Brassinolides and IAA induce the transcription of four α-expansin genes related to development in Cicer arietinum

O iginal a icle B assinolides and IAA induce he ansc ip ion o ou a-expansin genes ela ed o de elopmen in Cice a ie inum > M a Angeles Sánchez, Isabel Ma eos, Emilia Lab ado , Be a Dopico * Depa amen o de Fisiología Vege al, Facul ad de Biología, Cen o Hispano-Luso de In es igaciones Ag a ias, Uni e sidad de Salamanca, Pza Doc o es de la Reina s/n, Salamanca 37007, Spain Recei ed 17 Ma ch 2004; accep ed 15 July 2004 A ailable online 20 Augus 2004 Abs ac Fou di e en cDNAs encoding a-expansins ha e been iden i ied in Cice a ie inum (Ca-EXPA1,Ca-EXPA2,Ca-EXPA3 and Ca-EXPA4). The sha ed amino acid sequence simila i y among he ou a-expansin p o eins anged om 67 o 89%. All o hem display common cha ac e is ics such as molecula mass (a ound 24 kDa), amino acid numbe s, and also he p esence o a signal pep ide. The ansc ip ion pa e n o chickpea a-expansin genes in seedlings and plan s sugges s a speci ic ole o each o he ou a-expansins in di e en phases o de elopmen o in di e en plan o gans. High le els o Ca-EXPA2 ansc ip s coincide wi h maximum epico yl and s em g ow h, indica ing an impo an in ol emen o his pa icula a-expansin in elonga ing issues. Ca-EXPA3 would be ela ed o adicle de elopmen , while Ca-EXPA4 seems o be in ol ed in pod de elopmen . A conside able inc ease in he le el o all Ca-EXPA ansc ip s accompanied he indole ace ic acid (IAA) plus b assinolide (BR)-induced elonga ion o excised epico yl segmen s. This IAA + BR induc ion was seen e en o he chickpea expansin genes whose ansc ip ion was no a ec ed by IAA o BR alone. © 2004 Else ie SAS. All igh s ese ed. Keywo ds: B assinolides; Cell wall; Cice a ie inum; Epico yl; Expansin; G ow h 1. In oduc ion Plan cell g ow h is accompanied by he modi ica ion and expansion o cell walls, which a e composed o cellulose mic o ib ils e he ed oge he wi h hemicelluloses and em- bedded in a ma ix o pec ins and s uc u al p o eins [2]. A class o p o eins called expansins p omo e cell wall loosening in i o and ca alyze wall ex ension and s ess elaxa ion in a pH-dependen manne [31]. Biochemical and biophysical da a indica e ha expansins bind o he su ace o cellulose mic o ib ils, he eby dis up ing he hyd ogen bonds o med wi h xyloglucan molecules and allowing he cell wall o ex end ( o a e iew, see [12,15,27]). Expansin genes ha e been iden i ied om many species and a e highly conse ed in gymnospe ms and, among he angiospe ms, in bo h monoco s and dico s [11,21,26]. Thei pa e n o exp ession indica es ha hey a e closely ela ed o cell g ow h and issue di e en ia ion ( o a e iew, see [5]). The occu ence o mul igene amilies o expansins sugges s ha di e en expansins play unique de elopmen al o issue- speci ic oles [6,7,10,19,44]. I has been sugges ed ha mos o he expansin genes cha ac e ized could be in ol ed in cell expansion du ing issue g ow h [1,8,16,21,38]. Expansins a e also exp essed in issues whe e cell wall disassembly is occu ing ins ead o cell g ow h [4,9,40]. The egula ion o expansin ac i i y by plan ho mones has been well documen