Two euAGAMOUS genes control C-function in Medicago truncatula

Two eu AGAMOUS Genes Con ol C-Func ion in Medicago unca ula Joanna Se wa owska 1.¤a , Edelı ´n Roque 1. , Concepcio ´nGo ´mez-Mena 1 , Gab iela D. Cons an in 3 , Jiangqi Wen 2 , Ki ankuma S. Myso e 2 , Ole S. Lund 3¤b , Elisabe h Johansen 3¤b , Jose ´Pı ´o Bel a ´n 1 , Luis A. Can ˜as 1 * 1Ins i u o de Biologı ´a Molecula y Celula de Plan as (CSIC-UPV). Ciudad Poli e ´cnica de la Inno acion, Valencia, Spain, ´2Plan Biology Di ision, The Samuel Robe s Noble Founda ion, A dmo e, Oklahoma, Uni ed S a es o Ame ica, 3Depa men o Plan Biology, Danish Ins i u e o Ag icul u al Sciences, F ede iksbe g C, Denma k Abs ac C- unc ion MADS-box ansc ip ion ac o s belong o he AGAMOUS (AG) lineage and speci y bo h s amen and ca pel iden i y and lo al me is em de e minacy. In co e eudico s, he AG lineage is u he di ided in o wo b anches, he euAG and PLE lineages. Func ional analyses ac oss lowe ing plan s s ongly suppo he idea ha duplica ed AG lineage genes ha e di e en deg ees o sub unc ionaliza ion o he C- unc ion. The legume Medicago unca ula con ains h ee C-lineage genes in i s genome: wo euAG genes (M AGa and M AGb) and one PLENA-like gene (M SHP). This species is he e o e a good expe imen al sys em o s udy he e ec s o gene duplica ion wi hin he AG sub amily. We ha e s udied he espec i e unc ions o each euAG genes in M. unca ula employing exp ession analyses and e e se gene ic app oaches. Ou esul s show ha he M. unca ula euAG- and PLENA-like genes a e an example o sub unc ionaliza ion as a esul o a change in exp ession pa e n. M AGa and M AGb a e he only genes showing a ull C- unc ion ac i i y, concomi an wi h hei ances al exp ession p o ile, ea ly in he lo al me is em, and in he hi d and ou h lo al who ls du ing lo al de elopmen . In con as , M SHP exp ession appea s la e du ing lo al de elopmen sugges ing i does no con ibu e signi ican ly o he C- unc ion. Fu he mo e, he edundan M AGa and M AGb pa alogs ha e been e ained which p o ides he o e all dosage equi ed o speci y he C- unc ion in M. unca ula. Ci a ion: Se wa owska J, Roque E, Go ´mez-Mena C, Cons an in GD, Wen J, e al. (2014) Two euAGAMOUS Genes Con ol C-Func ion in Medicago unca ula. PLoS ONE 9(8): e103770. doi:10.1371/jou nal.pone.0103770 Edi o : Hec o Candela, Uni e sidad Miguel He na ´ndez de Elche, Spain Recei ed June 6, 2014; Accep ed July 2, 2014; Published Augus 8, 2014 Copy igh : ß2014 Se wa owska e al. This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal au ho and sou ce a e c edi ed. Da a A ailabili y: The au ho s con i m ha all da a unde lying he indings a e ully a ailable wi hou es ic ion. All ele an da a a e wi hin he pape and i s Suppo ing In o ma ion iles. All da a conce ning gene sequences ob ained in his wo k a e eely a ailable in he GenBank public da abase (h p://www.ncbi.nlm. nih.go /genbank) p o ided by he Na ional Cen e o Bio echnology In o ma ion (NCBI). The sequences a e iden i ied in he manusc ip wi h hei espec i e GenBank accession numbe s: M AGa cDNA: KF159804 M AGb cDNA: KF159805 M AGa genomic: KJ470633 M AGb genomic: KJ470634 M SHP in on: KJ470635. Funding: This wo k was unded by g an s BIO2009-08134 and BIO2012-39849-C02-01 om he Spanish Minis y o Economy and Compe i i eness and he Ramo ´n y Cajal P og am (RYC-2007-00627 o CGM). The unde s had no ole in s udy design, da a collec ion and analysis, decision o publish, o p epa a ion o he manusc ip . Compe ing In e es s: The au ho s ha e decla ed ha no compe ing in e es s exis . * Email: [email p o ec ed] .These au ho s con ibu ed equally o his wo k. ¤a Cu en add ess: Labo a o io Nacional de Geno ´mica pa a la Biodi e sidad (LANGEBIO), Cen o de In es igacio ´n y de Es udios A anzados del Ins i u o Poli e ´cnico Nacional (CINVESTAV), I apua o, Guanajua o, Me ´xico ¤b Cu en add ess: Depa men o Ag icul u e and Ecology, Facul y o Li e Sciences, Uni e si y o Copenhagen, Taas up, Denma k In oduc ion Gene ic egula ion o lowe de elopmen has been subjec o s udy o e he las decades, pa icula ly in he model species A abidopsis haliana and An i hinum majus. These s udies p o ided a gene al unde s anding o lo al o gan de elopmen in highe plan s and led o he p oposal o he ABCDE model, which pos ula es ha lo al o gan iden i y in each who l is de ined by i e unc ions named A, B, C, D and E ( o e iew [1,2,3,4]). In pa icula , C- unc ion is equi ed o p omo e s amen and ca pel iden i y, o es ablish he de e mina e na u e o he lo al me is em [5] and also o ep ess A- unc ion in he hi d and ou h who ls [6]. Cloning o ABCDE o gan iden i y genes in A abidopsis showed ha mos o hem