pls-09-01029 July 12, 2018 Time: 18:28 # 1
ORIGINAL RESEARCH
published: 16 July 2018
doi: 10.3389/ pls.2018.01029
Edi ed by:
C is ina Fe andiz,
Ins i u o de Biología Molecula y
Celula de Plan as (IBMCP), Spain
Re iewed by:
Pablo Daniel Jenik,
F anklin & Ma shall College,
Uni ed S a es
Michael Nicolas,
Cen o Nacional de Bio ecnología
(CNB), Spain
*Co espondence:
Ma ga ida Roche a
[email p o ec ed]
†These au ho s ha e con ibu ed
equally o his wo k.
Special y sec ion:
This a icle was submi ed o
Plan E olu ion and De elopmen ,
a sec ion o he jou nal
F on ie s in Plan Science
Recei ed: 11 May 2018
Accep ed: 25 June 2018
Published: 16 July 2018
Ci a ion:
Coi o JL, Sil a H, Ramos MJN,
Mon ez M, Cunha J, Amâncio S,
Cos a MMR and Roche a M (2018)
Vi is Flowe Sex Speci ica ion Ac s
Downs eam and Independen ly
o he ABCDE Model Genes.
F on . Plan Sci. 9:1029.
doi: 10.3389/ pls.2018.01029
Vi is Flowe Sex Speci ica ion Ac s
Downs eam and Independen ly o
he ABCDE Model Genes
João L. Coi o1†, Helena Sil a2†, Miguel J. N. Ramos1, Miguel Mon ez1, Jo ge Cunha3,
Sa a Amâncio1, Ma ia M. R. Cos a2and Ma ga ida Roche a1*
1Linking Landscape, En i onmen , Ag icul u e and Food (LEAF), School o Ag icul u e, Uni e si y o Lisbon, Lisbon,
Po ugal, 2Plan Func ional Biology Cen e, Biosys ems and In eg a i e Sciences Ins i u e, Uni e si y o Minho, B aga,
Po ugal, 3Ins i u o Nacional de In es igação Ag á ia e Ve e iná ia, Oei as, Po ugal
The mos disc imina ing cha ac e is ic be ween he cul i a ed Vi is ini e a subsp.
ini e a and he wild- o m Vi is ini e a subsp. syl es is is hei sexual sys em. Flowe s
o cul i a s a e mainly he maph odi ic, whe eas wild plan s ha e emale and male
indi iduals whose lowe s ollow a he maph odi ic pa e n du ing ea ly s ages o
de elopmen and la e de elop non- unc ional ep oduc i e o gans. In angiospe ms,
he basic de elopmen al sys em o lo al o gan iden i y is explained by he ABCDE
model. This model pos ula es ha egula o y gene unc ions wo k in a combina o ial
way o con e o gan iden i y in each who l. In wild Vi is no hing is known abou he
unc ion and exp ession p o ile o hese genes. He e we show an o e all iew o he
empo al and spa ial exp ession pa e n o he ABCDE genes as well as he pa e n o
V iSUPERMAN ha es ablishes a bounda y be ween he s amen and he ca pel who ls,
in he male, emale and comple e lowe ypes. The esul s show a simila pa e n in Vi is
species sugges ing ha he pa hway leading o unisexuali y ac s independen ly and/o
downs eam o B- and C- unc ion genes.
Keywo ds: Vi is ini e a syl es is,in si u hyb idiza ion, homeo ic genes, lowe ABCDE model, de elopmen ,
dioecious
INTRODUCTION
Flowe o gan de elopmen is unde he con ol o highly specialized gene ic ne wo ks ha ha e
been well s udied du ing he pas decades (Wellme and Riechmann, 2010;O’Maoileidigh e al.,
2014). The genus Vi is p esen s a no o ious a iabili y o lowe ypes. The lowe s o he cul i a ed
V. . ini e a a e mainly he maph odi e, while he wild ype, V. . syl es is is dioecious wi h male
plan s p oducing lowe s wi h e ec s amens bu wi hou pis ils and emale plan s displaying lowe s
wi h a ully o med pis il bu e lexed s amens wi h in e ile pollen (Valleau, 1916;Ca mona e al.,
2008;Ramos e al., 2014). The shi in sexual sys em om dioecy o he maph odi ism in Vi is
species is no ye comple ely unde s ood.
Se e al a emp s we e made o unde s and and p o ide insigh in o he molecula
mechanism ega ding he o igins o his sexual dimo phism p esen in V. . syl es is
indi iduals. Se e al gene ic mapping s udies based on he 8x e sion o he Vi is genome1
anno a ion (Dalbó e al., 2000;Riaz e al., 2006;Ma gue i e al., 2009) loca ed a locus
esponsible o sex de e mina ion a he icini y o he gene ic ma ke s V iMD34 and
V iIB23 (Dalbó e al., 2000;Riaz e al., 2006) on ch omosome 2 in he 8x e sion2.
1h p://www.genoscope.cns. /ex e ne/GenomeB owse /Vi is/en y_ggb.h ml
2h p://genomes.c ibi.unipd.i /g ape/
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Coi o e al. Vi is Flowe Homeo ic Genes Exp ession
Using hese ma ke s pu a i ely linked o he sex locus, a new
gene ic map was de eloped and e ined o es ic he sex locus o
143 kb in he ch omosome 2, be ween 4,907,434 and 5,050,616 bp
(Fech e e al., 2012). A mo e ecen s udy, ocusing on he 143 kb
egion o ch omosome 2 ex ended he sex locus egion o 158 kb
downs eam o he gene ic ma ke V iIB23 and encompassing
he p e ious 143 kb egion (Picq e al., 2014). This new locus
showed haplo ype di e si y, linkage disequilib ium, and se e al
genes seg ega ing o ypically associa ed X-Y sex de e mining
egion.
The canonical he maph odi e lowe s uc u e can be di ided
in o ou who ls. Fi s and second who ls comp ise he s e ile
pe ian h o he lowe , con aining sepals and pe als, espec i ely.
Rep oduc i e o gans a e o med in he inne mos who ls, he
s amens in he hi d who l and ca pel in he lowe cen e , he
ou h who l (Dellapo a and Calde on-U ea, 1993;Bowman
e al., 2012). The i s genes in ol ed in lowe o gan iden i y
we e desc ibed in he model plan s An i hinum majus and
A abidopsis haliana (Coen and Meye owi z, 1991). Func ional
analysis o hese genes allowed he pos ula ion o he ABC model
ha assumes ha h ee classes o p o eins ac in a combina o ial
way o con e o gan iden i y in each who l. In A. haliana,
he A- class homeo ic genes APETALA1 (AP1) and APETALA2
(AP2) speci y sepal iden i y and in combina ion wi h he B-
class genes, APETALA3 (AP3) and PISTILLATA (PI), speci y
pe al iden i y. When B- is combined wi h C- class, con e ed by
he gene AGAMOUS (AG), s amen iden i y is speci ied, whe eas
C- class alone speci ies ca pel iden i y and lo al de e minacy
(Coen and Meye owi z, 1991). La e , he ABC model was
expanded and classes D and E we e included. The D- class genes,
SEEDSTICK (STK) and SHATTERPROOF (SHP) 1 and 2, a e
equi ed o o ule iden i y wi hin he ca pel (Fa a o e al., 2003;
Pinyopich e al., 2003). The E- class genes SEPALLATA (SEP) ac
edundan ly in he speci ica ion o sepals, pe als, s amens, ca pels
and o ules by pa icipa ing in complexes wi h he A, B, C, and
D p o eins (Pelaz e al., 2000;Di a e al., 2004;Cas illejo e al.,
2005).
