RESEARCH ARTICLE Open Access
In ol emen o e hylene biosyn hesis and
signalling in ui se and ea ly ui de elopmen
in zucchini squash (Cucu bi a pepo L.)
Cecilia Ma ínez
1
, Susana Manzano
1
, Zo aida Megías
1
, Dolo es Ga ido
2
, Belén Picó
3
and Manuel Jamilena
1*
Abs ac
Backg ound: We ha e iden i ied a kind o pa henoca py in zucchini squash which is associa ed wi h an
incomple e and omonoecy, i.e. a pa ial con e sion o emale in o bisexual lowe s. Gi en ha and omonoecy in
his and o he cucu bi species is caused by a educ ion o e hylene p oduc ion in he emale lowe , he associa ed
pa henoca pic de elopmen o he ui sugges ed he in ol emen o e hylene in ui se and ea ly ui
de elopmen .
Resul s: We ha e compa ed he p oduc ion o e hylene as well as he exp ession o 13 e hylene biosyn hesis and
signalling genes in pollina ed and unpollina ed o a ies/ ui s o wo cul i a s, one o which is pa henoca pic
(Ca ili), while he o he is non-pa henoca pic (Tosca). In he la e , unpollina ed o a ies show an induc ion o
e hylene biosyn hesis and e hylene signal ansduc ion pa hway genes h ee days a e an hesis, which is
concomi an wi h he ini ia ion o ui abo ion and senescence. F ui se and ea ly ui de elopmen in pollina ed
lowe s o bo h cul i a s and unpollina ed lowe s o Ca ili is coupled wi h low e hylene biosyn hesis and signalling,
which would also explain he pa ial and omonoecy in he pa henoca pic geno ype. The educ ion o e hylene
p oduc ion in he o a y coseg ega es wi h pa henoca py and pa ial and omonoecy in he sel ing p ogeny o
Ca ili. Mo eo e , he induc ion o e hylene in an hesis (by e hephon ea men s) educed he pe cen age o
bisexual pa henoca pic lowe s in Ca ili, while he inhibi ion o e hylene biosyn hesis o esponse (by AVG and STS
ea men s) induces no only and omonoecy bu also he pa henoca pic de elopmen o he ui in bo h cul i a s.
Conclusions: Resul s demons a e ha a educ ion o e hylene p oduc ion o signalling in he zucchini lowe is
able o induce ui se and ea ly ui de elopmen , and he e o e ha e hylene is ac i ely in ol ed in ui se and
ea ly ui de elopmen . Auxin and TIBA ea men s, inducing ui se and ea ly ui de elopmen in his species,
also inhibi e hylene p oduc ion and he exp ession o e hylene biosyn hesis and esponse genes. A model is
p esen ed ha discusses he c oss alk be ween e hylene and auxin in he con ol o ui se and ea ly ui
de elopmen in zucchini squash.
Keywo ds: Cucu bi a pepo, F ui se , Pa henoca py, E hylene, Gene exp ession, Auxin
Backg ound
Despi e i s molecula simplici y, e hylene egula es a
numbe o de elopmen al and physiological p ocesses
[1,2], including lea and lowe abscission, ipening o
climac e ic ui and bio ic and abio ic s esses. In he
species o he Cucu bi aceae amily e hylene con ols
sexual exp ession and is he main de e minan o sexual
pheno ypes [3-5]. Thus, he e hylene biosyn hesis genes
CmACS7 and CsACS2 o melon and cucumbe , espec -
i ely, egula e he a es o s amen de elopmen in e-
male lowe s o monoecious cul i a s, and hei loss o
unc ion mu a ions lead o he con e sion o emale in o
bisexual lowe s, and he e o e he ans o ma ion o
monoecious in o and omonoecious cul i a s [5-7]. In
Cucu bi a pepo, e hylene also egula es he sexual ex-
p ession o monoecious cul i a s, con olling bo h he
p ecoci y and he numbe o emale lowe s [4,5,8,9]. In
* Co espondence: [email p o ec ed]
1
Depa amen o de Biología y Geología, Ag i ood Campus o In e na ional
Excellence (ceiA3), Uni e sidad de Alme ía, La Cañada de San U bano s/n,
04120 Alme ía, Spain
Full lis o au ho in o ma ion is a ailable a he end o he a icle
© 2013 Ma ínez e al.; licensee BioMed Cen al L d. 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 (h p://c ea i ecommons.o g/licenses/by/2.0), 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 wo k is p ope ly ci ed.