ed. In his sense, auxin-induced acid g ow h esponse elies on he ac i i y o expansins o wall loosening and cell elonga ion [28,31,32]. In addi ion, plan ho mones egula e a-expansin gene exp ession. Examples a e he oma o a-expansins Le-EXPA1 and Le-EXPA2; he Abb e ia ions: ABA, abscisic acid; BR, b assinolides; IAA, indole ace- ic acid; GA, gibbe ellin. > The nucleo ide sequences epo ed in his pape ha e been submi ed o EMBL/GenBank da abase unde accession numbe s: Ca-EXPA1, AJ291816;Ca-EXPA2,AJ291817;Ca-EXPA3,AJ489608;Ca-EXPA4, AJ489609. * Co esponding au ho . E-mail add ess: [email p o ec ed] (B. Dopico). Plan Physiology and Biochemis y 42 (2004) 709–716 www.else ie .com/loca e/plaphy 0981-9428/$ - see on ma e © 2004 Else ie SAS. All igh s ese ed. doi:10.1016/j.plaphy.2004.07.004 deepwa e ice a-expansin Os-EXPA4, and he soybean (Gly- cine max) ß-expansin Cim1, which accumula e in esponse o e hylene, auxin, gibbe ellin and cy okinin, espec i ely [8,14,39]. The egula ion o expansin genes by b assinolides (BR) has been less s udied, al hough some a-expansin genes, such as A -EXPA5 and A -EXPA8 om A abidopsis ha e been ecen ly e e ed as BR-inducible genes using DNA mic oa ay analysis [17,35]. The p esen pape epo s he isola ion and iden i ica ion o ou comple e cDNAs—Ca-EXPA1,Ca-EXPA2,Ca- EXPA-3 and Ca-EXPA4—encoding ou di e en a-expansin p o eins om chickpea. Thei ansc ip ion was in es iga ed du ing epico yl and oo de elopmen as well as in se e al plan issues. In o de o elucida e he ela ionship be ween he Ca-EXPA genes and ho mone- egula ed g ow h, hei ansc ip ion was s udied in epico yl sec ions unde ea men wi h gibbe ellin (GA), abscisic acid (ABA), indole ace ic acid (IAA) and BR. 2. Resul s 2.1. Chickpea ␣ -expansin clones Fou clones, named Ca-EXPA1,Ca-EXPA2,Ca-EXPA3 and Ca-EXPA4, wi h high sequence simila i y o se e al a-expansins we e ound in a chickpea cDNA lib a y cons- uc ed wi h RNA om 5-day-old chickpea epico yls and sc eened as desc ibed in Sec ion 4. All Ca-EXPAs we e ull-leng h clones wi h sizes o 1291, 1034, 1497 and 1270 bp, espec i ely, he non-coding e- gions being hose ha mainly con ibu ed o such di e ences. The p o eins deduced om he chickpea Ca-EXPA clones, named a-expansin 1, 2, 3 and 4 (EXPA1, EXPA2, EXPA3 and EXPA4), had a signi ican le el o sha ed amino acid sequence simila i y wi h one ano he and also wi h o he published plan a-expansins. Fig. 1 shows he alignmen o he ou amino acid sequences, whe e he high deg ee o simila i y can be obse ed. The ou chickpea a-expansin p o eins had he cha ac e is ic expansin mo i s; namely, conse ed Cys esidues in he N- e minal egion o he p o- ein (ma ked wi h an as e isk in he igu e), conse ed se- quences (ma ked wi h a line in he igu e) including a pu a- i e ca aly ic domain wi h he His-Phe-Asp (HFD) mo i in he cen al po ion o he p o ein, and conse ed T p esidues in he pu a i e cellulose-binding domain in he C- e minal egion (ma ked by “+” in he igu e). EXPA3 and EXPA4 a e he closes a amino acid sequence le el (89% simila i y). EXPA1 and EXPA2 show a lowe deg ee o simila i y: abou 67% be ween each o he and a ound 70–75% wi h