P e ious s udies o lowe de elopmen genes in g ape ine
we e based on he iden i ica ion and unc ional analysis o
Vi is ini e a subsp. ini e a homologous o he co esponding
ABCDE A abidopsis genes (Boss e al., 2001, 2002;Calonje
e al., 2004;S eekan an e al., 2006;Poupin e al., 2007). The
g ape ine AP1 homologous gene, V iAP1, is exp essed du ing
lowe de elopmen , becoming excluded om he sepal- o ming
egion, being p e e en ially exp essed in he g owing pe als,
s amens, and ca pels (Calonje e al., 2004). The A- class homeo ic
gene AP2 is he only non-MADS box gene ha ac s as a nega i e
egula o o AG, as obse ed in ap2 A abidopsis mu an lowe s
in which sepals a e eplaced by ca pels (Yan e al., 2010). In
he g ape ine genome he e a e se e al genes encoding AP2/ERF
p o eins exp essed in bo h ege a i e and ep oduc i e issues a
di e en de elopmen al s ages (Licausi e al., 2010).
Se e al s udies ha e shown ha he exp ession o V iPI and
V iAP3 in he cul i a ed Vi is is consis en wi h PI and AP3
exp ession in A. haliana, being es ic ed o he pe al and s amen
who ls (Poupin e al., 2007;Díaz-Riquelme e al., 2009). In he
he maph odi e g ape ine V iTM6, he homolog o TOMATO
MADS BOX GENE 6 (TM6), a gene closely ela ed o AP3
(K ame and I ish, 2000), is exp essed du ing lowe o gan
iden i y in he h ee inne who ls as well as du ing ui g ow h
and ipening (Díaz-Riquelme e al., 2009).
In cul i a ed g ape ine he exp ession pa e n o he E-
class V iSEP1 and V iSEP3 genes, is simila o he A abidopsis
coun e pa s. V iSEP1 is exp essed in all lo al who ls whe eas
V iSEP3 exp ession is only excluded om sepal who l (Boss e al.,
2002).
The ABCDE model genes ha e been long conside ed as
candida e genes o sex de e mina ion in monoecious and
dioecious species. In he dioecious species Spinacia ole acea, wi h
unisexual lowe s (Sa he e al., 2010), he exp ession o B- class
lo al iden i y genes is absen in emale lowe s whe eas in male
lowe s he genes a e s ongly exp essed (P en e al., 2005).
In male plan s, SpPI-silencing o igina es no mal emale lowe s,
indica ing ha sexual dimo phism occu s h ough he egula ion
o B- class gene exp ession ha , by supp essing he o ma ion o
he gynoecium ac as masculinizing genes (Sa he e al., 2010).
Con a y o Spinacia, in he dioecious Silene la i olia, unisexuali y
a ises by o gan abo ion (Ha denack e al., 1994). The exp ession
pa e ns o SlM2 and SlM3, he PI and AP3 homologous
genes, di e du ing male and emale lowe o ganogenesis. A
ea ly lowe de elopmen s ages SlM2 and SlM3 a e exclusi ely
exp essed in pe al and s amen p imo dia, bo h in male and
in emale lowe me is ems (Ha denack e al., 1994). Howe e ,
du ing he la e s ages o emale lowe de elopmen , SlM2 is no
exp essed in he p imo dial o he abo ed s amen (Kazama e al.,
2005). In he dioecious Rumex ace osa, C- class ansc ip s we e
de ec ed in he hi d and ou h who ls o young male and emale
lowe s. Howe e , in male lowe s he exp ession in he ca pel
who l is ansien , disappea ing om he a es ed ou h-who l,
while in emale lowe s he exp ession is e ained in he ca pel
bu is absen om he s amen p imo dia (Ainswo h e al., 1995,
2005).
Ano he impo an gene ac ing du ing lowe de elopmen
is SUPERMAN (SUP), necessa y o he es ablishmen o he
bounda ies be ween hi d and ou h who l. SUP is hough
o coo dina e he p oli e a ion o s amen and ca pel speci ic
me is ema ic cells, keeping he p ope s uc u e o he who ls
and main aining he bounda y be ween who l 3 and who l 4
a he igh posi ion (Sakai e al., 2000). The SUP homolog
o S. la i olia, SlSUPERMAN (SlSUP), shows gende -speci ic
exp ession. SlSUP is a emale lowe speci ic gene, exp essed in
he second and hi d who ls and in he o ules, sugges ing ha
SlSUP has a posi i e ole in emale lowe de elopmen (Kazama
e al., 2009). Also in Cucumis sa i us, a SUP o holog (CsSUP)
is p edominan ly exp essed in emale o gans, and absen in male
lowe s (Zhao e al., 2014), sugges ing a conse ed ole in lowe
o gan de e mina ion.
In emale and male lowe s o wild Vi is ini e a, he
in ol emen o homeo ic genes in he es ablishmen o dioecy o
lowe ype speci ica ion has no ye been deeply analyzed. RNA-
seq and qRT-PCR analysis du ing he in lo escence de elopmen
o male and emale plan s o he dioecious species (V. . syl es is)
and he he maph odi e plan s o V. . ini e a, showed ha he e
was no signi ican di e ences in he o e all le els o ABCDE
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Coi o e al. Vi is Flowe Homeo ic Genes Exp ession
gene exp ession ha could accoun o he speci ica ion o he
di e en lowe ypes (Ramos e al., 2014). Howe e , i is possible
ha a he han di e ences in he o e all le el o exp ession
o hese genes, a dis inc spa ial pa e n o exp ession in he
h ee lowe ypes du ing ep oduc i e o gan de elopmen could
be esponsible o con e ing di e en lowe o gan iden i y.
The e o e, in he cu en wo k we pe o med a de ailed analysis
o he spa ial pa e n o exp ession o he ABCDE genes du ing
he de elopmen o male, emale and he maph odi e lowe s o
Vi is. Also, we analyzed he exp ession p o ile o V iSUP, o
in e whe he he e was a di e en es ablishmen o he bo de s
be ween ep oduc i e who ls in hese lowe s. We obse ed he
exp ession p o ile o he genes analyzed is simila in he h ee
lowe ypes. The e o e, his wo k p o ides u he e idence o
sugges ha he ABCDE genes migh no be di ec ly in ol ed in
sex speci ica ion and may ac ups eam o he pa hways leading
o o gan abo ion in Vi is unisexual lowe s.
MATERIALS AND METHODS
Plan Ma e ial
In lo escences om emale and male plan s o Vi is . syl es is
and he maph odi e lowe s o Vi is . ini e a (Tou iga
Nacional) (Figu e 1) we e collec ed om he Po uguese
Ampelog aphic Collec ion (PRT051), p ope y o Ins i u o
Nacional de In es igação Ag á ia e Ve e iná ia, in Dois Po os
(Lisbon dis ic , Po ugal). In lo escence/ lo al buds a la e
s ages B o G [acco ding o phenological classi ica ion o
de elopmen al s age by Baggiolini (1952)] we e collec ed om
se e al male, emale and he maph odi e plan s du ing Ap il and
May (Figu e 1).
