, Hyshi et l. 28). While syntheti uxin inhiitors hve provie importnt informtion out the moleulr mehnisms involve in uxin tion, the in plnt role of these unnturl ompouns is questionle. More reently, nturlly ourring tryptophn erivtives suh s tryptophn onjugtes of jsmoni i n IAA hve een foun to interfere with ro rnge of uxin-meite proesses (Stswik 29). Our results show tht serotonin my lso t s n enogenous uxin inhiitor. We use the Ariopsis root system to test the effets of exogenously supplie serotonin on severl morphogeneti proesses inluing primry root growth, LR formtion, ventitious root formtion n root hir evelopment, most of whih re uxin-regulte proesses. We foun tht serotonin stimulte LR evelopment t onentrtions of 1 16 mm y inuing LRP mturtion (Figs. 2, ). This effet orrelte with erese expression of the uxin response mrker DR:uiA in LRPs (Fig. 9A). These results suggest tht uner norml growth onitions, uxin synthesis/response in eveloping LRPs is suproptiml for LR growth. Serotonin might thus inrese LRP mturtion y ntgonizing uxin signling in LRPs. However, it lso represse LR formtion n root hir growth t higher onentrtions ut inrese ventitious root formtion from intt plnts n from shoot explnts (Fig. 6, 7; Supplementry Fig. S1). The tivity of serotonin in moulting root growth ws lower thn tht oserve for uxins. Fig. 2 shows tht this ompoun only moestly slowe root growth. Even t 3 mm, primry root growth inhiition ws <% of the ontrol vlue. y omprison, uner similr growth onitions n using the sme growth meium, IAA or uxin-relte signls proue % inhiition t onentrtions of t lest two orers of mgnitue lower thn this (Contrers-Cornejo et l. 29). Intriguingly, nother niml neurotrnsmitter, glutmte, exerte 6% primry root growth inhiition in WT Ariopsis (Col-) seelings t mm onentrtion (Wlh-Liu et l. 26). Similrly to glutmte, serotonin i not interfere with LR initition ut potentite LR outgrowth. An importnt ifferene in the moe of tion of serotonin ompre with glutmte is the role plye y uxin signling in plnt responses to these signls. Two loss-of-funtion mutnts t the AXR1 lous (xr1-3 n xr1-12) were hypersensitive to glutmte in primry root growth inhiition, wheres ux1-7 ws resistnt, initing tht uxin trnsport n signling might e importnt for root responses to glutmte (Wlh-Liu et l. 26). In ontrst, primry root growth in oth xr1-3 n ux1-7 ws inhiite similrly to tht in WT seelings when trete with 4 mm serotonin (Supplementry Fig. S). The effets of serotonin on inhiiting primry root growth n repressing LR formtion re similr to those use y uxin influx or efflux inhiitors suh s TIA n NPA (Fujit n Syono 1996, Csimiro et l. 21). Serotonin effets lso resemle those use y pplition of yokonolie, n inhiitor of uxin tion isolte from Streptomyes isttohromogenes, whih stimulte LR formtion t low onentrtions, wheres t higher onentrtions it promote ventitious root evelopment (Hyshi et l. 23). Although serotonin ws etete t low levels in roots n shoots of solventtrete Ariopsis seelings (Fig. 8), it my still ply signifint role sine it inreses in onentrtion uner prtiulr evelopmentl