Ma ínez e al. BMC Plan Biology 2013, 13:139
h p://www.biomedcen al.com/1471-2229/13/139
ac , he applica ion o blocking agen s o e hylene p o-
duc ion (AVG) o pe cep ion (STS) is able o delay emale
lowe ing and educe he numbe o emale lowe s pe
plan , bu also o induce a con e sion o emale in o bisex-
ual lowe s [4]. Likewise, emale lowe s o zucchini p o-
duce much mo e e hylene han male lowe s h oughou
hei de elopmen and ma u a ion up o an hesis [4].
A e pollina ion and e iliza ion, ui se and ui
de elopmen is dependen on cell di ision and expan-
sion p omo ed by ho mones such as gibbe ellin (GAs),
auxin and cy okinin [10-12]. Auxin is he de e minan o
ui se , as has been demons a ed by he s udy o mu-
an s o ansgenic lines o ARF o IAA/Aux mul igene
amilies in oma o and A abidopsis [10-12]. Howe e ,
hey seem o be closely ela ed o gibbe ellin which is
able o igge ui ini ia ion wi hou changes in auxin
signalling genes [13,14]. In he Cucu bi aceae amily,
ui se and de elopmen depend mainly on auxin [15].
The applica ion o auxins induces pa henoca pic ui
se and de elopmen in cucumbe [16], al hough he ap-
plica ion o cy okinin also ac i a es cell di isions in ui s
[17], whe eas b assinos e oid inc ease ui se [10,18].
In zucchini auxin is also shown o be he mos e ec i e
ho mone o induce pa henoca pic ui de elopmen
[19], and his g ow h egula o is commonly applied o
p omo e ui se and g ow h in g eenhouse p oduc ion
o his ege able c op.
E hylene has been ela ed wi h lo al o gan senescence
and abscission a e pollina ion. Pollina ion induces
e hylene p oduc ion in he o a ies and pe als, and his
e hylene appea s o be esponsible o coo dina ing
o a y g ow h and pe al senescence [20-23]. The implica-
ion o e hylene in ui se and de elopmen was no
s udied in dep h un il a ew yea s ago. Recen s udies
ha e shown an in e connec ion be ween ea ly o ule
abo ion and he size o he silique in A abidopsis e hyl-
ene mu an s [24]. Meanwhile, pollina ion and gibbe ellin
ea men s a e esponsible o down egula ing e hylene
biosyn hesis and signalling genes in oma o immedia ely
a e ui se [12,22].
Since C. pepo mo pho ype zucchini has a la ge in e io
o a y (abou 6–8 cm long a an hesis), i is an ideal
species sui able o s udy ui se and ea ly ui de elop-
men . We ha e ecen ly obse ed ha a educ ion o
e hylene in emale lowe s o zucchini can no only
p omo e he de elopmen o s amens in he lowe ,
con e ing emale in o bisexual lowe s, bu also induce
he pa henoca pic ui de elopmen in absence o
pollina ion and e iliza ion [25]. This is also ue o
some cul i a s o zucchini squash g own unde high
empe a u e condi ions [25]. To s udy he ole o e hyl-
ene in ui se and ea ly ui de elopmen in zucchini
squash, he p esen pape compa es he p oduc ion o
e hylene and he exp ession o e hylene biosyn hesis and
esponse genes be ween pollina ed and unpollina ed
o a ies o a non-pa henoca pic cul i a , as well as be-
ween a pa henoca pic and a non-pa henoca pic cul i-
a o zucchini squash. Mo eo e , we analysed ui
g ow h a es as well as e hylene p oduc ion and he ex-
p ession o e hylene genes in esponse o auxin and
TIBA ea men s. Resul s indica e ha e hylene is di -
ec ly in ol ed in ui se and ea ly ui de elopmen in
his species. These wo de elopmen al p ocesses equi e
a low le el o e hylene p oduc ion and signalling wi hin
he ew days a e pollina ion, and he loss o pollina ion
and e iliza ion is accompanied by an induc ion o
e hylene biosyn hesis and signalling 3 days a e an hesis,
concomi an ly wi h ui abo ion.
Resul s
Di e en app oaches ha e been used o de e mine he im-
plica ion o e hylene in zucchini ui se and ea ly ui
de elopmen . Fi s ly we ha e de e mined e hylene p oduc-
ion and he exp ession o 13 e hylene biosyn hesis and
signalling genes in pollina ed and unpollina ed o a ies and
ui s o he non-pa henoca pic cul i a Tosca. Secondly,
we ha e compa ed e hylene p oduc ion and he exp es-
sion o e hylene genes in he ui s o wo con as ing cul-
i a s o pa henoca py: Tosca and Ca ili. Finally, he
same wo cul i a s we e used o s udy he c oss alk
be ween e hylene and auxins in he con ol o hese de el-
opmen al p ocesses.