RNA Ex ac ion, cDNA Syn hesis and
Cloning
To al RNA was ex ac ed om in lo escences using a plan RNA
ex ac ion ki , Spec umTM Plan To al RNA Ki (Sigma-Ald ich,
Inc) ollowing he manu ac u e ’s ins uc ions. cDNA syn hesis
was pe o med by e e se ansc ip ion wi h hea dena u a ion
acco ding o he Two S ep RT-PCR P ocedu e o RETROsc ip
Re e se T ansc ip ion Ki (Ambion, Li e Technologies, Spain).
Fo each sample 100 ng o o al RNA we e used. cDNA
ampli ica ion was pe o med h ough PCR in 25 µl o al olume
composed by 1 µg o cDNA, PCR bu e (20 mM T is–HCl
[pH 8.4], 50 mM KCl), 1.5 mM o MgCl2, 0.2 mM o dNTP
mix, 0.4 µM o each o wa d and e e se p ime s, 5 U o
Taq DNA polyme ase and au ocla ed MiliQ wa e . The ini ial
4 min dena u a ion occu ed a 94 C ollowed by 30 cycles
o 45 s a 94◦C (dena u a ion), 45 s a 55◦C (annealing), 90
s a 72◦C (ex ension), and a inal ex ension s ep o 4 min
a 72 C. PCR agmen s we e cloned in he linea ized ec o
pGEMR
-T Easy Vec o Sys em (P omega, Leiden, Ne he lands)
acco ding o he manu ac u e ’s ins uc ions. The ex ac ion o
plasmid DNA o Esche ichia coli cells was ca ied ou wi h he
Pu eLinkR
Quick Plasmid Minip ep Ki (In i ogenTM, Ca lsbad,
Cali o nia) ollowing he manu ac u e ’s ins uc ions.
Gene Sequence Iden i ica ion
The p o ein sequence o AP1, AP2, AP3, PI, AG, SUP, SEP1,
and SEP3 om A abidopsis we e e ie ed om TAIR3and TM6
om oma o was e ie ed om NCBI4and blas ed (Camacho
e al., 2009) agains he Vi is da abase5using he mo e ecen
anno a ion 12x 2.1. The same app oach was used o iden i y he
same genes in he o he species.
Phylogene ic analysis o p o ein sequences was pe o med
wi h he Maximum Likelihood me hod h ough MEGA
(Molecula E olu iona y Gene ics Analysis) e sion 6 (Tamu a
e al., 2013). The boo s ap consensus ee was in e ed om
1,000 eplica es.
RNA in Si u hyb idiza ion
Plan issue ixa ion, clea ing and in si u hyb idiza ion
expe imen s we e pe o med as p e iously desc ibed (Coen
e al., 1990;Coi o e al., 2017). P ime s o ibop obes syn hesis
used in he in si u hyb idiza ion we e design using he so wa e
P ime P emie 5.0 (P emie Bioso In e na ional) using a
p ime leng h o 20 ±2 bp (Supplemen a y Table 1). cDNA
p obes we e cloned in o he pGEMR
T-easy ec o sys em and
ampli ied by PCR wi h he M13 o wa d/ e e se p ime s and
speci ic o wa d/ e e se p ime s (Supplemen a y Table 1). The
PCR p oduc was pu i ied using he MinElu e PCR Pu i ica ion
Ki (QIAGEN, Valencia, CA, Uni ed S a es), acco ding o
he manu ac u e ’s ins uc ions, and used as empla e o he
ibop obe syn hesis, which was ca ied wi h SP6 and T7 RNA
polyme ase o ob ain he sense and an isense s ands. The
pa a in embedded ma e ial was sec ioned a 7 µm and he issue
slices moun ed wi h dis illed wa e . Images we e cap u ed wi h
a luo escence mic oscope (Wild Lei z, Labo lux S) wi h an
AxioCam HRM (Zeiss). Hyb idiza ions we e pe o med a 50◦C
wi h he excep ion o V iAP3 and V iTM6 ha we e pe o med
wi h highe s ingency a 55◦C due o ela i ely high homology
be ween bo h p obes. All in si u hyb idiza ion p ocedu es we e
made wi h sense (da a no show) and an i-sense p obes.
RESULTS AND DISCUSSION
The ABCDE model genes a e good candida es o be in ol ed
in he es ablishmen o male and emale lowe s in dioecious
species, pa icula ly he B- and C- class genes due o hei ole in
ep oduc i e o gan iden i y. The e o e, i is essen ial o analyze
he exp ession o hese genes in a sys ema ic way, ega ding he
empo al and spa ial dynamics h oughou lowe de elopmen
in male, emale and he maph odi e Vi is plan s.
Phylogene ic Analysis o ABCDE Model
Genes in Vi is
Se e al g ape ine genes in ol ed in lowe o gan iden i y ha e
been p e iously epo ed (Boss e al., 2001, 2002;Calonje
e al., 2004;Poupin e al., 2007;Díaz-Riquelme e al., 2009).
3h ps://www.a abidopsis.o g/
4h ps://www.ncbi.nlm.nih.go /
5h p://genomes.c ibi.unipd.i /gb2/gb owse/public/ i is_ ini e a_ 2/
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Coi o e al. Vi is Flowe Homeo ic Genes Exp ession
FIGURE 1 | In lo escence mo phology in he h ee Vi is lowe ypes. (A) Mo phological dispa i ies be ween he h ee lowe ypes ound be ween V. . syl es is and
V. . ini e a. Male plan s p oduce a la ge in lo escence ha display lowe s wi h e ec s amens bu educed pis il. Female plan s p oduce clus e s wi h ew lowe s
bea ing a comple e and unc ional pis il bu e lexed s amens wi h in e ile pollen. He maph odi e plan s p oduce a la ge in lo escence wi h bo h unc ional male and
emale o gans. (B) Phenological s ages o in lo escence de elopmen collec ed o pe o med in si u hyb idiza ion.
Howe e , in he Vi is genome da abase6 he e a e mo e han
one gene anno a ed as V iAP1,V iAP2,V iAG,V iSEP1, and
V iSEP3 (Supplemen a y Table 2). The e o e, o make su e
we we e analyzing he issue exp ession pa e n du ing lowe
de elopmen o he igh ABCDE homolog, phylogene ic ees
we e gene a ed (Supplemen a y Figu e 1) wi h he deduced
amino acid sequences o he MADS-box p o eins SHP1, STK,
AG, PI, AP1, AP3, FRUITFULL (FUL), TM6 and SEP, and as
well as he non MADS-box p o ein AP2 om he ollowing
species: Vi is ini e a (V i), A abidopsis haliana (A ) and o he
dico s plan s such as Ci us sinensis (Ci), Cucumis sa i us (Cs),
An i hinum majus (Am), Malus domes ica (Md), Pe unia hyb ida
(Ph), Populus ichoca pa (P ), P unus pe sica (Pp), Cas anea
mollissima (Cm), Solanum lycope sicum (Sl), Pinus adia e (P ),
and he monoco plan O ysa sa i a (Os) (Supplemen a y
6h p://genomes.c ibi.unipd.i /g ape/
Table 3). The esul ing phylogene ic ees (Supplemen a y
Figu e 1A) show ha V iAP1 p o eins (VIT_201s0011g00100,
VIT_214s0083g01030 and VIT_217s0000g04990) a e g ouped
in h ee clades. In he cu en wo k, we ha e chosen he
gene VIT_201s0011g00100 o wo k wi h (V iAP1), p e iously
iden i ied as he closes A AP1 homolog (Calonje e al., 2004).