encode MADS-box ansc ip ion ac o s. S udies o MADS-box genes in highe eudico yledoneous lowe ing plan s show ha hey a e key egula o s o lowe de elopmen . In A abidopsis and An i hinum, he C- unc ion is essen ially ep esen ed by a single gene espec i ely, AGAMOUS (AG) [5,7] and PLENA (PLE) [8]. Addi ional C- unc ion genes ha e been iden i ied: SHATTERPROOF1 (SHP1) and SHATTER- PROOF2 (SHP2) [9,10] genes in A abidopsis and FARINELLI (FAR) [11] in An i hinum. Phylogene ic s udies using a la ge da a se o AG-like sequences show ha AG and PLE ac ually ep esen pa alogous lineages de i ed om a duplica ion in a common ances o ea ly in he his o y o he co e eudico s. This duplica ion ga e ise o he euAG lineage which includes AG and FAR, and he PLENA lineage (PLE), whe e SHP1/SHP2 and PLENA a e placed [12,13,14,15]. An e en mo e ancien duplica ion occu ed be o e PLOS ONE | www.plosone.o g 1 Augus 2014 | Volume 9 | Issue 8 | e103770 he adia ion o ex an angiospe ms, p oducing he C lineage (AG lineage) and he o ule-speci ic D lineage (AGL11 lineage) [12,15]. AG, FAR and PLE all display e y simila exp ession pa e ns du ing he de eloping male and emale ep oduc i e o gans [5,8,11]. Mu a ions o AG and PLENA in hei espec i e species p oduce iden ical pheno ypes cha ac e ized by de eloping lowe s wi h pe als in who l 3 ins ead o s amens, and sepals in who l 4 ins ead o ca pels. In addi ion, he lo al me is em is no de e mina e [7,8]. Howe e , a mu a ion only a ec s male ep oduc i e o gans, causing pa ial male s e ili y [11]. Mean- while, SHP genes a e only exp essed in he o ules, in he de eloping pis il and show almos iden ical exp ession pa e ns in de eloping A abidopsis ui [16,17,18]. They unc ion edundan - ly in s yle and s igma de elopmen , no ably in he usion o he ca pel [19], and in seedpod sha e ing [10]. SHP and FAR genes do no con ibu e signi ican ly o he C- unc ion al hough FAR is exp essed a ea ly s ages o lowe de elopmen . These s udies showed a andom e olu iona y ajec o y o gene unc ions a e a duplica ion e en . AG and PLE, di e en membe s o a duplica ed gene pai , e ained he p ima y homeo ic unc ions in di e en lineages. Thei espec i e o hologs ha e aken comple ely new oles in ui dehiscence, as he case o SHP1/SHP2 genes, o con ibu e edundan ly in male ep o- duc i e de elopmen , as he case o FAR. Rep esen a i es o bo h euAG and PLE lineages ha e been iden i ied in di e en co e eudico species and se e al unc ional analyses a e a ailable o he pa alogs om pe unia, oma o, and Nico iana ben hamiana [20,21,22,23]. In addi ion o he ancien duplica ion e en s obse ed in he AGAMOUS sub amily, mo e ecen duplica ion e en s appea o ha e occu ed in he euAG lineage [12,15,24]. The e ha e been epo ed wo membe s o he euAG lineage in some species, as o example Populus ichoca pa [25], Cucumis sa i us [26], Ge be a hyb ida [27], and in he legume species Lo us japonicus [28], Pisum sa i um and Medicago unca ula ( his s udy). Howe e , in mos cases, unc ional analyses a e lacking. Bo h phylogene ic and unc ional da a o gene lineages a e e y impo an o unde s and he e olu ion o gene amilies [12,29]. Medicago unca ula (M ) has h ee C-lineage genes: wo euAG genes (M AGa and M AGb) and one PLENA-like gene (M SHP; [30]). To gain insigh in o he speci ic con ibu ion o he euAG and PLE-like pa alogous genes in he con ol o he C- unc ion, we compa ed he exp ession pa e ns o he h ee C-lineage genes du ing lowe de elopmen . We ha e also cha ac e ized M AGa and M AGb loss-o - unc ion mu an s and plan s whe e bo h genes ha e been simul aneously silenced. The pa icula capaci y o hese wo genes o p omo e ep oduc i e o gan iden i y has been es ed by ec opically exp essing hem in A abidopsis. Ou esul s indica e ha he membe s o euAG and PLE lineages in M. unca ula a e sub unc ionalized, whe e he C- unc ion is only p omo ed by he euAG pa alogs. They la gely o e lap in unc ion bu he o e all dosage o bo h gene p oduc s is c i ical o p omo e comple e s amen and ca pel iden i y and lo al me is em de e minacy in M. unca ula. Ma e ial and Me hods Plan ma e ial and g ow h condi ions Medicago unca ula c . Jemalong lines A17, SA1335 and R108, and A abidopsis haliana c . Columbia plan s we e used in his s udy. Plan s we e g own in he g eenhouse, a 22uC (day) and 18uC (nigh ) wi h a 16 h ligh /8 h da k pho ope iod, in soil:sand (3:1) i iga ed wi h Hoagland N o .1 solu ion supplemen ed wi h oligoelemen s [31]. The m aga mu an allele (p e iously ma ag-2) was isola ed in a p e ious sc eening [32] and homozygous plan s we e used in his s udy. Iden i ica ion o Tn 1 inse ion si es in M AGb and co- seg ega ion es