E hylene p oduc ion and e hylene genes exp ession in
pollina ed and unpollina ed o a ies/ ui s o zucchini
I has been shown ha he ui s o many zucchini
cul i a s can ini ia e hei g ow h in he absence o pol-
lina ion and ho mone applica ion. In ac , in he non-
pa henoca pic c . Tosca he g ow h a e o pollina ed
and unpollina ed ui s we e e y simila o he i s
h ee days (Figu e 1A), which highligh s he na u al pa -
henoca py o his species. A e he hi d day, howe e ,
mos unpollina ed ui s abo ed, and some o hem
g ew a a signi ican ly slowe a e han pollina ed ui s
(Figu e 1A). In he i s 5 DPA, he p o iles o e hylene
p oduc ion in pollina ed and unpollina ed ui s we e
e y dissimila (Figu e 1B). While e hylene p oduc ion
dec eased sligh ly in pollina ed ui s, in unpollina ed
ui s i inc eased sha ply 3 DPA (Figu e 1B). These e-
sul s indica e ha he dec ease in he g ow h a e o
unpollina ed ui s ha occu s a 3 DPA is co ela ed
wi h a bu s o e hylene in he ui , and ha he main-
enance o ui g ow h likely equi es a low le el o
e hylene.
To de ec pu a i e genes egula ing e hylene biosyn-
hesis du ing ui se and ea ly ui de elopmen , we
ha e analysed he exp ession o six ACS genes and one
ACO gene om C. pepo (Addi ional ile 1: Table S1,
Ma ínez e al. BMC Plan Biology 2013, 13:139 Page 2 o 14
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Figu e 2). CpACS1 was one o he i s ACS genes iso-
la ed om plan s [3]; CpACS2, and CpACS4 o CpACS6
a e ou unigenes iden i ied by new gene a ion sequen-
cing (NGS) o cDNA om di e en squash issues [26;
a ailable a Cucu bigene.ne ]; and CpACS7 and CpACO1
ha e been isola ed by PCR s a egy wi h degene a ed
p ime s (unpublished). Exp ession was s udied h ough
qPCR a an hesis (0 DPA) and 3 DPA (Figu e 2).
CpACS1 was no exp essed du ing his pe iod (Figu e 2).
A an hesis, he exp ession o he o he 6 genes was e y
low, excep o CpACS4, which could explain he basal
p oduc ion o e hylene a his poin . This basal exp es-
sion was main ained in pollina ed ui s a 3 DPA
(Figu e 2). Ne e heless, in unpollina ed ui s he ex-
p ession o he i e ACS genes and CpACO1 was
up egula ed a 3 DPA. The highes induc ion was
obse ed in CpACS4,CpACS6 and CpACS7 (Figu e 2),
which appea o be he main gene esponsible o he
e hylene p oduced a his poin .
To de e mine he molecula mechanisms behind he
ac ion o e hylene in ui se and ea ly ui de elop-
men , he ela i e exp essions o six addi ional e hylene
pe cep ion and signalling genes we e also s udied in pol-
lina ed and unpollina ed ui s o e 5 DPA. As occu ed
o CpACSs and CpACO1, o he genes in ol ed in e hyl-
ene pe cep ion such as CpETR1 and CpERS1, as well as
in e hylene signalling (CpCTR1,CpCTR2,CpEIN3.1 and
CpEIN3.2), also showed an exp ession p o ile simila o
ha o e hylene p oduc ion (Figu e 3). In pollina ed
ui s, exp ession le els emained low du ing ea ly de el-
opmen , while in non-pollina ed ui s, all pe cep ion and
esponse genes we e signi ican ly up egula ed a 3 DPA
(Figu e 3), coinciding wi h he peak o e hylene and he
abo ion o ui g ow h (Figu e 1).
A
b
bb
a
a
a
a
a
a
0
50
100
150
200
250
012345
F ui lengh (mm)
Days pos an hesis (DPA)
pollina ed
unpollina ed
a
B
aaaa
aa
b
b
b
a
0
5
10
15
20
25
30
012345
E hylene p oduc ion (nLg -1 FW)
Days pos an hesis (DPA)
Figu e 1 E olu ion o he size and e hylene p oduc ion in pollina ed and unpollina ed o a ies/ ui s o zucchini c . Tosca. (A) F ui size.