Th ee V iAP2 (VIT_207s0031g00220, VIT_208s0040g03180 and
VIT_213s0019g03550) genes we e ound in he Vi is genome.
Ne e heless, VIT_207s0031g00220 displays highe p o ein
homology wi h AP2 genes wi h p o en unc ions (Supplemen a y
Figu e 1A) and also, RNA-seq da a ( lowe s om V. . syl es is
and V. . ini e a) showed ha VIT_207s0031g00220 is exp essed
in dis inc lowe de elopmen al s ages (Ramos e al., 2014)
sugges ing ha V iAP2 could be a wo hy candida e o ul ill he
AP2 canonical unc ion.
As a as B- class genes a e conce ned, he wo V iAP3 genes
anno a ed (VIT_218s0001g13460 and VIT_204s0023g02820)
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Coi o e al. Vi is Flowe Homeo ic Genes Exp ession
we e g ouped in he AP3 and TM6 clade, espec i ely
(Supplemen a y Figu e 1B). In a p e ious s udy
VIT_204s0023g02820 was assigned as V iTM6 (Poupin
e al., 2007). In o de o cla i y hese anno a ions, we ook
a close look in o he V iAP3 genes (VIT_218s0001g13460
and VIT_204s0023g02820). The B- class gene AP3/TM6,
o m a di e gen linage wi h C- e minal speci ic mo i s.
The mo i DLTTFALLE de ine he euAP3 linage p esen in
highe eudico plan s such as A abidopsis haliana,Pe unia
hyb id, and An i hinum majus while he mo i DLRLA is
p esen in he paleoAP3 linage in lowe eudico s, dico s
and monoco s (Vandenbussche e al., 2003). Compa ison
o VIT_218s0001g13460 and VIT_204s0023g02820 p o ein
sequences allowed he iden i ica ion o he DLRLA mo i in
VIT_204s0023g02820, while VIT_218s0001g13460 shows he
euAP3 mo i : DLTFTLLE (Supplemen a y Figu e 2). The e o e,
in his s udy we conside ed VIT_218s0001g13460 gene as V iAP3
and VIT_204s0023g02820 gene as V iTM6 (Poupin e al., 2007).
Rega ding V iPI (VIT_218s0001g01760), also a B- class gene,
alls in he PI clade (Supplemen a y Figu e 1B).
The C- class gene, V iAG (VIT_210s0003g02070), and he
D- class genes, V iSHP1 (VIT_212s0142g00360) and V iSTK
(VIT_218s0041g01880), belong o he same sub- amily, as
sugges ed by a p e ious wo k (Becke and Theissen, 2003;
Pinyopich e al., 2003). V iSHP1 (VIT_212s0142g00360) p esen
in ch omosome 12 was subsequen ly conside ed o be he AG
homolog (Joly e al., 2004) (Supplemen a y Figu e 1C). In he
cu en wo k we decided o ca y ou a de ailed analysis o hese
h ee p o ein sequences. The da a show ha he V iSHP1 p o ein
sequence sha es highe simila i y wi h A SHP2 (82%) han wi h
A SHP1 (70%) (Supplemen a y Figu es 3A,B). V iAG, is p esen
on ch omosome 10 (VIT_210s0003g02070) was chosen, ha is
he one wi h highes homology o A AG.
The genes associa ed wi h he E- class, V iSEP1
(VIT_214s0083g01050) and V iSEP3 (VIT_201s0010g03900)
a e highly ela ed wi h SEPALLATA homologs o o he species,
(Supplemen a y Figu e 1D) and we e p e iously desc ibed as
V iSEP1 and V iSEP3, espec i ely (Boss e al., 2002;Joly e al.,
2004).
Exp ession o Flo al Iden i y Genes
Du ing Vi is Flowe De elopmen
A-Class Genes: V iAPETALA1 and V iAPETALA2
In A abidopsis haliana,AP1 oge he wi h AP2, con ibu es o
sepal and pe al iden i y in he i s and second who ls (Theissen,
2001;K izek and Fle che , 2005). De ec i e mu an s in AP1
exhibi a ious de ec i e pheno ypes due o he ole o his gene
in o gan and lo al me is em iden i y along wi h LEAFY (Wagne
e al., 1999). ap1 lowe s ha e sepals con e ed in o b ac s and
addi ional lowe s a e o med in he axis o he b ac s (I ish
and Sussex, 1990) sugges ing ha AP1 no only speci ies he
iden i ies o sepals and pe als bu also de e mines he iden i y o
lo al me is em (Theissen and Saedle , 2001). S udies o eudico
species poin o a conse a ion o he ole o he AP1 gene in
he lo al me is em speci ica ion. Howe e , hei in ol emen
in he de elopmen o pe ian h o gans is unclea (Li , 2007;
Rijpkema e al., 2010). E en in some A abidopsis ap1 mu an s
sepals a e s ill o med p o ing he dubious ole o AP1 pe ian h
iden i y (Bowman e al., 1993;Yu e al., 2004;Cas illejo e al.,
2005).
In Vi is, exp ession o V iAP1 occu s ea ly in lo al
me is em de elopmen in he h ee lowe ypes (Figu es 2A–C),
in acco dance wi h i s po en ial lo al me is em iden i y
ole (Mandel e al., 1992). Howe e , when s amens s a o
de elop, V iAP1 exp ession pe sis s in he cen e o he lowe
p imo dium, ins ead o being es ic ed o he i s and second
who ls (whe e sepals and pe als o m) (Figu es 2D–F). The
V iAP1 exp ession pa e n is simila in he h ee lowe ypes
om ea ly o la e lowe de elopmen al s ages and is de ec ed
in pe al, s amen and ca pel p imo dia (Figu e 2). Howe e , i
is almos absen in pe als, when hey encapsula e he s amens
and ca pel (Figu es 2G–I). The absence o V iAP1 exp ession
in he sepal egions and i s exp ession in he lowe cen e was
no expec ed, conside ing ha in A abidopsis he exp ession o
A AP1 is es ic ed o he sepal and pe al who ls and absen
om he de eloping s amens and ca pels (Sunds om e al.,
2006). Howe e , he Vi is exp ession pa e n in no comple ely
un o eseen. The exp ession o an AP1 homolog in he ca pel
egion was also epo ed o he SQUAMOSA (SQUA) gene in
A. majus (Huijse e al., 1992) as well as o he AP1-like gene
in Ge be a hyb ida (Yu e al., 1999). Also, in g ape ine i has
been epo ed he p esence o V iAP1 ansc ip s in he hi d
and ou h who ls (Calonje e al., 2004). AP1 is canonically
c edi ed wi h wo unc ions in lowe de elopmen : (1) lowe
me is em ini ia ion and (2) pe ian h iden i y (Huijse e al.,
1992). Howe e , a iabili y in pe ian h o gans iden i y exis s and
seems o be a consequence o a mul iple e en s o e olu ion
wi hin angiospe ms, as has been achie ed h ough phylogene ic
econs uc ions using A- unc ion genes (Zanis e al., 2003;
Hileman and I ish, 2009). In he case o Vi is he absence o
V iAP1 exp ession in he lowe i s who l ein o ce he unclea
ole o his gene in sepal speci ica ion in species o he hen
A abidopsis.