trnsitions n in response to pthogen ttk (Ishihr et l. 28, Kng et l. 29). Auxin is very importnt for root rhiteture remoeling n it is highly regulte y omplex network of interting mehnisms; therefore, serotonin s n enogenous uxin inhiitor might e expete to remin low in most tissues uner onitions of norml growth. Downloe from y guest on 4 Septemer 218 Fig. 7 Continue the primry root of -y-ol Ariopsis seelings grown on the surfe of gr pltes supplemente with the inite onentrtions of serotonin. ( n C) Dt points inite the men ± SD for root hir length () or root hir numer (C) of 1 epierml ells lote in fully ifferentite zone of the primry root from 2 seelings nlyze. (D) AtExp7:uiA expression in response to serotonin tretments. These experiments were repete twie with similr results. Different letters inite sttistil ifferenes t P <.. Sle rs in (A) = mm n in (D) = 1 mm. Plnt Cell Physiol. 2(3): 49 8 (211) oi:1.193/pp/pr6! The Author

11 R. Pelgio-Flores et l. A Serotonin (mg-g FW) Root Shoot Aunne 24 E C G Interestingly, supplementtion of serotonin in the growth meium rmtilly inrese serotonin onentrtions in oth root n shoot tissues, initing tht Ariopsis seelings re le to tke up serotonin from the meium n trnsport it within ifferent plnt tissues (Fig. 8). To test whether serotonin my t on uxin trnsport or signling, we exmine the effet of serotonin on DR:uiA n A3:uiA gene expression inue y uxins with ifferent trnsport properties. IAA is sustrte for uxin influx rriers, while NAA freely iffuses through memrnes (Delrre et l. 1996, Mrhnt et l. 1999). Serotonin similrly ntgonize Serotonin C 1 H 12 N 2 O MW = 176 Aunne + HO 19 O 7 C NH NH HO NH 21 3 NH M D m/z Aunne F 24 N-Aetylserotonin 24.6 HO Time (min) Time (min) NH H NH 2 Aeti nhyrie N-Aetylserotonin C 12 H 14 N 2 O 2 NH MW = 218 HO N-Aetylserotonin Fig. 8 Determintion of serotonin from ontrol or serotonin-trete Ariopsis seelings y GC-MS. Ariopsis WT seelings were germinte n grown for 22 in.2 MS meium supplemente with the solvent or with inrese onentrtions of serotonin. (A) Serotonin quntifition in root n shoot. () Aetyltion retion of serotonin with eti nhyrie. (C) The 7 ev eletron impt full sn mss spetr from m/z to of the N-etylserotonin stnr. (D) N-Aetylserotonin in plnt extrt. (E) Totl ion hromtogrm of N-etylserotonin from solvent-trete shoots or roots (F), or from shoot n roots of plnts trete with 4 mm serotonin, respetively (G H) uxin-inuile gene expression revele y oth mrker lines in response to oth IAA n NAA (Figs. 9, 1), suggesting tht serotonin oes not pertur uxin trnsport. Auxin lters the stility of AUX/IAA repressors, n therefore serotonin my t y loking AUX/IAA protein egrtion, thus explining the inhiitory effets of this ompoun on DR:ui n A3:uiA gene expression. To test this possiility, we nlyze the effets of serotonin on uxin-inue egrtion of n AUX/IAA protein. The Ariopsis HS::AXR3NT-GUS trnsgeni line strongly expresses n IAA17/ AXR3 trnsltionl fusion protein uner ontrol of het shok NH O Downloe from y guest on 4 Septemer 218 Plnt Cell Physiol. 2(3): 49 8 (211) oi:1.193/pp/pr6! The Author 211.

