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
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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
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Duplica ed euAG Genes in Medicago unca ula
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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.
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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
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