(B) E hylene p oduc ion. Di e en le e s indica e signi ican di e ences be ween he means a each da a poin be ween pollina ed and
unpollina ed o a ies/ ui s (p ≤0.05; n = 12 o ui leng h, and n = 4 o e hylene p oduc ion).
0
5
10
15
20
25
30
35
03030303030303
CpACS1 CpACS2 CpACS4 CpACS5 CpACS6 CpACS7 CpACO1
Rela i e exp ession
Days pos an hesis (DPA)
pollina ed
unpollina ed
Figu e 2 Rela i e exp ession o 7 e hylene biosyn hesis genes in pollina ed and unpollina ed o a ies/ ui s o zucchini c . Tosca a
an hesis (0 DPA) and 3 DPA. In pollina ed lowe s, exp ession a he day o an hesis was de e mined 8 h a e hand pollina ion. Each da a poin
ep esen s he mean o 3 eplica es wi h 3 ui s each. E o ba s indica e SE.
Ma ínez e al. BMC Plan Biology 2013, 13:139 Page 3 o 14
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E hylene is ac i ely in ol ed in ui se and ea ly ui
de elopmen in zucchini
In o de o asce ain whe he he di e ences in e hylene
p oduc ion be ween pollina ed and unpollina ed ui s a 3
DPA a e he consequence o ui se o ui abo ion, o
a he a signal ha is ac i ely in ol ed in ui se and de-
elopmen , we ha e compa ed e hylene p oduc ion and sig-
nalling in one pa henoca pic and one non-pa henoca pic
a ie y, as well as in a seg egan ing popula ion de i ed om
he pa henoca pic one, and de e mined he e ec s o
e hylene eleasing and blocking agen s on ui se and ea ly
ui de elopmen .
The hyb id cul i a Ca ili is able o de elop pa heno-
ca pic ui s o comme cial size in absence o pollina ion
o ho monal ea men s [25]. The pa henoca py o his
cul i a is associa ed wi h an incomple e and omonoecy,
i.e. a pa ial con e sion o emale in o bisexual lowe s,
and a delay in lo al o gan ma u a ion (Figu e 4). O a y-
bea ing lowe s o his cul i a can be classi ied in o
emale lowe s wi h no s amen de elopmen (Figu e 4A),
0
5
10
15
20
25
30
35
0235
Rela i e Exp ession
Days pos an hesis (DPA)
CpCTR1
0
5
10
15
20
25
30
35
0235
Rela i e exp ession
Days pos an hesis (DPA)
CpCTR2
0
2
4
6
8
10
12
14
0235
Rela i e exp ession
Days pos an hesis (DPA)
CpEIN3.1
0
1
2
3
4
5
6
0235
Rela i e exp ession
Da
y
s pos an hesis (DPA)
CpEIN3.2
0
5
10
15
20
25
30
0235
Rela i e exp ession
Days pos an hesis (DPA)
CpETR1
0
20
40
60
80
100
120
140
160
180
0235
Rela i e Exp ession
Days pos an hesis (DPA)
CpERS1
0
1
2
3
4
5
6
7
0235
Rela i e exp ession
Days pos an hesis (DPA)
CpACS4
pollina ed
unpollina ed
Figu e 3 Rela i e exp ession o e hylene biosyn hesis (CpACS4), pe cep ion (CpETR1 and CpERS1) and signalling (CpCTR1,CpCTR2,
CpEIN3.1 and CpEIN3.2) genes o e 5 DPA in pollina ed and unpollina ed o a ies/ ui s o c . Tosca. Each da a poin ep esen s he mean
o 3 eplica es wi h 3 o a ies/ ui s each. E o ba s indica e SE.
Ma ínez e al. BMC Plan Biology 2013, 13:139 Page 4 o 14
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o bisexual lowe s showing a ce ain deg ee o s amen
de elopmen (Figu e 4B), bu ne e eaching he size o
s amen in male lowe s (Figu e 4C). A he same s age o
de elopmen , bisexual lowe s always showed a highe
o a y and ui size (Figu e 4D). We compa ed he longi-
udinal g ow h a e o o a ies/ ui s be ween emale and
bisexual lowe s o a o al o 22 days, s a ing wi h lo al
buds o abou 4 mm in leng h (Figu e 4E and F). The
g ow h a e o o a y leng h in bisexual lowe s de ia ed
om ha o emale lowe s a wel e days, immedia ely
a e an hesis o emale lowe s (Figu e 4F). By his ime
pe als o bisexual lowe s we e s ill imma u e and closed
bu hei o a ies kep g owing a a much as e a e han
hose o emale lowe s. Many o he o a ies in bisexual
lowe s eached a comme cial size be o e an hesis. In
ac , many o he bisexual lowe s did no each an hesis
in he 24 days o s udy, and in o he s an hesis was delayed
wi h espec o emale lowe s because o a lowe g ow h
a e o pe als (Figu e 4F). These esul s indica e ha he
pa henoca py o his cul i a is no only co ela ed wi h
s amen de elopmen , bu also wi h a lowe g ow h a e o
pe als, which delays ma u a ion o pe als and an hesis.