The M. unca ula popula ion used o he sc eening o mu an s was desc ibed in de ail [33,34,35,36] (h p://bioin o4.noble.o g/ mu an /). The m agb allele was iden i ied by PCR sc eening o a seg ega ing popula ion o app oxima ely 10,000 independen lines, using p ime s annealing o he M AGb sequence (AGb-F, Table S2) in combina ion wi h p ime s annealing o he LTR bo de s o he Tn 1 e oelemen (Tn 1-F; Table S2 and Figu e S3). We iden i ied a line (NF4908) wi h an inse ion o he e oelemen loca ed in he i s in on a 277 bp o he s a ing codon (Figu e 1A). The R1 plan s we e geno yped by PCR using he Tn 1-F p ime in combina ion wi h he gene-speci ic p ime s M AGb-F and M AGb-Rgenomic (Table S2 and Figu e S3B). App oxima ely 70% o he plan s showed a mu an pheno ype and co-seg ega ed wi h he Tn 1 inse ion. Isola ion and sequence analysis M AGa and M AGb cDNAs we e isola ed om a lib a y o M. unca ula A17 in lo escence apices [37], using he MADS-box agmen o he M. unca ula PISTILLATA gene as a p obe. Sequence alignmen s and simila i y compa isons o he in e ed p o eins we e pe o med using Align and Clus alW ools [38]. The deduced amino acid sequences we e aligned using Clus alW and u he e ined by hand. Genomic sequences sea ch was pe o med using BLAST [39]. The second in on sequences we e ob ained by PCR using genomic DNA and he p ime s M AGa-in on-F and M AGa- in on-R o M AGa and M SHP-in on- F and M SHP-in on-R o M SHP (Table S2). To p edic he p obabili y o he p esence o LFY binding si es in he in on sequences, we used he Mo pheus webpage acili y (h p://biode .cea. /mo pheus/ De aul .aspx) wi h he LFY ma ix. The sco e ool pe mi s localiza ion o he bes ansc ip ion ac o binding si es in DNA agmen s. Phylogene ic ee The phylogene ic ee was in e ed by Neighbo -Joining using Poisson-Co ec ed amino acid dis ances. Reliabili y o in e nal nodes was assessed using boo s ap wi h 10000 pseudo- eplica es. T ee in e ence was conduc ed using MEGA e sion 4 [40]. The da a se comp ised 28 p e iously epo ed C- and D-class genes ob ained om GenBank and he wo new sequences ha we isola ed (M AGa and M AGb). The ee was oo ed using he A abidopsis D-class gene SEEDSTICK (STK) sequence. All sequences used in his analysis, wi h hei espec i e species and accession numbe s, a e included in Table S1. Sou he n blo hyb idiza ion Plan genomic DNA was ex ac ed om lea es as desc ibed by [41]. Ten mic og ams o DNA we e diges ed wi h es ic ion enzymes, sepa a ed on 0.7% T is-bo a e EDTA 1X aga ose gels o e nigh a 1V/cm and ans e ed o a nylon memb ane. Sou he n blo hyb idiza ion was pe o med by s anda d me hods a 65uC. A 241 bp agmen o M AGa cDNA (posi ions 572-813) and a 215 bp agmen o he M AGb cDNA (posi ions 558-773) we e ampli ied wi h he speci ic p ime s M AGadi , M AGa e , M AGbdi and M AGb e 1 (Table S2) and used as p obes. Duplica ed euAG Genes in Medicago unca ula PLOS ONE | www.plosone.o g 2 Augus 2014 | Volume 9 | Issue 8 | e103770 Exp ession analyses To al RNA was isola ed om ozen plan ma e ial using he RNeasy Plan mini Ki (Qiagen, Ge many) acco ding o he manu ac u e ’s ins uc ions. Fo No he n blo analysis o al RNA (15 mg) om ozen lea es, oo s, s ems, lowe s and pollina ed o a ies (young ui s) was used. RNA elec opho esis was ca ied ou in o maldehyde- aga ose gels, ans e ed o Hybond N + memb anes (Ame sham Figu e 1. Two eu AGAMOUS genes in Medicago unca ula .(A) Gene s uc u e o M AGa and M AGb. Coding sequences a e ep esen ed as boxes and in ons as do ed lines. Black iangles localize he posi ion o he Tn 1 inse ions p esen in he mu an lines m aga (NF13380) and m agb (NF4908) used in his s udy. (B) Neighbo -Joining T ee o euAG and PLENA homologs om a selec ion o di e se species. The numbe s nex o he nodes e e o boo s ap alues om 10000 pseudo- eplica es.(C) Dis ibu ion o pu a i e LFY binding si es in he i s in on o M AGa,M AGb and M SHP genes as iden i ied wi h he use o a posi ion-speci ic sco ing ma ix using a cu o alue o 220. doi:10.1371/jou nal.pone.0103770.g001 Duplica ed euAG Genes in Medicago unca ula PLOS ONE | www.plosone.o g 3 Augus 2014 | Volume 9 | Issue 8 | e103770 Biosciences, USA), and hyb idized wi h 32 P-labeled p obes unde s anda d condi ions. The p obes we e gene a ed om he same gene agmen s used o Sou he n blo analysis. Fo Real Time RT-PCR analysis o al RNA was ea ed wi h DNaseI o he DNase T ea men and Remo al Ki (Ambion, Li e Technologies, USA). Fo i s -s and syn hesis, o al RNA (1 mg) was e e se- ansc ibed in a 20 ml eac ion mix u e using he P ime Sc ip 1 s s and cDNA Syn hesis Ki (Taka a, Japan). One mic oli e o RT eac ion was used o a Real Time RT-PCR analysis wi h 300 