In A abidopsis,AP2 is he o he A- class gene ha plays a
ole in speci ying sepals and pe als (Huala and Sussex, 1992;
Jo uku e al., 1994;Husbands e al., 2009). A AP2 is also exp essed
in he hi d and ou h who ls (Jo uku e al., 1994), whe e i
is pos - ansc ip ionally a ge ed by miRNA172 (Chen, 2004).
ap2 lowe s ha e lea -like s uc u es o ca pels ins ead o sepals
and s amens o s amenoid pe als ins ead o pe als (Bowman
e al., 1989, 1991;Kuns e al., 1989). In Vi is,V iAP2 exp ession
is de ec ed in ea ly lowe me is em and becomes excluded
om ea ly sepal p imo dia (Figu es 3A–C). The exp ession o
V iAP2 in ea ly lowe p imo dia sugges s a conse ed ole
in lowe me is em iden i y (Huala and Sussex, 1992). When
pe al p imo dia ini ia e, V iAP2 is excluded om his who l
(Figu es 3D–F) and emains in he hi d and ou h who l
h oughou la e s ages o lowe de elopmen . In A abidopsis,
AP2 is exp essed in all who ls h oughou lowe de elopmen
(Jo uku e al., 1994;Wu schum e al., 2006;Zhao e al., 2007)
bu mo e ecen ly, he AP2 exp ession pa e n was eanalyzed
epo ing a dis inc beha io om he one p e iously desc ibed
(Wollmann e al., 2010). These au ho s epo ha AP2 is
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Coi o e al. Vi is Flowe Homeo ic Genes Exp ession
FIGURE 2 | In si u hyb idiza ion o A- class gene V iAPETALA1 (V iAP1) in de eloping lowe s. V iAP1 (VIT_201s0011g00100) exp ession analysis on longi udinal
sec ions o wild and he maph odi e Vi is in lo escences. Male: A,D,G; Female: B,E,H; He maph odi e (He ): C,F,I. The exp ession pa e n is simila in he h ee lowe
ypes h oughou de elopmen and is i s de ec ed in he cen al dome o he lowe (A–C) when sepal o ma ion s a de eloping. When pe als eme ge, V iAP1
emaining in he second, hi d, and ou h who l (D–F). A la e de elopmen al s ages, V iAP1 exp ession weakens a he pe al base bu emains in he hi d and
ou h who l (G–I). The diag ams ep esen c oss-sec ions wi h he co esponding igu e indica ed on each line. Abb e ia ions a e as ollows: m, lowe me is em;
se, sepals; pe, pe als; s , s amens; c, ca pel. Scale ba , 100 µm.
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Coi o e al. Vi is Flowe Homeo ic Genes Exp ession
FIGURE 3 | In si u hyb idiza ion o A- class gene V iAPETALA2 (V iAP2) in de eloping lowe s. V iAP2 (VIT_207s0031g00220) exp ession analysis on longi udinal
sec ions o wild and he maph odi e Vi is in lo escences. Male: A,D; Female: B,E; He maph odi e (He ): C,F.V iAP2 exp ession is simila in he h ee lowe ypes
h oughou lowe de elopmen and is i s de ec ed in he cen al dome o he lowe me is em (A–C) bu absen in he sepal p imo dia. When pe als and s amens
s a o eme ge he exp ession is es ic ed o he hi d and ou who ls (D–F) whe e i emains. The diag ams ep esen c oss-sec ions wi h he co esponding igu e
indica ed on each line. Abb e ia ions a e as ollows: m, lowe me is em; se, sepals; pe, pe als; s , s amens; c, ca pel. Scale ba , 100 µm.
exp essed in sepals bu is absen om he cen e o lowe
p imo dia. Subsequen ly, A AP2 mRNA is excluded om he
i s who l and appea s in s amen and pe al p imo dia. La e ,
A AP2 signal emains in pe als, s amens and ca pels including
he o ules (Wollmann e al., 2010). The p esence o A AP2 in
he s amens and ca pels shows ha no only A AP2 has a ole
in o ule de elopmen (Wollmann e al., 2010) bu also ha A AG
does no an agonize A AP2 ansc ip s in he s amens (Wollmann
e al., 2010). To pe o m his wo k, Wollmann e al. (2010) used
a p obe agains he 30 egion o he ansc ip o a oid c oss
hyb idiza ion wi h o he AP2 homologs. In Vi is he ull-leng h
V iAP2 ansc ip was used and i is possible ha he de ec ion
o mRNA in he dome o he lowe me is em is a esul o
c oss hyb idiza ion. Howe e , Wollmann e al. (2010) also es ed
he ull-leng h cDNA o A AP2 and he accumula ion pa e n o
mRNA was simila wi h he exp ession pa e n using only a 30
egion RNA p obe.
In Vi is he exp ession o V iAP2 sugges s (1) ha his gene
may no be undamen ally necessa y o sepal o pe al o ma ion;
(2) i s ansien exp ession du ing p imo dia ini ia ion could be
he igge o i s and second who l iden i ies o (3) may be
pa o a complex se o genes ha ac edundan ly o es ablish
he iden i y o he i s and second who l. The esul s ob ained
in Vi is seem o co obo a e he esul s epo ed in A. haliana
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Coi o e al. Vi is Flowe Homeo ic Genes Exp ession
(K ogan e al., 2012) being sugges i e o a ole o V iAP2 in lo al
me is em iden i y, as i s mRNA accumula ion is obse ed ea ly
in lowe de elopmen (Figu es 3A–C) and la e may be ac ing
syne gis ically wi h B- and C- class genes in he hi d and ou h
who l, espec i ely, o speci y he ep oduc i e o gans.
Al hough AP1 and AP2 show a widesp ead ole in lowe
me is em ini ia ion, he associa ion ega ding hei ole in o gan
iden i y has been oublesome ( e iewed in Li and K ame ,
2010). The esul s, ega ding Vi is AP1 and AP2 con ibu e o
he mys e y su ounding he ole o A- unc ion genes ega ding
pe ian h iden i y ac oss he co e eudico s (Theissen e al., 2000;
Maes e al., 2001;Shepa d and Pu ugganan, 2002;Smy h, 2005;
Mo el e al., 2017) and ein o ce he in icacy o hese se o genes.