espec o he o he chickpea a-expansins. The mos di e en ia ed e- gion was ound a he N- e minal egion, whe e he p edic ed N- e minal hyd ophobic signal pep ide is loca ed, as de e - mined by Signal P [36]. A e elease o he signal pep ide (ma ked by an a ow in Fig. 1), he ou ma u e p o eins we e qui e simila in hei molecula mass (24.14–24.51 kDa) and in hei amino acids numbe (224–228). A basic p edic ed isoelec ic poin (9.13, 7.89, 9.15 and 8.80 o EXPA1, EXPA2, EXPA3 and EXPA4 espec i ely) was ano he com- mon cha ac e is ic o he ou chickpea a-expansins. Chickpea EXPA2, EXPA3 and EXPA4 did no p esen any N-glycosyla ion si e. Al hough he lack o N-glycosyla ion si e is a cha as e is ic o mos a-expansins desc ibed, EXPA1 had a pu a i e N-glycosyla ion si e a amino acid 202 (asn-se - h -leu). 2.2. Phylogene ic ee The di e ences among chickpea a-expansins a e clea ly seen in he phylogene ic ee compiled using he ou dedu- ced p o eins and o he plan a-expansins based on p o ein sequence alignmen (Fig. 2). a-Expansins appea sepa a ed in ou di e en phylogene ic b anches named A, B, C and D acco ding o Link and Cosg o e [28]; chickpea Ca-EXPA1, Ca-EXPA3 and Ca-EXPA4 appea in g oup C. This clade con ains a-expansin genes wi h he e ogeneous exp ession pa e ns. Some membe s a e ansc ibed in mul iple issues, such as Le-EXPA5 [1] and A -EXPA5 [42], while o he s appea o ha e mo e specialized oles, as A -EXPA10 [12]. Fig. 1 . Alignmen o he ou deduced a-expansin amino acid sequences o he Ca-EXPA clones. Sequences we e aligned using he Clus al me hod in he MegAlign p og am, pa o he Lase gene sequence analysis so wa e p og am a ailable om DNASTAR (Madison, WI). Shading indica es amino acid iden i y wi h espec o he majo i y. An a ow indica es he pu a i e signal sequence clea age si e. Conse ed Cys esidues a e ma ked by an as e isk. Conse ed sequences a e ma ked by a line. Conse ed T p esidues a e ma ked by “+”. 710 M.A. Sánchez e al. / Plan Physiology and Biochemis y 42 (2004) 709–716 Ca-EXPA2 appea s in b anch A, oge he wi h many a-expansin genes ha a e exp essed in apidly g owing is- sues (Fig. 2). Thus, Cs-EXPA1 and Le-EXPA2 a e exp essed in elonga ing hypoco yl in cucumbe o oma o. O he a-expansins wi hin he A clade a e exp essed in expanding ui issue in species such as che y, ap ico , s awbe y and oma o. 2.3. Tissue speci ici y in seedlings and adul plan s In o de o de e mine he ansc ip ion le el o each chick- pea expansin gene, speci ic p obes consis ing mainly o he 3′ un ansla ed egions we e p epa ed o each cDNA wi h a iew o minimizing any po en ial c oss-hyb idiza ion, acco - ding o Sou he n blo analysis (da a no shown). S udy o expansin gene ansc ip ion in seedling o gans disclosed a speci ic pa e n o ansc ip ion o each gene (Fig. 3). Ca- EXPA1 ansc ip le el was e y low in all seedling o gans. A simila low ansc ip ion was ound o Ca-EXPA4, al hough unlike Ca-EXPA1 no ansc ip was de ec ed in hooks. Ca- EXPA2 and Ca-EXPA3 ansc ip we e mainly de ec ed in epico yls and adicles, espec i ely. None o he expansin genes showed ansc ip ion in co yledons, excep o a e y low le el o Ca-EXPA3 mRNA. I should be no