nM o each p ime mixed wi h he Powe SYBR G een PCR Mas e Mix (Applied Biosys ems) acco ding o he manu ac u e ’s ins uc ions. The eac ion was ca ied ou in o 96 well-op ical eac ion pla es using an ABI PRISM 7500 Sequence De ec ion Sys em and app op ia e so wa e (Applied Biosys ems). The ela i e le els we e de e mined by he 2 –DDC Me hod [42]. To no malize he a iance among samples, Sec e Agen (O-linked N-ace yl glucosamine ans e ase: TC77416; [43]) was used as an endogenous con ol. All eac ions we e pe o med by iplica e using a biological eplica e o each sample. P ime s we e designed using PRIMER EXPRESS so wa e (Applied Biosys ems, USA) using de aul pa ame e s and a e lis ed in Table S2. RNA in si u hyb idiza ion was pe o med on 8 mM pa a in sec ions o M. unca ula in lo escences as desc ibed by [16], using digoxigenin-labelled p obes. The RNA sense and an isense p obes we e gene a ed om he same gene agmen s used o Sou he n blo analysis. Bo h agmen s we e cloned in o he pGEM T-easy ec o (P omega, USA) and he p obes we e syn hesized using SP6 o T7 RNA polyme ases. Vi us Induced Gene Silencing in Medicago unca ula pCAPE1 and pCAPE2 de i a i es we e used as ec o s o gene silencing [44]. Two DNA agmen s om he 39 egion o he M AGa (310 bp) and M AGb (338 bp) genes we e ob ained by PCR using p ime s (M AGaVIGSdi , M AGbVIGSdi , M AGa- VIGS2 e , M AGbVIGS2 e ) ha added es ic ion si es o bo h ends o he agmen s (Table S2). The amplicons we e cloned in o pGEM T-easy (P omega, USA), diges ed using he app op ia ed es ic ion enzymes and cloned in o a simila ly diges ed pCAPE2 ec o [44]. The esul ing plasmid (pCAPE2-M AGab) was con i med by sequencing be o e being in oduced in o Ag obac- e ium ume aciens s ain C58/pMP90. Ag obac e ium inocula ion o M. unca ula lea es was pe o med as desc ibed by [44]. Gene a ion o ansgenic RNAi plan s T ans o ma ion o M. unca ula R108 was pe o med as desc ibed p e iously [33]. The 35S::RNAi-M AG cons uc was pe o med using a 215 bp agmen om M AGb (posi ions 557– 772 om he ATG codon), ampli ied using p ime s M AGb- RNAiD and M AGb-RNAiR (Table S2) ha inco po a e wo es ic ion si es ha a e used o cloning in o he pHANNIBAL ec o [45]. A abidopsis ans o ma ion M AGa and M AGb cDNA agmen s we e ampli ied using AGaSBXdi , AGaSBX e , AGbSBXdi and AGbSBX e p ime s (Table S2) and cloned in o he pBINJIT60 ec o [46], a pBIN19 de i a i e (Clon ech, Palo Al o, CA, USA), which placed gene ansc ip ion unde he con ol o a andem epea o he CaMV 35S p omo e . A abidopsis plan s we e ans o med by lo al dipping acco ding o s anda d p ocedu es [47] and selec ed on kanamycin. Pho og aphy, mic oscopy and c yo-SEM Ligh pho og aphs we e made wi h a s e eomic oscope Leica MZ16F a ached o a DFC300 FX came a (Leica Mic osys ems, Ge many). A e in si u hyb idiza ions, sec ions we e obse ed and pho og aphed wi h a Nikon Eclipse E-600 mic oscope equipped wi h a digi al came a. Fo c yo-SEM, samples we e ozen in slush ni ogen and a ached o he specimen holde o a C yoT ans 1500 C yo- P epa a ion Sys em (Ox o d Ins umen s, UK) in e aced wi h a JEOL JSM-5410 scanning elec on mic oscope. The samples we e hen ans e ed om c yos age o he mic oscope sample s age, whe e he condensed su ace wa e was sublimed by con olled wa ming o -85uC. A e wa ds, he sample was ans e ed again o he c yos age in o de o gold coa i by spu e ing. Finally he sample was pu back on he mic oscope sample s age o be iewed a an accele a ing ol age o 15 KeV. Resul s Iden i ica ion o wo euAGAMOUS genes in Medicago unca ula To iden i y MADS-box genes in ol ed in lowe de elopmen , we sc eened a cDNA lib a y o M. unca ula lo al apices using a se o MADS-box agmen s om di e en species as a p obe [48,49]. Among he isola ed clones, h ee co esponded o ull- leng h sequences ha p esen signi ican simila i y o genes o he AGAMOUS sub amily. One o hese clones p esen s high sequence simila i y wi h he A abidopsis SHP1 and SHP2 genes [17,18,50] and wi h he An i hinum PLENA gene (PLE; [8]) and co esponds o he M SHP gene [30]. The o he wo clones isola ed p esen high sequence simila i y wi h he C-lineage genes AGAMOUS o A abidopsis [5] and FARINELLI o An i hinum [11] and ha e been named M AGa and M AGb. The M AGa clone is 1208 bp long, wi h an ORF o 780 bp and a deduced p o ein o 260 amino acids. The M AGb clone is 1099 bp long, wi h an ORF o 732 bp and he deduced p o ein has 244 amino acids. M AGa shows 81% amino acid iden i y wi h M AGb, being mo e simila in he N- e minal domains (98% in he MADS domain, 76% in he I egion and 80% in he K domain) han in he C- e minal egion (68% o iden i y). M AGa and M AGb show espec i ely 