Gi en ha male lowe s equi e wice as long as emale
ones o ma u e and each an hesis (Figu e 4F), i is likely
ha he delay in he ma u a ion o bisexual lowe s is as-
socia ed wi h hei masculinisa ion, i.e. he p esence o
s amens.
To assess whe he pa henoca py and pa ial an-
d omonoecy had he same gene ic egula ion, we
pheno yped he F2 popula ion de i ed by sel -pollina ion
o he F1 hyb id Ca ili, which seg ega es o he wo
ai s. A comple e coseg ega ion be ween he wo
ai s has been ound in he F2 gene a ion. O a o al
o 95 plan s, 23 we e comple ely monoecious, and
p oduced only emale lowe s,while72we epa ially
and omonoecious and p oduced bo h emale and bi-
sexual lowe s (Table 1). Al hough he numbe o bisexual
lowe s in he la e a ied om 20% o 100%, all o hem
C
aaaaa
b
c
a
bbba
aaaaa
a
F
0
10
20
30
40
50
60
70
80
90
100
0 2 4 6 8 1012141618202224
Pe als leng h (mm)
Time (days)
an hesis bisexual lowe s
an hesis
emale
lowe s
an hesis
male
lowe s
aaa
a
a
a
aaaaaa
b
b
b
b
bE
0
50
100
150
200
250
300
350
400
0 2 4 6 8 101214161820
O a y leng h (mm)
Time (days)
Ca ili Female
Ca ili Bisexual
Ca ili Male
ABD
50 mm
10 mm
10 mm 10 mm
Figu e 4 G ow h a es o o a ies/ ui s and pe als in emale, bisexual and male lowe s o he pa henoca pic cul i a Ca ili. (A) Female
lowe . (B) Bisexual lowe wi h a pa ial de elopmen o s amen. (C) Male lowe . (D) O a y size o emale and bisexual lowe s wi h he same
ime o de elopmen . Flowe s we e agged when hey we e 4 mm in leng h ( ime 0), and allowed o g ow o 20 days in he case o emale and
bisexual lowe s, and 24 days in he case o male lowe s. (E) Compa ison o o a y/ ui longi udinal g ow h a es be ween emale and bisexual
lowe s. (F) Compa ison o pe al longi udinal g ow h a es o emale, bisexual and male lowe s. No e ha pe al g ow h is delayed in bisexual
lowe s, bu no as much as in male lowe s. An hesis in he bisexual lowe s is delayed e en mo e han in male lowe s. S a is ical analysis was
pe o med using he LSD me hod (p ≤0.05; n = 15).
Ma ínez e al. BMC Plan Biology 2013, 13:139 Page 5 o 14
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de eloped in o pa henoca pic ui s, which eached com-
me cial size e en be o e an hesis (Table 1). The 3:1 seg e-
ga ion a io (χ
2
= 0,014, p alue = 0,91) indica ed ha he
pa ial and omonoecy and pa henoca py o Ca ili appea s
o be con olled by a leas one dominan gene.
Sex de e mina ion and emale lowe ma u a ion in
zucchini is known o be egula ed by e hylene in he
ea lies s ages o lowe de elopmen [4,8,9]. The e o e,
he pa henoca py o Ca ili could be he esul o a educ-
ion o e hylene in emale lowe s. We ha e ound ha he
unpollina ed Ca ili o a ies/ ui s p oduce signi ican ly less
e hylene han hose o he non-pa henoca pic Tosca du -
ing he days immedia ely a e an hesis (Figu e 5B). The
p oduc ion o e hylene was also measu ed in he o a ies
o 25 emale and 25 bisexual sample lowe s de i ed om
he F2 popula ion o Ca ili. Resul s indica ed ha he
highe g ow h a e o ui s in bisexual lowe s
coseg ega ed wi h a signi ican educ ion o e hylene p o-
duc ion in he o a y a 3 DPA (Table 1).