12 Role of serotonin in root evelopment A Solvent 1 mm Serotonin Solvent mm laa 4 mm Ser C DR:uiA A3:uiA Solvent mm laa 4 mm Ser Fig. 9 Effet of serotonin on uxin-regulte gene expression. Twelve hour GUS stining of DR:uiA or A3:uiA Ariopsis seelings grown for 7 on gr pltes ontining.2 MS meium or meium supplemente with IAA or serotonin. (A) DR:uiA expression in lterl root primori. Comprtive effet of IAA n serotonin on DR:uiA () n A3:uiA expression (C) Notie the erese in GUS expression in LRPs, shoots n roots in the tretments with serotonin. Photogrphs re representtive iniviuls of t lest 2 stine seelings. The experiment ws repete twie with similr results. Sle rs in (A) = mm n in (, C) = mm. Downloe from y guest on 4 Septemer 218 promoter (Gry et l. 21). The egrtion rte of the AUX/ IAA fusion protein is rpi n ws enhne y IAA tretment (Fig. 11). In ontrst, serotonin file to inue egrtion of the fusion protein, initive of the lk of n uxin tivity. When IAA n serotonin re supplemente together, the egrtion rte of the AUX/IAA fusion protein is rpi n resemles the effets of pplying IAA lone (Fig. 11). These t suggest tht serotonin my not ompete for uxin ining to its reeptors or tht it ts ownstrem of uxin reeptors, whih moulte the egrtion of the AXR3 protein. Serotonin i not suppress the inhiition of primry root growth use y IAA or NAA tretment (Fig. 12, Supplementry Fig. S4). However, it stimultes LR growth t low onentrtions in the sene of exogenous uxin n Plnt Cell Physiol. 2(3): 49 8 (211) oi:1.193/pp/pr6! The Author

13 R. Pelgio-Flores et l. A DR:uiA A3:uiA Solvent Solvent mm IAA 4 mm Ser mm IAA + 4 mm Ser 1 mm IAA 1 4 mm Ser 4 mm Ser + 1 mm IAA Solvent 1 mm NAA 4 mm Ser 4 mm Ser + 1 mm NAA DR:uiA A3:uiA Fig. 1 Serotonin ntgonizes the uxin-inuile expression moulte y IAA or NAA tretment. Twelve hour GUS stining of DR:uiA n A3:uiA Ariopsis seelings grown for 7 on gr pltes ontining.2 gr MS meium n then trnsferre to.2 MS liqui meium supplemente with uxins, with serotonin or oth. Note the erese in GUS expression in shoots n roots in oth IAA (A) n NAA () tretments y serotonin. Photogrphs re representtive iniviuls of t lest 2 stine seelings. The experiment ws repete twie with similr results. Sle rs = 2 mm. exertes the effets of NAA y inuing growth of LRs (Fig. 12C, D). High serotonin onentrtions (i.e. 4 mm) loke the stimultion of lterl rooting in response to exogenous uxin (Supplementry Fig. S4). Auxin ntgonists vry signifintly in how they ffet root rhiteture. For exmple, terfesttin A n syntheti uxin inhiitor hving n lkyl sustitution t the -position of IAA stimulte primry root growth, whih ws ttriute to the inhiition of enogenous uxin (Ymzoe et l. 2, Hyshi et l. 28). In ontrst, oth PCI n yokonolie suppresse primry root growth, n this ws epenent on TIR1 n AUX/IAA7, suggesting it ws not toxi effet (Hyshi et l. 23, Oono et l. 23). y using trnsgeni Ariopsis seelings expressing AtHistH2: YFP (yellow fluoresent protein) n vitl stining with propiium ioie, we etermine tht the primry root growth inhiitory effet of serotonin ws not ue to toxiity, s 3 6 Min Fig. 11 Anlysis of AUX/IAA stility with HS::AXR3NT-GUS fusion. Trnsgeni seelings expressing the HS::AXR3NT-GUS onstruts were het shoke t 37 C for 2 h. After het inution, the seelings were trete with IAA, serotonin or IAA plus serotonin for ifferent times t the inite onentrtions, n stine overnight for GUS tivity. Representtive photogrphs of otyleons from t lest 2 stine seelings re shown. Similr results were otine in two inepenent experiments. Sle rs = 2 mm. serotonin-trete seelings i not show meristem ell mge (Supplementry Fig. S7). Inste, it oul e ue to serotonin moulting ell ivision n elongtion (Fig. 4). Severl uxin-resistnt Ariopsis mutnts suh s xr1 n ux1-7, whih re efetive in uxin signling n trnsport, respetively, exhiit fewer LRs n reue root hir formtion. Sine similr phenotypes were oserve in WT Ariopsis (Col-) roots trete with serotonin, it is possile tht high levels of serotonin in the plnt impir the ellulr uxin response n therey inhiit the initition of LRs n root hirs. To etermine whether uxin-relte mutnts were lso resistnt to serotonin, we teste the effets of this ompoun on LR evelopment, primry root growth inhiition n ventitious root formtion in WT Ariopsis seelings n in xr2-1, xr4-1, ux1-7 n xr1-3 uxin-relte mutnts. We Downloe from y guest on 4 Septemer Plnt Cell Physiol. 2(3): 49 8 (211) oi:1.193/pp/pr6! The Author 211.