71 and 68% amino acid iden i y wi h he euAG clade p o ein FAR and ei he one 67% amino acid iden i y wi h AG (Figu e S1). The M AGb genomic sequence was ound in wo BAC clones: m h2-30e7 (GenBank AC137837.4) and m h2-76i7 (GenBank AC153460.24). This gene is o ganized in se en exons and six in ons (GenBank KJ470634; Figu e 1A). The genomic sequence o he M AGa gene was no a ailable in he da abases and was ob ained in his s udy (GenBank KJ470633). M AGa gene is also o ganized in se en exons and six in ons (Figu e 1A). Bo h genes a e p esen as single copy in he genome as con i med by Sou he n blo analysis (Figu e S2A). Genomic sequences om M AGa and M AGb genes we e used as BLASTN que ies, and displayed using he Ch omosome Visualiza ion Tool (CViT, h p://www. medicagohapmap.o g/ ools/blas o m). We ound ha M AGb gene is loca ed on ch omosome 8 (Figu e S2B). Howe e , no loca ion was ob ained o he M AGa gene. Phylogene ic analyses showed ha M AGa and M AGb a e ela i ely ecen pa alogs wi hin he euAG subclade (Figu e 1B). O he model legumes such as Pisum,Lo us and Glycine used in his analyses, also displayed wo pa alogs in his subclade. In mos o he s udied plan s, he euAG subclade is ep esen ed by one Duplica ed euAG Genes in Medicago unca ula PLOS ONE | www.plosone.o g 4 Augus 2014 | Volume 9 | Issue 8 | e103770 single gene, such as AG o A abidopsis,FAR o An hi inum o TAG1 o oma o. Genes om he euAG- and PLE-subclades p esen a well conse ed gene s uc u e and cha ac e is ic la ge in ons essen ial o hei co ec exp ession pa e n [51,52]. In M. unca ula, bo h M AG genes show a la ge i s in on (4607 bp in M AGa and 3007 bp in M AGb). We sea ched in hese sequences o he p esence o pu a i e LFY binding si es ha a e cha ac e is ic o C- unc ion genes. We included in his s udy he M SHP gene and hen we ha e sequenced i s i s in on o 4281 bp (GenBank KJ470635). We used a bioin o ma ics ool a ailable a he Mo pheus webpage acili y (h p://biode .cea. /mo pheus/ De aul .aspx) which examines he dis ibu ion o indi idual LFY binding si es. The p esence o absence o LFY binding si es iden i ied by his app oach appea s o be help ul in p edic ing o wha ex en a C-clade gene ac s as a ue C- unc ion gene [53]. Ou esul s show di e en binding si e landscapes o he h ee genes analysed (Figu e 1C). In he case o he M AGb i s in on, he e is a main single binding si e o e y high sco e simila o he p o ile ound o he PLE gene [53]. This si e co esponds o wo ully in ac CCAAT-boxes. In he i s in on o M AGa, lowe a ini y si es a e p esen bu he binding seems o be compensa ed h ough he ac ion o se e al nea by si es as epo ed o he si es p esen in he second in on o AG (Figu e 1C). The p edic ion o binding si es o LFY in he i s in on o M SHP ga e only low sco e alues (Figu e 1C) indica ing a low p obabili y o be egula ed by LFY a ea ly s ages o lo al de elopmen . Globally, hese da a sugges ha he duplica ed euAG genes M AGa and M AGb migh con ibu e o C- unc ion speci ica ion in M. unca ula and play a ole du ing ea ly s ages o lowe de elopmen . Exp ession pa e ns compa ison o h ee M. unca ula C-lineage genes du ing lo al de elopmen The exp ession pa e ns o M AGa,M AGb and M SHP we e analysed by No he n blo in di e en plan issues and he h ee genes a e exclusi ely exp essed in lo al and young ui issues (Figu e 2). We pe o med de ailed in si u hyb idiza ion expe i- men s o show he dis ibu ion o M AGa,M AGb and M SHP mRNAs du ing lo al de elopmen (Figu e 3). Bo h M AGa and M AGb ansc ip s began o accumula e a s age 2 o lowe de elopmen . A his s age, M AGb accumula es in he cen e o he lo al p imo dia while M AGa signal was de ec ed h oughou he lo al me is em (Figu e 3F and 3A). A s age 4, M AGb ansc ip was loca ed in he egion o he common p imo dia ha will gi e ise o he s amens and also in he cen al pa o he lo al apex whe e he ca pel is de eloping (Figu e 3G). Howe e , M AGa exp ession is s ill obse ed on he whole lo al me is em, including pe al and sepal p imo dia (Figu e 3B). F om s age 5, exp ession o bo h pa alogs was dis ibu ed uni o mly in who ls 3 and 4 (Figu e 3C-E and 3H-J), al hough hyb idiza ion signal o M AGb was s onge on he abaxial egion o he ca pel. In la e s ages, exp ession o bo h ansc ip s was obse ed in he de eloping o ules, in he dis al egion o he ca pel and on he ilamen o he an he s (Figu e 3E and 3J). In con as , M SHP mRNA began o accumula e la e on lowe de elopmen and can be de ec ed a s age 6 in he inne cells o he de eloping ca pel (Figu e 3M). Since la e s age 7, M SHP exp ession is exclusi ely de ec ed in he o ules (Figu e 3N). We conclude ha he exp ession o he wo M. unca ula euAG