All he analyzed ACS genes, including CpACS4,
CpACS6 and CpACS7, which we e hose ha mainly
egula e e hylene p oduc ion in unpollina ed o a ies in
he days immedia ely a e an hesis, showed no signi i-
can di e en ial exp ession be ween Ca ili and Tosca
un e ilized o a ies a 3DPA o showed highe exp es-
sion in Ca ili (Figu e 5C). Only he exp ession o
CpACO1 was lowe in Ca ili (Figu e 5C). I appea s
he e o e ha he educ ion in e hylene p oduc ion ob-
se ed in he unpollina ed o a y o he pa henoca pic
cul i a Ca ili is no egula ed a he le el o ansc ip-
ion. Fo e hylene pe cep ion and esponse genes, only
he ansc ip s o CpERS1 showed a lowe accumula ion
o ansc ip s in Ca ili (Figu e 5C), sugges ing ha pe -
cep ion o e hylene could also be al e ed in his pa -
henoca pic cul i a .
To con i m whe he a educ ion in e hylene p oduc-
ion du ing he de elopmen o emale lowe s was
enough o induce he pa henoca pic de elopmen o
he zucchini o a y, we de e mined he e ec s o e hylene
eleasing and blocking agen s on ea ly ui de elop-
men . Con ol plan s o Tosca p oduced no bisexual pa -
henoca pic lowe s, while hose o Ca ili p oduced 60%
(Figu e 5D and Addi ional ile 2: Figu e S1). The applica-
ion o e hephon signi ican ly educed he p oduc ion o
bisexual pa henoca pic lowe s in Ca ili, while he
applica ion o he e hylene blocking agen s STS and AVG
inc eased he p oduc ion o bisexual pa henoca pic
lowe s no only in he pa henoca pic Ca ili, bu also in
Tosca (Figu e 5D and Addi ional ile 2: Figu e S1). These
esul s demons a e ha e hylene is ac i ely in ol ed in
ui se and ea ly ui de elopmen in zucchini squash,
and indeed a educ ion o ei he e hylene biosyn hesis o
signalling in he de eloping emale lowe o zucchini can
no only inhibi he a es o s amens, p omo ing he con-
e sion o emale in o bisexual lowe , bu also o induce
he pa henoca pic de elopmen o he ui .
E ec s o auxins on ui g ow h a es and e hylene
biosyn hesis and signalling
I is known ha ex e nal applica ion o auxins and
TIBA, he la e an inhibi o o auxin pola anspo ,
can induce ui se and ea ly ui g ow h in di e en
species. In zucchini, syn he ic auxins a e commonly used
o s imula e he pa henoca pic de elopmen o ui in
o -season g eenhouse p oduc ion. We ha e s udied he
e ec s o NAA + NAAmide and TIBA (applied a an he-
sis in he o a y) on ui de elopmen and e hylene p o-
duc ion in he cul i a s Ca ili and Tosca. In he
pa henoca pic c . Ca ili nei he auxins no TIBA we e
able o al e he longi udinal g ow h a e o he ui
(Figu e 6A), sugges ing ha he o a ies o his cul i a
could ha e a high concen a ion o auxins. Howe e , in
he ui s o he non-pa henoca pic c . Tosca bo h
ea men s p omo ed longi udinal g ow h o he ui , al-
hough TIBA was mo e e ec i e han NAA + NAAmide
(Figu e 6C). E hylene p oduc ion in con ol and ea ed
ui s was nega i ely co ela ed wi h ea ly ui g ow h
a e. In Ca ili, e hylene dec eased p og essi ely h ough-
ou he i s 4 DPA in bo h con ol and ea ed ui s
(Figu e 4B), while in Tosca he only ui s whe e e hyl-
ene was induced a 3 DPA we e he unpollina ed con ol
ui s, which we e hose which showed he lowes
g ow h a e and inally abo ed (Figu e 6D).