B- Class Genes: V iAPETALA3, V iPISTILLATA, and
V iTM6
Ea ly in de elopmen o A abidopsis lowe s, PI is exp essed in
he cen al dome o he lowe me is em (Go o and Meye owi z,
1994). La e , he exp ession o AP3/PI is de ec ed in he second
and hi d who ls whe e he pe als and s amens a e speci ied
(Bowman e al., 1989). TM6, conside ed a B-class homeo ic
gene (Poupin e al., 2007), was i s iden i ied in oma o and
is exp essed in s amens and in ca pels (Pnueli e al., 1994;de
Ma ino e al., 2006). AP3/PI a e esponsible o pe al (along
wi h AP2) and s amen ( oge he wi h AG) iden i y. Mu a ions in
B- class genes lead o sepaloid s uc u es o med in he second
who l and ca peloid s uc u es in he hi d who l (Hill and Lo d,
1989;Jack e al., 1992;Wues e al., 2012). In A abidopsis, he AP3
gene is exp essed only a e sepal iden i y has been es ablished
and is con ined o he second and hi d who l. E en in ea ly
s ages o de elopmen , he AP3 mRNA is no de ec ed in he
cen al egion o he lowe me is em (Go o and Meye owi z,
1994). In g ape ine, in si u hyb idiza ion showed ha V iAP3
is exp essed in he ca pel who l in he h ee lowe ypes. In
ea ly s ages o Vi is lowe de elopmen , be o e sepal p imo dia
s a o eme ge, high le els o V iAP3 we e de ec ed in he
cen al egion o he lowe me is em in he cells ha will
con ibu e o he o ma ion o pe al, s amen and ca pel p imo dia
(Figu e 4, Uppe panel). When sepal p imo dia become isible,
V iAP3 is s ill de ec ed in he cen al dome o he lowe
p imo dia (Figu es 4A–C). A e he eme gence o he pe al
p imo dia, V iAP3 exp ession is p esen in he second and hi d
who ls and i s absence om he ca pel who l is inconclusi e
(Figu es 4D–F). This pa e n is common o he h ee lowe
ypes (Figu es 4G–I). Howe e , p e ious wo k, using RT-qPCR,
showed ha he exp ession o V AP3 is es ic ed o pe als and
s amens in a he maph odi e a ie y o Vi is . ini e a (Poupin
e al., 2007). Due o he sequence simila i y be ween V iAP3
and V iTM6 (Supplemen a y Figu e 2) and in o de o a oid
c oss hyb idiza ion wi h V iTM6, i was necessa y o inc ease
he s ingency o V iAP3 p obe hyb idiza ion (see Ma e ial and
Me hods). Ne e heless, we do no ule ou he possibili y ha
some signal in he hyb idiza ion wi h he V iAP3 p obe may ha e
he con ibu ion om he V iTM6 RNA. In all hyb idiza ions
wi h highe s ingency he esul s we e consis en and he e is a
s ong possibili y ha V iAP3 ansc ip does no accumula e in
he ca pel who l (Figu e 4).
TM6 is conside ed a B- class homeo ic gene, and was i s
iden i ied as being exp essed in pe als, s amens and ca pels o
L. lycope sicum lowe s (Pnueli e al., 1994;de Ma ino e al.,
2006). In Vi is, in ea ly s ages o emale, male, and he maph odi e
g ape ine lowe p imo dia wi h al eady eme ging sepals, he
highes le els o V iTM6 exp ession was de ec ed in he cen e
o he lowe me is em (Figu es 5A–C), being excluded om
he sepal who l (Figu es 5A–C). A e he de elopmen o pe al
and s amen p imo dia (Figu es 5D–F), V iTM6 was con ined o
he second, hi d and ou h who ls in he h ee lowe ypes.
V iTM6 seems equally exp essed in all h ee lowe ypes when
s amen p imo dia s a eme ging (Figu es 5G–I), which does
no sugges a p e e en ial ole in s amen de elopmen as seen
by he analysis o he TM6 unc ion in oma o (de Ma ino
e al., 2006). As epo ed in oma o (de Ma ino e al., 2006),
he gene silencing o TM6 by RNAi gene a es lowe s wi h
a comp omised s amen de elopmen , howe e , no change in
V iTM6 exp ession was obse ed be ween unc ional s amens o
male and e lexed s amens o emale lowe s in Vi is sugges ing
ha V iTM6 is no in ol ed in s amen abo ion. In he Pe unia
ap3 mu an ( ha lacks pe als and s amens) is complemen ed
wi h 35S-d i en PhTM6, pe al de elopmen is es o ed (Rijpkema
e al., 2006), sugges ing a ole in pe al de elopmen . In g ape ine,
he p esence o V iTM6 mRNA in he h ee inne who ls poin s
o i s pa icipa ion in he de elopmen o pe als, s amens, and
ca pels.
PISTILLATA in A abidopsis is exp essed in cells ha will
gi e ise o pe als, s amens, and ca pel p imo dia in ea ly
s ages o lowe de elopmen (Hill and Lo d, 1989;Sunds om
e al., 2006), and i s exp ession is con ined o he second
and hi d who ls only in la e s ages (Go o and Meye owi z,
1994). In ea ly s ages o Vi is lowe de elopmen , V iPI is
exp essed in he cen e o he lowe me is em (Figu e 6, Uppe
panel) simila o wha was desc ibed o A abidopsis (Go o
and Meye owi z, 1994). When sepal p imo dia s a o eme ge,
V iPI has a high exp ession in he cells ha will de elop in o
pe als and s amens and s a s o ade om he egion ha
will de elop in o ca pels (Figu es 6A–C). As soon as pe al
p imo dia s a s o eme ge, V iPI is comple ely excluded om
he ou h who l (Figu es 6D–F) emaining con ined o he
second and hi d who l du ing he la e s ages o de elopmen
(Figu es 6G–I). V iPI seems o be mo e exp essed in s amen
han in pe al p imo dia, bo h in male and in emale lowe s.
This exp ession pa e n is simila in emale and male lowe s and
did no ollow he pa e n o o gan abo ion ounded in Silene
la olia, whe e a la e s ages o emale lowe de elopmen PI
exp ession is absen om he abo ed s amens (Kazama e al.,
2005).
Toge he , he exp ession o V iPI and V iAP3 B- class genes
is simila o wha was desc ibed o he B- class A abidopsis
homologs (Go o and Meye owi z, 1994). The ea ly dynamics o
bo h V iAP3 and V iPI (Figu es 4,6) aises ques ions ega ding
he molecula mechanism unde lying he ac i a ion o bo h genes
in he lo al me is em in Vi is. Ou esul s sugges ha he
bounda ies o V iAP3 and V iPI a e iden ical du ing he onse o
Vi is lowe me is em sugges ing, a leas in pa , ha bo h genes
could be unde he egula ion o iden ical ups eam ac o s.
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Coi o e al. Vi is Flowe Homeo ic Genes Exp ession
FIGURE 4 | In si u hyb idiza ion o B- class gene V iAPETALA3 (V iAP3) in de eloping lowe s. V iAP3 (VIT_18s0001g13460) exp ession analysis on longi udinal
sec ions o wild and he maph odi e Vi is in lo escences. Uppe panel: in ea ly s ages o in lo escence de elopmen he exp ession pa e n is de ec ed in he cen al
dome o he lowe me is em (a owhead), be o e sepal p imo dia eme ge, being simila in he h ee lowe ypes. Male: A,D,G; Female: B,E,H; He maph odi e (He ):
C,F,I.V iAP3 exp ession is simila in he h ee lowe ypes h oughou lowe de elopmen and is i s de ec ed in he cen al dome o he lowe me is em (A–C).