14 Role of serotonin in root evelopment A 4. C Primry root length (m) Lterl root numer C NAA (mm).1.2 Ser + NAA (mm) foun tht 1 mm serotonin signifintly inrese LR numer n ensity in WT, xr2-1, xr4-1 n ux1-7, ut not in xr1-3 (Fig. 13, C). Surprisingly, the ominnt xr2-1 mutnt, with gin of funtion in IAA7/AXR2, use inrese LR formtion oth uner norml growth onitions n in response to serotonin. This result suggests tht IAA7/AXR2 plys positive role in LR evelopment, in greement with previously pulishe informtion (Ngpl et l. 2). The lk of response of xr1-3 to serotonin inites tht this ompoun requires n intt AXR1 protein to tivte LR evelopment. Tretment with 4 mm serotonin lso showe exerte ventitious root proution in xr2-1 n erese ventitious root response in xr1-3 when ompre with WT plnts C NAA (mm).1.2 Ser + NAA (mm) Lterl root length (mm) D NAA (mm) 1 C Solvent (Supplementry Fig. S6). Although the ext mehnism of tion of serotonin is still unler, severl lines of eviene inite tht serotonin proly ts s nturl uxin inhiitor. (i) Serotonin is present in Ariopsis tissues t low onentrtions. (ii) Serotonin tretment stimultes LRP mturtion y eresing uxin responses uring LRP evelopment. (iii) Exogenous pplition of serotonin inhiite root evelopmentl proesses whih re uner uxin ontrol, suh s primry root growth, LR formtion n root hir evelopment. (iv) Serotonin loke uxin-responsive DR:uiA n A3:uiA gene expression n uxin-regulte LR formtion. (v) Mutnt nlyses inite tht serotonin inhiits primry root growth n promotes ventitious root formtion NAA (mm) Ser + NAA (mm) 1 mm Ser Fig. 12 Serotonin inues lterl growth in omintion with uxin. Ariopsis (Col-) seelings were germinte n grown for 7 on gr soliifie.2 MS meium supplemente with NAA, 1 mm serotonin or oth ompouns in omintion. (A) Primry root length. () Lterl root numer per plnt. (C) Lterl root length. (D) Representtive photogrphs of lterl roots grown in the inite tretments. Dt points inite the men ± SD from 2 seelings nlyze. Different letters inite sttistil ifferenes t P <.. The experiments were repete twie with similr results. Sle r = 1 mm. Downloe from y guest on 4 Septemer 218 Plnt Cell Physiol. 2(3): 49 8 (211) oi:1.193/pp/pr6! The Author

15 R. Pelgio-Flores et l. A Primry root length Lterl root numer 4 2 C Lterl root ensity C 1 mm Ser inepenently of t lest three uxin-relte loi, nmely xr2-1, xr4-1 n ux1-7. Plnt neuroiology hs reently emerge s n integrte view of plnt signling. Plnts proess the informtion from hnging environment to evelop n reproue. Communition etween ells n tissues is essentil for plnt fitness, whih involves n integrte signling system tht inlues long-istne eletril signls, vesile-meite trnsport of IAA n proution of hemils known to e neuronl in nimls (lusk et l. 2, renner et l. 26). Among the niml neurotrnsmitters, etylholine, teholmines, histmine, serotonin, opmine, meltonin n glutmte re the most ommon in the niml nervous system, plying roles in informtion proessing n evelopment. It is of interest tht eh of these ompouns is present in plnts. Similrly to IAA, serotonin n meltonin re tryptophn erivtives. Interestingly, IAA, whih is trnsporte from ell to ell hs some hrteristis reminisent of neurotrnsmitters, suh s poorly unerstoo vesile-se proess tht involves IAA trnsporters n reyling etween the plsm memrne n enosomes (Gelner et l. 23, Shliht