ed ha he le el o ansc ip ion o Ca-EXPA1 and Ca-EXPA4 was low in seedlings as compa ed wi h ansc ip ion in adul plan s, while Ca-EXPA3 mRNA le els in adicles eached he hi- ghes alue among all he plan o gans s udied. Ca-EXPA1 and 2 ansc ip ion along he s em in e nodes om 11-day-old plan s showed a co ela ion wi h he in e - node elonga ion a e, he highes mRNA le els being obse - ed a he 4 h in e node, jus below he apical one, he ea e dec easing owa ds he basal one (Fig. 3). By con as , he mRNA le els o Ca-EXPA3 and 4 ollowed he opposi e pa e n, dec easing om he basal in e node (1s ) o he apical young one (5 h). The ansc ip ion pa e n o Ca-EXPA clones in chickpea plan s was b oad. Ca-EXPA1 did no show signi ican ans- c ip ion in any pa excep he in e nodes. The o he h ee expansin genes showed high ansc ip le els in he ep oduc- i e o gans, he mos signi ican being he lowe s o Ca- EXPA2 and pods o Ca-EXPA3 and 4. The imma u e pod was he o gan wi h he highes mRNA le els o Ca-EXPA4. The highe ansc ip ion in imma u e pods compa ed wi h ma u e ones should be no ed. A e y low signal o e en no signal a all was de ec ed o all he genes s udied in o he plan o gans, including he lea es, d y seeds and oo s. Ca-EXPA3 Fig. 2 . Phylogene ic ee o he deduced amino acid sequences encoded by plan a-expansin genes. Alignmen s was made using he Clus al me hod. Sou ces o he a-expansin sequences a e as ollows: A -EXPA5,A -EXPA6 and A -EXPA10 om A abidopsis haliana;Bn-EXPA om B assica napus; Ca-EXPA1,Ca-EXPA2,Ca-EXPA3 and Ca-EXPA4 om C. a ie inum;Cs- EXPA1 and Cs-EXPA2 om Cucumis sa i us;Fa-EXPA2 om F aga ia ananassa;Le-EXPA1,Le-EXPA2,Le-EXPA4,Le-EXPA5,Le-EXPA8,Le- EXPA9,Le-EXPA10 and Le-EXPA18 om L. esculen um;N -EXPA1 and N -EXPA4 om Nico iana abacum;Os-EXPA1,Os-EXPA3,Os-EXPA7 and Os-EXPA12 om O yza sa i a;P -EXPA om Pinus aeda;Ps-EXPA1 om Pisum sa i um;Pa-EXPA1 and Pa-EXPA2 om P unus a meniaca;Pa - EXPA1 and Pa -EXPA2 om P unus a ium;Pc-EXPA5 om P unus ce a- sus;Rp-EXPA13 om R. palus is and T -EXPA3 om T. e sicolo .The b anches we e named A, B, C, and D acco ding o Link and Cosg o e [28]. Fig. 3 . No he n analysis o Ca-EXPAs mRNA ansc ip ion le els in di e- en pa s o C. a ie inum seedlings and adul plan s. H, hook; E, epico yl; M, mesoco yl; C, co yledon; R, adicle. In e nodes a e numbe ed 1–5 om he basal o he apical one. L1, young lea es; L2, ma u e lea es; P1, imma u e pods; P2, ma u e pods; F, lowe s; S1, imma u e seed; S2, d y ma u e seed; , adul oo s. Signals we e quan i ied and no malized acco - ding o 18S RNA hyb idiza ion. In eg a ed op ical densi y (IOD). 711M.A. Sánchez e al. / Plan Physiology and Biochemis y 42 (2004) 709–716 and 4, unlike Ca-EXPA1 and 2, we e exp essed in imma u e seeds (Fig. 3). 2.4. T ansc ip ion pa e n du ing epico yl g ow h Since Ca-EXPA2 ansc ip le el was high in epico yls, he empo al ansc ip ion pa e n h ough epico yl g ow h was examined. RNA was ex ac ed om epico yls anging in age om 2-day-old (when epico yl eme gence s a s) o 8-day- old (when he a e o g ow h s a s o all) (Fig. 4). The Ca-EXPA2 ansc ip le el inc eased wi h he epico yl elon- ga ion a e, being lowe in 2-day-old epico yls and eaching he highes alue in 5-day-old epico yl (Fig. 4). The Ca- EXPA1,Ca-EXPA3 and Ca-EXPA4 mRNAs le els we e e y low h oughou epico yl g ow h and no a ia ions we e ob- se ed wi h epico yl age (da a no shown). 