When pe als eme ge he exp ession s a s o be es ic ed o he second and hi d who l being excluded om sepals (D–I). The diag ams ep esen c oss sec ions
wi h he co esponding igu e indica ed on each line. Abb e ia ions a e as ollows: m, lowe me is em; se, sepals; pe, pe als; s , s amens; c, ca pel. Scale ba ,
100 µm.
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Coi o e al. Vi is Flowe Homeo ic Genes Exp ession
FIGURE 11 | Homeo ic genes exp ession in Vi is and hei ole in lowe sex de elopmen . (A) Diag ams o lowe ing genes exp ession. Exp ession pa e n o he
ABCDE model genes and he cadas al gene V iSUPERMAN. Homeo ic genes V iAPETALA (V iAP1) and V iAP2 (V iAP2) om he A- unc ion ha e no exp ession
in he i s who l o sepals. They a e exp essed in pe als, s amens and ca pel p imo dia. The genes V iAPETALA (V iAP3) and V iPISTILATA (V iPI) om he B-
unc ion o m pe als and s amens in he second and hi d who l. V iSUPERMAN es ic s B- unc ion exp ession genes in lowe me is ems in di e en lowe
de elopmen al s ages o V. . ini e a and a V. . syl es is. V iAGAMOUS (V iAG) is he only gene in he C- unc ion esponsible o ca pel o ma ion in he ou h
who l. Ou da a sugges ha he e a e no an agonism be ween A- and C- unc ion in Vi is. E- unc ion V iSEPALLATA (V iSEP) genes ac edundan ly and a e
exp essed in he h ee inne who ls and equi ed o he co ec o ma ion o all lowe o gans. (B) P oposed model o lowe de elopmen . The model sugges s ha
lowe de e mina ion is es ablished a he momen o ep oduc ion by ye unknown ac o s. The lowe de elopmen is egula ed h ough he onse o he ABCDE
model genes un il a la e s age o de elopmen , as obse ed in his wo k. Downs eam o homeo ic genes ha es ablish lowe o gan iden i y and ini ia ion, o he
egula o s migh be in ol ed in lowe ype speci ica ion. These egula o s can ei he (I) ac unde he con ol o he ini ial unknown sexual de e mina ion ac o s o (II)
he ini ial unknown sexual de e mina ion ac o s a e di ec ly in ol ed in lowe speci ica ion in o male and emale. Bo h scena ios can be exclusi ely o ac oge he
p omo ing o gan abo ion in V. . syl es is o a he maph odi e lowe de elopmen in V. . ini e a. se, sepals; pe, pe als; s , s amens; c, ca pel.
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Coi o e al. Vi is Flowe Homeo ic Genes Exp ession
The Cad as al Gene: V iSUPERMAN
The SUP gene es ablishes he bounda ies be ween s amen and
ca pel who ls by ac ing as a cadas al ac o es ic ing he B-
class genes exp ession (Hi a su e al., 2002). In sup A abidopsis
mu an s, he exp ession o AP3/PI expands o he ou h who l
whe e, s aminoid s uc u es a e o med ins ead o a ca pel
(Bowman e al., 1991, 1992). SUP ac s by inhibi ing he exp ession
o AP3 and PI in he ou h who l o he de eloping lowe
(Jack e al., 1992;Sakai e al., 2000). In Vi is in lo escences, he
exp ession o V iSUPERMAN (V iSUP) is simila du ing lowe
de elopmen o he h ee lowe ypes (Figu e 7). The ea lies
exp ession o V iSUP is de ec ed in he cen al egion o he lo al
me is em adjacen o he bounda y be ween he hi d and ou h
who ls (Figu es 7A–C). In la e s ages o lo al de elopmen ,
V iSUP is exp essed in he inne egion o he de eloping s amen
p imo dia (Figu es 7D–F). When s amen p imo dia a e clea ly
de ined, V iSUP exp ession emains in he egion adjacen o he
ca pel p imo dia (Figu es 7G–I).
The in si u esul s ob ained wi h V iAP3/V iPI and hei
egula o V iSUP show ha V iAP3 exp ession appea s i s in
he cen al egion o lowe me is em and is, in a la e s age,
ollowed by V iSUP exp ession, which goes in ag eemen wi h
wha has been obse ed in A abidopsis mu an s, in which SUP
exp ession is de ec a e he ini ia ion o AP3 exp ession (Sakai
e al., 1995).
Ou da a sugges ha ea ly V iSUP exp ession ac in a simila
way o wha has been desc ibed in A abidopsis, main aining he
who l 3 and 4 bounda y a e he who l p epa e n has been
es ablished. In A abidopsis,SUP gene appea s o ac ansien ly,
only equi ed o a sho pe iod be o e he cells in he lo al
who ls unde go ex ensi e di isions o p oduce o gan p imo dia
(Sakai e al., 2000), a b ie exp ession ha is su icien o
ul ill SUP unc ion in who l bounda y main enance (Bowman
e al., 1991, 1992;Jack e al., 1992). Howe e , in g ape ine, he
exp ession o V iSUP pe sis s in he inne pa o who l 3 un il
la e lowe de elopmen al s ages (Figu es 7G–I) sugges ing ha
i may no be he igge bu could be in ol ed in main ain he
gene ic signaling by con olling he balanced p oli e a ion o wo
adjacen lo al who ls.
C- Class Genes: V iAGAMOUS
AGAMOUS is a C- class homeo ic MADS-box gene exp essed
bo h in he hi d who l, ac ing oge he wi h PI and AP3 in
speci ying s amen iden i y, and in he ou h who l, whe e i
is esponsible o ca pel iden i y and, o some deg ee, o ule
iden i y (Bowman e al., 1989, 1991). Mu an plan s o AG do
no ha e ep oduc i e o gans, and ins ead exhibi a pheno ype
desc ibed as “a lowe wi hin a lowe ” wi h he absence o
s amens and ca pels (Yano sky e al., 1990). In Vi is,V iAG
exp ession (VIT_210s0003g02070) ollows a empo al and spa ial
pa e n (iden ical in all he h ee lowe ypes) ha i s wi h
wha was p oposed by he ABCDE model. V iAG is exp essed
in he cen e o he Vi is lowe me is em (Figu es 8A–C)
and is con ined o he hi d and ou h who ls when s amen
p imo dia eme ge (Figu es 8D–F). This exp ession pa e n
is main ained h oughou la e s ages o lowe de elopmen
(Figu es 8G–I). A simila exp ession p o ile has been epo ed
in he he maph odi e Riesling a ie y, in which V iSHP1 is
exp essed in la e s ages o lowe de elopmen (Joly e al.,
2004) whe e i is equi ed o con ol o ule iden i y on issues
ha de elop wi hin he ca pels (Pinyopich e al., 2003). In he
dioecious Rumex ace osa,AG ansc ip accumula ion dec ease
in he abo ed o gans (Ainswo h e al., 1995), howe e , in
g ape ine he simila exp ession p o ile o V iAG in all lowe
ypes (Ramos e al., 2014) may exclude he di ec in ol emen
o V iAGAMOUS in ca pel abo ion o he o ma ion o e lexed
s amens.
E- Class Genes: V iSEPALLATA1 and
V iSEPALLATA3
The E- class SEPALLATA (SEP) play a cen al ole in lowe
me is em de e minacy and o gan iden i y (Di a e al., 2004). The
SEP genes, al hough unc ionally edundan , a e equi ed o he
o ma ion o pe als, s amens and ca pels as he iple mu an
(sep 1/2/3) has an inde e mina e lowe wi h all lowe o gans
con e ed in o sepals (Pelaz e al., 2000;Honma and Go o, 2001).