genes emained la gely es ic ed o he de eloping male and emale o gans om ea ly s ages and h oughou lowe de elop- men . M AGa and M AGb showed nea ly iden ical spa ial exp ession pa e ns om s age 4 o la e de elopmen al s ages. Di e ences we e mainly obse ed a ea ly s ages, whe e M AGa exp ession appea ed o be wide sp ead in he lo al me is em han M AGb. Essen ially, he duplica ed M AGa and M AGb genes showed simila spa ial and empo al exp ession pa e ns o hose desc ibed o C- unc ion genes om he euAG subclade such as AG and PLE. In con as , M SHP was only exp essed in he de eloping ca pel and in he o ules, simila o he pa e n desc ibed o A abidopsis SHP genes ha do no signi ican ly con ibu e o C- unc ion. Figu e 2. Exp ession pa e ns o M AGa, M AGb and M SHP genes in a ious plan issues o M. unca ula .No he n blo analyses we e pe o med using o al RNA p epa ed om lea es (L), lowe s (Fl), young ui s (F ), s ems (S) and oo s (R). doi:10.1371/jou nal.pone.0103770.g002 Duplica ed euAG Genes in Medicago unca ula PLOS ONE | www.plosone.o g 5 Augus 2014 | Volume 9 | Issue 8 | e103770 M AGa and M AGb loss-o - unc ion analyses To in es iga e he speci ic con ibu ion o M AGa and M AGb genes in he M. unca ula lo al de elopmen , we looked o e o ansposon inse ion mu an s [32]. These mu an s we e a ailable o bo h genes, each con aining a single Tn 1 elemen inse ed in he N- e minal pa o he gene (Figu e 1A). m agb Tn 1 inse ion was iden i ied as desc ibed in he expe imen al p ocedu es and i was loca ed in he i s in on a 277 bp om he s a codon (Figu e 1A). m aga (p e iously m ag-2), was isola ed as a case s udy o demons a e he u ili y o he e e se gene ic pla o m in he model legume Medicago unca ula [32]. Using quan i a i e RT-PCR we ha e con i med ha M AGa o M AGb ansc ip s we e nea ly unde ec able in he co esponding homo- zygous mu an plan s wi hou a ec ing he exp ession le els o he co esponding M AG pa alog (Figu e S4). m aga and m agb mu an s exhibi e y simila lo al pheno ypes: lowe s a e wild- ype in appea ance, showing only mild de elopmen al de ec s on he hi d and ou h-who l o gans (Figu e 4). Incomple e usion o he s aminal ube was obse ed and occasionally, some pe aloid p olonga ion in he an he ip (Figu e 4B and 4C). Who l 4 is equen ly composed by mul iple ca pels (2-3) o by modi ied ca pels wi h s igma ic p o ube ances and exposed o ules (Fig- u e 4B and 4C). Flowe s we e gene ally s e ile, al hough occasionally a ew seeds we e p oduced. To in es iga e he e ec o he combined loss-o - unc ion o M AGa and M AGb, we pe o med i us induced gene silencing (VIGS) expe imen s. We used a VIGS ec o based on PEBV (Pea ea ly b owning i us) ini ially de eloped o induce gene silencing in Pisum [44]. The silencing e iciency o his ec o is lowe in M. unca ula cul i a s han in P. sa i um [54]. We gene a ed he pCAPE2-M AGab cons uc o simul aneously silence bo h genes (see Ma e ials and Me hods). Twen y one plan s we e inocula ed wi h he pCAPE2-M AGab cons uc and we analysed he pheno ype o 150 lowe s. 10% o he M AGab-VIGS lowe s showed homeo ic ans o ma ions in who ls 3 o 4. Only 1% o he M AGab-VIGS lowe s showed a nea comple e loss-o -C- unc ion pheno ype (Figu e 4 D). In who l 3 some an he s a e comple ely con e ed in o pe al-like o gans, and in he ou h who l, sepal-like s uc u es a ise ins ead o ca pels (Figu e 4 D). These lowe s also showed inde e minacy o he lo al me is em e ealed by he p esence o mul iple pe aloid o sepaloid concen ic s uc u es in he cen e (Figu e 4D, a ows). This pheno ype sugges s ha bo h M AG genes we e s ongly down- egula ed bu , because o lo al issue limi a ion, we could no de e mine he exp ession le els o he a ge ed genes. In pa allel, we also de eloped ansgenic M. unca ula lines in which M AG genes we e down egula ed by RNA in e e ence (RNAi) and h ee independen lines we e ob ained. Only one o hese lines (line 5.7) showed lo al pheno ypes simila o hose o single m ag mu an s (Figu e S5). These lowe s showed clea mo phological abe a ions consis ing o mild homeo ic ans o - ma ion o s amens o pe als and mul iple ca pels wi h exposed Figu e 3. Exp ession pa e n o M AGa , M AGb and M SHP genes du ing ea ly lo al de elopmen . In si u localiza ion o M AGa (A-E), M AGb (F-J) and M SHP (K-N) ansc ip s in M. unca ula wild- ype lowe buds. De elopmen al s ages we e de ined acco ding o [74]. M AGa ansc ip s a e localized in he whole lo al me is em a s ages 2 (A) and 4 (B). A s age 5 (C) M AGa mRNA is de ec ed in s amen and ca pel p imo dia. A s age 7 (D-E) exp ession loca es in s amens, ca pel and he de eloping o ules. M AGb ansc ip s a e localized in he cen e o