et l. 26). Our results showing tht serotonin regultes root rhiteture in similr wy to nother neurotrnsmitter, glutmte, n tht these ompoun n lso ffet uxin-meite responses in Ariopsis re in greement with the propose role of IAA in the plnt neuroiologil perspetive. The possile roles plye y other neurotrnsmitter signls in plnt proesses my e further lrifie y using the moleulr tools ville in A. thlin n other moel plnts. Mterils n Methos Plnt mteril n growth onitions Ariopsis (A. thlin Col-), the trnsgeni Ariopsis lines HS::AXR3NT-GUS (Gry et l. 21), DR:uiA (Ulmsov et l. 1997), A3:uiA (Oono et l. 1998), pprz1:uia (Sieerer et l. 23) n CyC1:uiA (Colón-Crmon et l. 1999), histone H2:YFP (iosnr-lorig et l. 21), n the mutnt lines xr1-3 (Linoln et l. 199), ux1-7 (Piket et l. 199), xr2-1 (Timpte et l. 1994) n xr4-1 (Hoie n Estelle 199) were use for the ifferent experiments. Sees were surfe sterilize with 9% (v/v) ethnol for min n with 2% (v/v) leh for 7 min. After five wshes in istille wter, sees were germinte n grown on gr pltes ontining.2 MS meium. The MS meium (Murshige n Skoog sl Slts Mixture, tlog no. M24) ws purhse from Sigm. Phytgr Downloe from y guest on 4 Septemer 218 Col- xr2-1 xr4-1 ux1-7 xr1-3 Fig. 13 Effets of serotonin on primry root growth n lterl root formtion in WT Ariopsis (Col-) seelings n uxin-relte mutnts. Ariopsis WT n mutnt seelings were germinte n grown for 12 in.2 MS meium supplemente with the solvent or 1 mm serotonin. (A) Primry root length. () Lterl root numer per seeling. (C) Lterl root ensity. Vlues shown represent the mens of 3 seelings ± SD. The experiment ws repete three times with similr results. 4 Plnt Cell Physiol. 2(3): 49 8 (211) oi:1.193/pp/pr6! The Author 211.

16 Role of serotonin in root evelopment (ommeril gre) ws purhse from Gio-RL. Pltes were ple vertilly t n ngle of 6 to llow root growth long the gr surfe n to llow unimpee eril growth of the hypootyls. Plnts were ple in plnt growth hmer (Perivl AR-9L) with photoperio of 16 h light/8 h rkness, light intensity of 1 mmol m 2 s 1 n temperture of 22 C. Anlysis of growth Growth of primry roots ws registere using ruler. LR numers were etermine y ounting the LRs present in the primry root, from the tip to the root/stem trnsition. LR ensities were etermine y iviing the LR numer y the primry root length n expresse s LRP m 1. Root hirs were mesure in mm region from the primry root tip. The verge length of root hirs ws etermine y mesuring 1 hirs for eh root, tking s referene the root protoxylemti plne to lote the ril hir se in the epierml ell. Determintion of evelopmentl stges of LRP LRPs were quntifie 7 fter germintion. Seeling roots were first lere to enle LRPs t erly stges of evelopment to e visulize n ounte. Eh LRP ws lssifie oring to its stge of evelopment s reporte y Mlmy n enfey (1997). The evelopmentl stges re s folllows, Stge I: LRP initition. In the longituinl plne, pproximtely 8 1 short periyle ells re forme. Stge II: the LRP is ivie into two lyers y perilinl ivision. Stge III: the outer lyer of the primorium ivies perilinlly, generting three-lyer primorium. Stge IV: n LRP with four ell lyers. Stge V: the LRP is miwy through the prent ortex. Stge VI: the LRP hs psse through the prent ortex lyer n hs penetrte the epiermis. It egins to resemle the mture root tip. Stge VII: the LRP ppers to e just out to