In A. haliana, SEP1 is exp essed h oughou lowe de elopmen
(Sa idge e al., 1995). In he h ee Vi is lowe ypes, V iSEP1 is
exp essed in he cen e o he lowe me is em (Figu es 9A–C),
bu excluded om he sepal p imo dia. When pe al p imo dia
eme ge, V iSEP1 emains in he second, hi d and ou h who ls
(Figu es 9D–F) and a la e de elopmen al s ages, V iSEP1 is s ill
exp essed in he hi d and ou h who ls bu has lowe exp ession
in he pe als (Figu es 9G–I).
SEP3 is in ol ed in sepal, pe al, s amen, ca pel, and o ule
de elopmen and i s ec opic exp ession is enough o ac i a e
A AP3 and A AG (Pelaz e al., 2000;Fa a o e al., 2003;Di a
e al., 2004). In Vi is, V iSEP3 exp ession is de ec ed e y ea ly
in he lo al me is em in all lowe ypes (Figu es 10A–C). When
pe al and s amen p imo dia eme ge, V iSEP3 is excluded om
sepals (Figu es 10D–F). In la e s ages o lowe de elopmen ,
V iSEP3 is weakly exp essed in bo h he base and adaxial side
o pe als (Figu es 10G–I). No di e ences we e ound in he
exp ession o V iSEP3 in he h ee lowe ypes (Figu e 10). In
A abidopsis SEP1 is exp essed sligh ly ea lie han SEP3, which is
exp essed in a egion co esponding o he inne h ee who ls jus
be o e he ini ia ion o lo al p imo dia (Flanagan and Ma, 1994;
Sa idge e al., 1995;Di a e al., 2004). In Vi is, he ac i i ies o
V iSEP1/3 span he h ee inne who ls simila o wha happens in
A abidopsis (Pelaz e al., 2000). Howe e , and con a ily o wha
happens in A abidopsis, ou da a show ha V iSEP1 exp ession is
spa ially and empo ally e y simila o V iSEP3.
CONCLUSION
The esul s o he p esen s udy ein o ce he g ea complexi y
o he e en s and molecula cascades ha occu du ing
de e mina ion and speci ica ion o lo al o gan iden i y in Vi is.
P e ious esul s (RNA-seq and qRT-PCR) showed simila le els
o ABCDE gene exp ession in male, emale and he maph odi e
Vi is lowe s (Ramos e al., 2014), sugges ing ha hese genes
may no be di ec ly in ol ed in sex speci ica ion in his species.
Howe e , simila le els o exp ession can be p oduced by
F on ie s in Plan Science | www. on ie sin.o g 17 July 2018 | Volume 9 | A icle 1029
pls-09-01029 July 12, 2018 Time: 18:28 # 18
Coi o e al. Vi is Flowe Homeo ic Genes Exp ession
di e en pa e ns o issue exp ession and, he e o e, he e was
s ill a possibili y ha hese ABCDE genes could media e he
speci ica ion o he h ee di e en Vi is lowe ypes.
Despi e some sligh di e ences, he homeo ic genes exhibi
a spa ial exp ession p o ile simila in he h ee lowe ypes
and analogous o wha has been desc ibed o A abidopsis
homologs. Addi ionally, none o hese genes all in o he egion
o ch omosome 2 esponsible o sex de e mina ion (Fech e
e al., 2014;Picq e al., 2014;Coi o e al., 2017;Zhou e al.,
2017). Mo eo e , ou esul s show ha despi e hese genes
being in ol ed in lowe o gan iden i y hey a e no di ec ly
esponsible o lowe ype speci ica ion in Vi is. One ou come
ha eme ges om his wo k conce ns he iden i y o sepals
(Figu e 11A). The homeo ic genes, V iAP1 and V iAP2, ha e
no exp ession in he i s who l whe e hey a e supposed o
egula e he o ma ion o sepals (Figu e 11A). This unexpec ed
absence o exp ession aises wo hypo heses: hese genes may
no be in ol ed in sepal iden i y speci ica ion in his species o
he s uc u e known as sepals has, in ac , an iden i y di e en
om canonical sepals. Addi ionally, hei exp ession in lowe
de elopmen a ini ial s ages also poin o a ole in lowe
me is em iden i y.
A model o lowe de elopmen , shown in Figu e 11B,
p oposes ha lowe de e mina ion is es ablished a he ime
o e iliza ion by ac o s s ill unknown (Figu e 11B) p esen
in au osomal ch omosomes. The subsequen lowe o gan
de elopmen may be egula ed h ough he onse o he ABCDE
homeo ic genes ha ac a e sex de e mina ion and ups eam o
lowe o gan abo ion egula o s, such as he al eady s udied gene
V iAPRT3 (Coi o e al., 2017). These pu a i e egula o s can ac
unde he con ol o he ini ial unknown sexual de e mina ion
ac o s o each o he la e may be di ec ly con olling he lowe
speci ica ion p omo ing he abno mal de elopmen o male o
emale ep oduc i e o gans in la e s ages o lowe de elopmen .
Bo h scena ios can be exclusi e o ac oge he leading o
o gan abo ion as in V. . syl es is lowe s o he maph odi e
lowe s as in V. . ini e a.
AUTHOR CONTRIBUTIONS
MR, JLC, HS, and MMRC concei ed and designed he
expe imen s. JLC, HS, MM, and MJNR pe o med he
expe imen s. HS, JLC, MJNR, MMRC, and MR analyzed he
da a. HS, JLC, MJNR, SA, MMRC, and MR w o e he pape .
JC, JLC, and MR es ablished Vi is ini e a syl es is collec ion
and collec ed plan issues acco ding phenological de elopmen al
s age.
FUNDING
This wo k was suppo ed by he unded p ojec PTDC/AGR-
GPL/119298/2010 om Fundação pa a a Ciência e Tecnologia
(FCT, Po ugal), by UID/AGR/04129/2013 cen e g an om
FCT, Po ugal ( o LEAF), and by UID/MULTI/04046/2013
cen e g an om FCT, Po ugal ( o BioISI), and JLC,
MJNR, MMRC, and MR a e suppo ed by FCT ellowships,
SFRH/BD/85824/2012, SFRH/BD/110274/2015, SFRH/BSAB/
113781/2015, SFRH/BPD/64905/2009, espec i ely.
ACKNOWLEDGMENTS
We a e also g a e ul o Eng. Ei as-Dias, cu a o o he Po uguese
Ampelog aphic Collec ion (p ope y o Ins i u o Nacional de
In es igação Ag á ia e Ve e iná ia, Dois Po os) whe e sampling
was pe o med, o he collabo a ion in his wo k allowing he
access o he Vi is collec ion.
SUPPLEMENTARY MATERIAL
The Supplemen a y Ma e ial o his a icle can be ound online
a : h ps://www. on ie sin.o g/a icles/10.3389/ pls.2018.01029/
ull#supplemen a y-ma e ial
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F on ie s in Plan Science | www. on ie sin.o g 20 July 2018 | Volume 9 | A icle 1029