The e ec o NAA and TIBA on he exp ession o
e hylene genes was s udied in ui s a 3 DPA, when di -
e ences in e hylene p oduc ion we e e iden be ween
pollina ed and unpollina ed ui s, and be ween pa -
henoca pic and non-pa henoca pic cul i a s. In con-
co dance wi h e hylene p oduc ion da a, ACS and ACO
genes we e down egula ed by NAA and TIBA in he
Table 1 F ui size and e hylene p oduc ion o emale and bisexual lowe s among monoecious and pa ially
and omonoecious plan s in he sel ing p ogeny o Ca ili (F2 popula ion)
F2 seg ega ion (No. plan s) Flowe pheno ype O a y leng h a an hesis (mm) E hylene p oduc ion a 3 DPA (nL/g FW)
Monoecious (23 plan s) Female 78.10±4.23 a 10.90±2.94 a
Pa ially and omonoecious (72 plan s) Female 77.43±1.00 a 10.90±2.94 a
Bisexual 139.07±4.75 b 2.51±1.23 b
O a y size and e hylene p oduc ion da a we e ob ained om 25 eplica es o each lowe pheno ype. Di e en le e s wi hin he same column indica e
signi ican di e ences be ween emale and bisexual lowe s ( -analysis, p<0,01).
Ma ínez e al. BMC Plan Biology 2013, 13:139 Page 6 o 14
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ui s o bo h Tosca and Ca ili a 3 DPA (Figu e 7),
indica ing ha auxins egula e nega i ely he p oduc-
ion o e hylene in he ui du ing he days immedi-
a ely a e an hesis. TIBA ea men was mo e
e ec i e han ea men wi h NAA + NAAmide in
educing he exp ession o e hylene biosyn hesis
genes (Figu e 7). Mo eo e , he down egula ion o
CpACS4 and CpACS7, he genes which con ibu e
mos o he p oduc ion o e hylene in unpollina ed
ui s du ing he days immedia ely a e an hesis,
was highe han ha obse ed o CpACS5,CpACS6
and CpACO1 (Figu e 7). The exp ession o he e hyl-
ene ecep o CpETR1 was simila in con ol o a ies
o Tosca and Ca ili, and was signi ican ly educed in
esponse o bo h ea men s in Ca ili, and in
esponse o TIBA in Tosca. The exp ession o he
o he ecep o gene CpERS1 was highe in he
non-pa henoca pic cul i a Tosca, and i was
down egula edbyNAAandTIBAin hiscul i a
(Figu e 7). Rega ding e hylene esponse genes, he
exp ession o CpCTR1 was also down egula ed by
TIBA in he wo cul i a s (Figu e 7), bu no by
NAA; and al hough he ho monal ea men s did no
signi ican ly change he exp ession o CpEIN3.1 and
CpEIN3.2 in Tosca (Figu e 7), in Ca ili NAA
down egula ed he exp ession o bo h, whe eas TIBA
only educed he exp ession o he o me . In con-
clusion, many o he analysed e hylene biosyn hesis,
pe cep ion and signalling genes we e down egula ed
by NAA and TIBA, wo ea men s ha induce he
g ow h a e o he zucchini ui in he days immedi-
a ely a e an hesis.
b
b
b
b
b
a
a
a
a
a
0
50
100
150
200
250
300
350
400
02468
F ui leng h (mm)
Days pos an hesis (DPA)
A
Ca ili
Tosca
a
C
a
A
ab
B
b
C
0
10
20
30
40
50
60
70
80
90
100
Tosca Ca ili
Pe cen age o
bisexual lowe s/ pa henoca pic ui
Con ol
E ephon
STS
AVG
D
B
aa
aa
a
b
b
0
2
4
6
8
10
12
14
16
18
20
01234
E hylene p oduc ion 6 h (nL g -1 FW)
Days pos an hesis (DPA)
Ca ili
Tosca
C
0
1
2
3
4
5
6
7
Rela i e exp ession 3DPA
Ca ili
Tosca
Figu e 5 In ol emen o e hylene in he pa henoca pic de elopmen o Ca ili ui . (A) Compa ison o o a y/ ui g ow h be ween he
pa henoca pic c . Ca ili (bisexual and unpollina ed lowe s) and he non-pa henoca pic c . Tosca ( emale and unpollina ed lowe s). (B) Compa ison
o e hylene p oduc ion in unpollina ed o a ies/ ui s o Ca ili and Tosca (C) Exp ession o e hylene biosyn hesis, pe cep ion and esponse genes in
unpollina ed o a ies o Ca ili and Tosca a 3 DPA. (D) E ec s o e hylene eleasing (e hephon) and blocking eagen s (AVG and STS) on he pe cen age
o bisexual pa henoca pic lowe s in Ca ili and Tosca. Di e en le e s indica e s a is ical di e ences be ween ea men s wi hin each cul i a (p ≤0.05;
n = 15; lowe case Tosca; uppe case, Ca ili).