he lo al me is em a s age 2 (F). A s age 4 (G) exp ession loca es in he ca pel p imo dia and he hal o he common p imo dia ha will gi e ise o he s amens. A s age 5 (H) M AGb mRNA is de ec ed in s amen and ca pel p imo dia. A s age 7 (I-J) M AGb mRNA is de ec ed in s amens, ca pel and de eloping o ules. M SHP ansc ip s a e no de ec ed a s ages 2 (K) and 5 (L). A s age 6 M SHP ansc ip s a e localized in he inne cells o he de eloping ca pel (M). A s age 7 (N) exp ession can be de ec ed in he de eloping o ules. F: lo al me is em; S: sepal; C: ca pel; CP: common p imo dia; S : s amen; P: pe al; Fi: ilamen ; A: an he ; O : o ule. doi:10.1371/jou nal.pone.0103770.g003 Duplica ed euAG Genes in Medicago unca ula PLOS ONE | www.plosone.o g 6 Augus 2014 | Volume 9 | Issue 8 | e103770 Duplica ed euAG Genes in Medicago unca ula PLOS ONE | www.plosone.o g 7 Augus 2014 | Volume 9 | Issue 8 | e103770 o ules. We analyzed he exp ession o bo h M AGa and M AGb genes in he h ee RNAi lines. These lines showed a di e en deg ee o silencing o each a ge ed gene (Figu e S5J). In silenced line 1.3, we ound M AGb le els conside ably educed (,15% o wild- ype le el), while M AGa le els a e somewha dec eased (,90% o wild- ype le el). S ong silencing o one pa alogous gene did no p oduce homeo ic lo al de ec s. Howe e , lo al de ec s in line 5.7 co ela e wi h simul aneous down egula ion o bo h genes (,40% o wild- ype le el) (Figu e S5J). M AGa and M AGb gain-o - unc ion analyses Ec opic exp ession o AG esul s in he homeo ic con e sion o sepals and pe als in o ca pels and s amens, espec i ely [55,56]. O he epo ed pheno ypes include ea ly lowe ing and cu ling o lea es. To in es iga e whe he M AGa and M AGb p o eins di e in hei abili y o induce ep oduc i e o gan a e we o e exp essed hese wo genes in A abidopsis. The coding sequence o hese genes was loca ed unde he con ol o he CaMV 35S cons i u i e p omo e . We analysed 75 independen p ima y ansgenic (T1) lines o e e y cons uc (35S::M AGa and 35S::M AGb plan s) and ound plan s wi h di e en lo al pheno ypes ha we classi ied as mild, medium and s ong pheno ype (Figu e 5B-D). T ansgenic plan s wi h mild pheno ype showed wax accumula ions on he su ace o he sepals (Figu e 5I) ha a e cha ac e is ic o he ca pel (Figu e 5H). The pe als a e na ow and asymme ic (Figu e 5B and 5J) wi h pa ial homeo ic con e sions o s amens (Figu e 5K). Flowe s wi h medium pheno ypes showed cu ed sepals (Fig- u e 5C) wi h abundan accumula ion o wax (Figu e 5L). T1 plan s wi h medium and s ong pheno ypes showed nea comple e homeo ic ans o ma ions o sepals o s amens (Figu e 5M and 5N). Finally, lowe s wi h s ong pheno ype showed hickened and cu ed sepals (Figu e 5D) ha con ain ca pelloid s uc u es wi h ec opic o ules and s igma ic papillae (Figu e 5P). Al hough hese h ee pheno ypical classes we e obse ed o bo h cons uc s, we no ice a highe p opo ion o plan s wi h medium/s ong pheno ypes (80.3%) in he M AGb o e exp essing lines (Fig- u e 5Q). In con as , 59.5% o M AGa o e exp essing lines showed mild homeo ic ans o ma ions. In summa y, cons i u i e exp ession o M AGa and M AGb in A abidopsis a e able o cause equi alen homeo ic al e a ions in he lowe s ha a e essen ially iden ical o hose caused by cons i u i e exp ession o o he euAG genes [56]. Howe e M AGb p o ein seems o be mo e e icien han M AGa o induce ep oduc i e o gan a e when o e exp essed in a he e ologous sys em. Discussion The model legume Medicago unca ula ha bo s wo membe s o he euAG sub-clade (M AGa and M AGb) and one membe o PLENA (M SHP) [30]. In his s udy, we epo he unc ional cha ac e iza ion o he euAG homologs om his species o achie e a mo e ho ough unde s anding o he e olu ion o he AGAMOUS sub amily in co e eudico s. Two euAG genes in Medicago unca ula The inding o wo euAG duplica ed genes (M AGa and M AGb) in M. unca ula, as well as in o he legume species (see Figu e 1B) sugges s ha each pai o pa alogs a ose by a duplica ion e en p io o he specia ion o legumes. They could ha e been o igina ed du ing he whole-genome duplica ion (WGD) e en ha p eda ed specia ion o M. unca ula and o he legumes a ound 50-60 Mya [57,58]. Gene duplica ions allowed he eme gence o c i ical changes in se e al MADS-box gene lineages ha con ol lo al o gan iden i y. In Medicago, B- unc ion genes a e also duplica ed: M AP3-like genes we e o igina ed by an ancien duplica ion concomi an wi h he base o he co e eudico adia ion [49], whe eas he wo duplica ed PI-like genes we e p obably o igina ed du ing he WGD e en ha also o igina ed he wo M AG genes ([48]; Roque e al. unpublished). The changes o hese genes a e duplica ion may ha