2.5. T ansc ip ion pa e n du ing adicle g ow h Because o he high Ca-EXPA3 ansc ip ion ound in 4-day-old chickpea oo s, we decided o e alua e he changes in Ca-EXPA mRNA le els in adicles a di e en de elop- men al s ages in o de o de e mine whe he one o hese expansins migh be in ol ed in adicle elonga ion. To al RNAs we e ex ac ed om adicles o 2, 3, 4 and 5-day-old seedlings and we e subjec ed o RNA No he n blo analysis. Olde oo s we e no s udied since seconda y oo s we e ini ia ed on day 5 a e ge mina ion. As shown in Fig. 5, Ca-EXPA3 mRNAs we e de ec ed a each de elopmen al s age s udied, wi h no signi ican changes along adicle g ow h. Ca-EXPA1 and Ca-EXPA4 ansc ip le els, al hough low, inc eased wi h adicle age; con a iwise, Ca-EXPA2 ansc ip s dec eased in ha pe iod. Owing o he low alues ound in Ca-EXPA1,2and 4 ansc ip ion in adicles, mRNA con ols om he ou h in e node ( o Ca-EXPA1 and 2)o imma u e pods ( o Ca-EXPA4) we e used o check ha e icien hyb idiza ion had aken place. 2.6. E ec o ho mone-induced elonga ion on ␣ -expansin gene ansc ip ion To de e mine he ela ionship be ween g ow h- egula ing ho mones and chickpea a-expansin ansc ip accumula ion, excised subapical epico yl segmen s (10 mm long) we e allowed o elonga e in i o, in ei he he p esence o absence o IAA, BR, GA 3 and ABA. The g ow h o excised chickpea epico yl segmen s was seen o be signi ican ly s imula ed by exogenous applica ion o IAA and BR. G ow h p omo ion eached 11.7% wi h IAA a e 8ho ea men . BR induced he elonga ion o epico yl segmen s by i sel (8.8%) and also enhanced IAA-induced elonga ion (17.9%), al hough i s e - ec on elonga ion was mo e p onounced a e 8ho ea - men . Nei he GA 3 no ABA caused changes in he a e o chickpea epico yl elonga ion [34]. IAA ea men o epico yl segmen s elici ed a signi ican inc ease o Ca-EXPA2 and Ca-EXPA4 ansc ip le els a 4and8ho ea men (Fig. 6). Li le o no e ec was de ec ed o Ca-EXPA1 and Ca-EXPA3, excep o Ca- EXPA3 a 8 h. Nei he GA 3 no ABA a ec ed he le els o chickpea a-expansin ansc ip s. We also in es iga ed he e ec o BR and he combina ion o IAA and BR. Like IAA, ea men wi h BR alone induced he ansc ip ion o he co esponding Ca-EXPA2 and Ca-EXPA4 genes bu had li le e ec on Ca-EXPA1 and Ca-EXPA3 gene ansc ip ion. Des- pi e his, i is impo an o no e ha a conside able inc ease in he le el o all Ca-EXPA ansc ip s accompanied he IAA plus BR-induced elonga ion o excised epico yl segmen s (a e bo h 4 and8ho ea men ). This induc ion was obse - ed e en o chickpea a-expansin genes whose exp ession was no a ec ed by IAA o BR alone (Fig. 6). 