emerge from the prent root. Histohemil nlysis For histohemil nlysis of GUS tivity, Ariopsis seelings were stine n inute overnight t 37 C in GUS retion uffer (. mg ml 1 -romo-4-hloro-3-inolyl--dgluuronie in 1 mm soium phosphte, ph 7). The stine plnts were lere n fixe with.24 N HCl in 2% methnol (v/v) n inute for 6 min t 62 C. The solution ws sustitute y 7% NOH (w/v) in 6% ethnol (v/v) for 2 min t room temperture. Plnts were ehyrte with ethnol tretments t 4, 2 n 1% (v/v) for 24 h perio eh, n fixe in % glyerol (v/v). The proesse roots were ple on glss slies n sele with ommeril nil vrnish. For eh mrker line n for eh tretment, t lest 2 trnsgeni plnts were nlyze. Aux/IAA protein egrtion ssy Six-y-ol HS::AXR3NT-GUS trnsgeni Ariopsis seelings were inute on.2 liqui MS meium for 2 h t 37 C, followe y trnsfer of the seelings into.2 liqui MS meium supplemente with IAA or serotonin or oth for 1, 3 or 6 min t 22 C. The seelings were wshe with fresh.2 MS meium n, h lter, histohemilly stine for GUS tivity. Serotonin etermintion y GC-MS The extrtion of serotonin from leves n roots of A. thlin Col- (.1 g) ws one with 3 ml of methnol with ontinuous gittion for 3 h. The extrt ws evporte to omplete ryness uner strem of nitrogen. Serotonin ws etylte with eti nhyrie (1. ml) n 1 ml of ihloromethne, n then sonite for 1 min n hete t 7 C for 1. h. The N-etylserotonin ws evporte uner strem of nitrogen n reissolve in ml of ihloromethne. N-Aetylserotonin ws nlyze in n Agilent 68 Series II gs hromtogrph equippe with n Agilent MS etetor moel 973, n 3 m.2 mm.2 mm, % phenyl methyl silione pillry olumn (HP- MS). Operting onitions use helium s rrier gs, 1 ml min 1 ; etetor temperture of 3 C n injetor temperture of 2 C. The volume of injete smple ws 1 ml. The olumn ws hel for 3 min t 1 C n progrmme t 6 C min 1 to finl temperture of 278 C for min. N- Aetylserotonin ws ientifie y omprison with mss spetr lirry (NIST/EPA/NIH, Chem Sttion Hewlett Pkr). The ientity of the N-etylserotonin ws further onfirme y the omprison of the retention time in the tissue extrt with smple of the pure serotonin stnr (Sigm) erivtize following the sme proeure mentione ove. A SIM nlysis ws use to verify the presene of this ompoun in the smple. The moleulr ions were monitore fter eletron impt ioniztion (7 ev). They were m/z 218, m/ z 19 n m/z 146. To estimte the mount of ompoun proue y the plnt, we onstrute n iniviul lirtion urve for the erivtize stnr using onentrtions from.1 to 3 mg. Mirosopy The A. thlin root system ws nlyze with stereosopi mirosope (Lei MZ6, Lei Mirosystems). Totl LRs were ounte t 3 mgnifition. Imges were pture with Sony Cyer-shot DSC-S7 igitl mer (Sony Eletronis In.) pte to the mirosope n proesse with the Zeiss Axio Vision 4AC softwre (Crl Zeiss). For onfol mirosopy, solvent- or serotonin-trete trnsgeni Ariopsis seelings expressing the histone H2:YFP onstrut (oisnr-lorig et l. 21) were mounte on mirosope slies into solution of propiium ioie (1 mg ml 1 ). Primry root meristems were nlyze y imging mounte smples with n inverte onfol mirosope (Zeiss Axiovert 2 LSM). For propiium ioie etetion, wvelengths employe were n exittion line of 68 nm with n emission winow of 8 61 nm. YFP ws exite with 488 nm line n emission etete t nm. Downloe from y guest on 4 Septemer 218 Plnt Cell Physiol. 2(3): 49 8 (211) oi:1.193/pp/pr6! The Author 211.