e played an impo an ole in he e olu ion o lo al mo phology and on ogeny in legumes. The p esence o wo euAG genes is no only ound in legumes. P e ious phylogene ic s udies ha e e ealed he exis ence o wo membe s o he euAG-lineage in se e al co e eudico s [12]. This is he case o he pa alogous CUM1 and CUM2 om Cucumis sa i us,PTAG1 and PTAG2 om Populus ichoca pa, and GAGA1 and GAGA2 om Ge be a hyb ida. These s udies a e mainly ocused on he analyses o hei exp ession pa e ns and limi ed unc ional in o ma ion is a ailable. Recen ly, phylogene ic analyses using AG p o ein sequences om se e al As e aceae species, ha e e ealed ha some o hem, ha bo wo o h ee membe s wi hin he euAG-lineage [24]. M AGa and M AGb p o eins conse e cha ac e is ic domains o he C-lineage membe s, as he p esence o an N- e minal ex ension p eceding he MADS domain [59]. The leng h o his sequence in M AG p o eins alls wi hin he ange p e iously desc ibed o o he membe s o his clade ( om 13 o 52 amino acids) [59] (Figu e S1). Howe e , ega ding o DNA sequence, bo h genes possess six in ons a he han he ypical eigh o he o he AG homologs [7,8,11,25,60,61]. This genomic o ganiza ion is obse ed in bo h angiospe m and gymnospe m genes sugges ing ha he p esence o eigh in ons is likely o be p imi i e in AG sub amily [12]. The genomic o ganiza ion o M. unca ula euAG genes could ha e aken place du ing he WGD e en ha p eda es legume specia ion ollowed by gene ea angemen . Wi hin he highly simila gene s uc u e o AGAMOUS genes, i has been shown ha he i s and second in ons a e essen ial o hei co ec exp ession [24]. Among he iden i ied enhance s, he ansc ip ion ac o LFY is essen ial o AG ac i a ion and binds o cis-elemen s loca ed in he second in on [52,62]. The p esence o LFY-binding si es has been epo ed o be c i ical o es ablish ea ly exp ession o C- unc ion genes du ing lowe de elopmen [63]. We iden i ied high a ini y binding si es o LFY in he i s in on o bo h M AGa and M AGb. Al hough he binding si e landscapes obse ed o each euAG-pa alogous gene a e di e en , expe i- men al analyses ha e demons a ed ha bo h ypes o dis ibu ion o LFY binding si es a e unc ionally ele an [53]. In con as , in ou analyses M SHP was no ound o be a likely LFY a ge and acco dingly, M SHP exp ession is exclusi ely de ec ed in he o ules a la e s ages o lo al de elopmen . Simila ly, SHP o Figu e 4. Flo al pheno ypes o M. unca ula m agb and m aga mu an s and M AGab-VIGS silenced plan s. (A) Dissec ed wild- ype M. unca ula lowe showing he ou lo al who ls (W1 o W4). Flo al pheno ypes a e simila in m aga. (B) and m agb (C) mu an s. Mu an lowe s (le ) we e opened o show he inne who ls. In W3 he s aminal ubes a e un used and occasionally pe aloid s uc u es appea (a ow) eplacing he an he s. Ca pels in W4 p esen s igma ic p o ube ances (a ow) o mul iple un used ca pels and exposed o ules (boxed). (D) M AGab-VIGS lowe wi h se e e homeo ic ans o ma ion o s amens in o pe als and ca pels in o sepaloid s uc u es (a ows). Ba s indica e 1 mm. doi:10.1371/jou nal.pone.0103770.g004 Duplica ed euAG Genes in Medicago unca ula PLOS ONE | www.plosone.o g 8 Augus 2014 | Volume 9 | Issue 8 | e103770 STK, which play la e oles in ui and o ule de elopmen , we e no ound o be LFY a ge s in ChIP-seq expe imen s [53]. Toge he , ou analyses sugges ha bo h M AG genes ha e he same p obabili y o be di ec ly ac i a ed by LFY, con e ing o gan iden i y on lo al me is ems ha a ise a e he ansi ion o ep oduc i e de elopmen . Figu e 5. O e exp ession o M AGa and M AGb in A abidopsis haliana : lo al pheno ypes. Wild- ype A abidopsis lowe (A). Flo al pheno ypes obse ed in A abidopsis plan s o e exp essing M AGa o M AGb genes ha e been classi ied in o mild (B), medium (C) and s ong (D). Scanning elec on mic og aphs showing he cha ac e is ic cellula ypes o wild- ype sepals (E), pe als (F), s amens (G) and ca pels (H). Scanning elec on mic og aphs o sepals om o e exp ession lines wi h mild pheno ype (I) showing wax accumula ion (a ow). Na ow pe als om o e exp essing lines (J) showing s aminoid cellula ypes (K). Wax accumula ion in sepals (L) o plan s wi h medium and s ong pheno ypes. Nea comple e homeo ic con e sion o pe al in o s amen (M) showing cha ac e is ic cellula ypes om an he (N) and ilamen (O). Flowe wi h s ong pheno ype showing ec opic o ules and s igma ic issue (P) in he i s who l. Rela i e p opo ion o pheno ypes obse ed in plan s o e exp essing M AGa o M AGb genes (Q). Ba s indica e: 1mm in A, B, C, and D. doi:10.1371/jou nal.pone.0103770.g005 Duplica ed euAG Genes in Medicago unca ula PLOS ONE | www.plosone.o g 9 Augus 2014 | Volume 9 | Issue 8 | e103770