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Discussion
F ui se and ea ly ui de elopmen is co ela ed wi h a
down egula ion o e hylene biosyn hesis and signalling
genes
E hylene p oduc ion and exp ession da a om 13 genes
co e ing e hylene biosyn hesis and signal ansduc ion
pa hway in C. pepo ha e demons a ed ha he loss o
pollina ion in a non-pa henoca pic geno ype o his spe-
cies is accompanied by a sha p inc ease in e hylene bio-
syn hesis and signalling in he o a y a 3 DPA. This is
ue o he 5 ACS-like genes showing exp ession in he
o a y (one o he analysed genes, CpACS1, showed no
exp ession in he o a y du ing his ime o de elopmen )
and CpACO1, as well as o wo e hylene ecep o s
(CpETR1 and CpERS1), wo CTR1-like genes (CpCTR1
and CpCTR2) and wo EIN3-like genes (CpEIN3.1 and
CpEIN3.2). This e hylene signalling in he o a y ew days
a e an hesis is associa ed wi h a decline in g ow h and
inally wi h he abo ion o ui . Pollina ed lowe s, on
he o he hand, main ain a e y low le el o e hylene
biosyn hesis and signalling in he ui du ing he days
immedia ely a e an hesis, concomi an ly wi h ui se
and de elopmen . This lowe e hylene signalling is also
obse ed in pa henoca pic ui s o he zucchini cul i a
Ca ili, as well as in he pa henoca pic plan s o he seg-
egan sel ing p ogeny o Ca ili. Taken as a whole, hese
da a indica e ha he p oduc ion o e hylene in he
o a y ew days a e an hesis is nega i e co ela ed wi h
ui se and g ow h.
Al hough he unc ion o e hylene in ui se and ea ly
ui de elopmen has no been s udied in dep h, i has
been epo ed ha he inhibi ion o e hylene p oduc ion
o esponse by ex e nal ea men s wi h AVG, STS o
1-me hylcyclop opene (1-MCP), induces ui se in pea
a
a
a
a
a
A
0
50
100
150
200
250
300
350
400
02468
F ui leng h (mm)
Days pos an hesis (DPA)
Con ol
NAA+NAAmide
TIBA
a
a
a
C
b
b
b
a
a
c
c
c
0
50
100
150
200
250
300
350
400
02468
F ui leng h (mm)
Days pos an hesis (DPA)
Con ol
NAA+NAAmide
TIBA
aa
b
b
a
D
a
a
0
5
10
15
20
25
1234
E hylene p oduc ion (nL g -1 FW)
Days pos an hesis (DPA)
a
ab
a
a
B
a
a
a
ab
0
2
4
6
8
10
12
1234
E hylene p oduc ion (nL g -1 FW)
Days pos an hesis (DPA)
Tosca Ca ili
F ui leng h E hylene P oduc ion
Figu e 6 E ec s o NAA + NAAmide and TIBA on ui g ow h a es and he e olu ion o e hylene p oduc ion in Ca ili and Tosca.
(A) F ui g ow h a es in Ca ili. (B) E hylene p oduc ion in Ca ili. (C) F ui h g ow h a es in Tosca. (D) E hylene p oduc ion in Tosca. O a ies we e
ea ed a an hesis. Di e en le e s a he same ime poin indica e signi ican di e ences be ween ea men s (p ≤0.05; n = 15 o o a y g ow h
and n = 4 o e hylene p oduc ion).
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0
0.5
1
1.5
2CpACS7
Rela i e exp ession 3 DPA
0
0.5
1
1.5
2CpEIN3.1
0
0.5
1
1.5
2CpCTR1
0
1
2
3
4
5CpERS1
0
1
2
3CpACO1
0
0.5
1
1.5
2CpACS4
C
NAA
TIBA
0
0.5
1
1.5
2CpACS5
0
0.5
1
1.5
2CpACS6
Tosca ToscaCa ili Ca ili
0
0.5
1
1.5
2
2.5
3CpEIN3.2
0
0.5
1
1.5
2CpETR1
Figu e 7 E ec o NAA and TIBA on he exp ession o e hylene genes. Flowe s we e ea ed a an hesis, and he exp ession o e hylene
biosyn hesis, pe cep ion and signalling genes was de e mined in he o a ies/ ui a 3 DPA, when e hylene p oduc ion is induced in unpollina ed
o a ies. Each da a poin ep esen s he mean o 3 eplica es wi h 3 o a ies/ ui s each. E o ba s indica e SE.
Ma ínez e al. BMC Plan Biology 2013, 13:139 Page 9 o 14
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