3. Discussion Sc eening o a Cice a ie inum cDNA lib a y e ealed he p esence o a leas ou di e en cDNAs encoding Fig. 4 . No he n analysis o Ca-EXPA2 mRNA ansc ip ion le els in epi- co yls om seedlings g own in wa e . Numbe s e e o days a e sowing. Signals we e quan i ied and no malized acco ding o 18S RNA hyb idiza- ion. IOD. Fig. 5 . No he n analysis o Ca-EXPA mRNA ansc ip ion le els in adicles om seedlings g own in wa e . Numbe s e e o days a e sowing. RNA con ol om he ou h in e node (4I) o imma u e pod (P1) we e used o check he co ec hyb idiza ion o Ca-EXPA1 and 2, and o Ca-EXPA4, espec i ely. Blo s we e hyb idized wi h 18S RNA as a loading con ol. 712 M.A. Sánchez e al. / Plan Physiology and Biochemis y 42 (2004) 709–716 a-expansins: Ca-EXPA1,Ca-EXPA2,Ca-EXPA3 and Ca- EXPA4. The p esence o mul igene amilies o expansins has been epo ed in se e al plan species. The la ge numbe o expansins poses in iguing ques ions ega ding he unc ion o hese p o eins and he signi icance o hei edundancy, and de e mining he pa e n and con ol o each expansin gene exp ession is he i s s ep in elucida ing he unc ion o indi idual expansins. Se e al s udies on a-expansin gene exp ession ha e been published ecen ly; he esul s sugges ha expansin genes a e exp essed di e en ly, depending on he issue used and he s imuli imposed, and ha hey a e mainly associa ed wi h g ow h. Thus, W obel andYode [43] epo ed he di e en ial ansc ip ion o h ee a-expansins in T iphysa ia e sicolo . Likewise, se e al a-expansins ha e been iden i ied in co on [18] and in oma o, s awbe y and pea ui s, wi h a di e en pa e n o ansc ip ion du ing ui g ow h and ipening [1,19,20]. The ou a-expansins analyzed in C. a ie inum show common cha ac e is ics. In ac , mos plan expansins a e e y homogeneous as ega ds bo h leng h and weigh [44]. Howe e , i should be no ed ha unlike mos a-expansins, which a e no glycosyla ed p o eins, EXPA1 om C. a ie i- num ha e a pu a i e N-glycosyla ion si e. EXPA-3 om Ly- cope sicon esculen um has also been epo ed o ha e an N-glycosyla ion si e [1]. To da e, we do no know whe he hey a e glycosyla ed, and he ole o glycosyla ion, i indeed i occu s, is no clea as ega ds he unc ion o a-expansins [13]. F om ou esul s in ansc ip ion s udies, i seems clea ha he co esponding chickpea EXPA2 gene is he mos closely ela ed o epico yl g ow h, as seen om i s high le el o ansc ip in his o gan (Fig. 3). Addi ionally, a ela- ionship be ween Ca-EXPA2 mRNA le el and epico yl g ow h was obse ed (Fig. 4). In ag eemen wi h he pu a i e unc ion o EXPA2 in epico yl g ow h, i should be no ed ha i is he only chickpea a-expansin ha is included wi hin phylogene ic g oup B (Fig. 2), acco ding o he classi ica ion o Link and Cosg o e [28]. This g oup con ains a-expansins ela ed o as -g owing issues, such as Cs-EXPA1,Le-EXPA2 and P -EXPA, which a e exp essed in elonga ing hypoco yls o cucumbe , oma o and pine, espec i ely [3,21,42]. The ansc ip ion pa e n o chickpea a-expansin genes in seedlings and plan s could indica e a speci ic ole o each o he ou a-expansins in di e en phases o de elopmen o in di e en plan o gans. Thus, in seedlings hese a-expansins would be ela ed o elonga ing o gans bu no o me is ema ic o ese e issues, and while Ca-EXPA2 shows he highes ansc ip ion in epico yls, Ca-EXPA3 is highly exp essed in adicles (Fig. 3). In plan s, an impo an ole o EXPA1 in in e node g ow h could be specula ed since no signi ican hyb idiza ion was de ec ed in any o