Involvement of ethylene biosynthesis and signalling in fruit set and early fruit development in zucchini squash (Cucurbita pepo L.)

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 h p://www.biomedcen al.com/1471-2229/13/139 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 h p://www.biomedcen al.com/1471-2229/13/139 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 h p://www.biomedcen al.com/1471-2229/13/139 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 h p://www.biomedcen al.com/1471-2229/13/139 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 h p://www.biomedcen al.com/1471-2229/13/139 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). Ma ínez e al. BMC Plan Biology 2013, 13:139 Page 7 o 14 h p://www.biomedcen al.com/1471-2229/13/139 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). Ma ínez e al. BMC Plan Biology 2013, 13:139 Page 8 o 14 h p://www.biomedcen al.com/1471-2229/13/139 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 h p://www.biomedcen al.com/1471-2229/13/139