he o gan s udied, ei he om seedlings o om adul plan s (Fig. 3). Finally, s ess should be placed on he s ong ansc ip ion o some o he chickpea Ca-EXPA in ep oduc i e o gans (Fig. 3). The high le el o Ca-EXPA4 ansc ip ion in imma u e pods, oge he wi h he di e ence in ansc ip le els be ween ma u e (P2) and imma u e pods (P1), indica es he possible in ol emen o EXPA4 in pod de elopmen . Al hough i has long been specula ed ha cell elonga ion in oo s is caused by expansins, as a as we know he e is only one epo add essing a oo -speci ic a-expansin gene in soybean Gm-EXPA1 [25] ha could be esponsible o oo elonga ion. In chickpea, al hough no a-expansin ansc ip was de ec ed in adul oo s, Ca-EXPA3 showed a high le el o ansc ip s in adicles (Fig. 3). Also, EXPA3 shows a high deg ee o simila i y wi h expansins in ol ed in oo g ow h such as EXPA13 om Rumex palus is (Q8L5S1, 83.6% simila i y) o EXPA3 om T. e sicolo (Q9M517, 83.5% simila i y) [43]. No a ia ions we e de ec ed in Ca-EXPA3 ansc ip ion along adicle g ow h (Fig. 5), indica ing ha his a-expansin gene may be necessa y o oo de elopmen , al hough no di ec ly ela ed o oo elonga ion. An in e es ing esul was he esponse o chickpea a-expansin genes o ea men wi h g ow h- egula ing ho - mones. The e ec o ho mones on expansins ha e been s udied in di e en plan ma e ials [28,31,32], and a ela- ionship o some ho mones wi h he induc ion o expansin genes has been epo ed [8,14,39]. In chickpea, BR induces epico yl subapical segmen elonga ion and also enhances auxin-induced elonga ion [33], as has been epo ed o o he Fig. 6 . No he n analysis o Ca-EXPA mRNA ansc ip ion le els in suba- pical epico yl sec ions o C. a ie inum a e 4 and8ho ea men wi h plan g ow h egula o s, as desc ibed in Sec ion 4. C, un ea ed sec ions used as con ol.B+I,B assinolides plus IAA. Signals we e quan i ied and no ma- lized acco ding o 18S RNA hyb idiza ion. IOD. 713M.A. Sánchez e al. / Plan Physiology and Biochemis y 42 (2004) 709–716 ma e ials such as azuki bean epico yls [29] o cucumbe hypoco yls [22]. BR g ow h induc ion in chickpea was well co ela ed wi h he induc ion o Ca-EXPA ansc ip ion, his induc ion being highe when BR and IAA we e p esen simul aneously (Fig. 6), sugges ing ha expansin genes a e in ol ed in BR-induced elonga ion. This e ec was common o all ou chickpea a-expansins cDNAs, e en hough some o hem had low le els o ansc ip ion in epico yl sec ions. Al hough some a-expansin genes, such as A -EXPA5 and A -EXPA8 om A abidopsis ha e been desc ibed as BR- inducible genes using DNA mic oa ay analysis [17,35], he e a e ew epo s o he induc ion o a-expansin gene ansc ip ion by BR in ela ion o o gan elonga ion. By con as , ea men o oma o hypoco yl segmen s wi h BR, which p omo es issue elonga ion, causes no inc ease in Le-EXPA2 mRNA le els [3]. Fu he s udies a e necessa y o es ablish whe he o he a-expansin gene(s) may be ela ed o b assinos e oid-induced elonga ion. Chickpea a-expansin genes we e also induced by IAA in epico yl segmen s, he mos signi ican e ec being obse ed in he case o Ca-EXPA2; his suppo s he in ol emen o chickpea EXPA2 in epico yl g ow h. The induc ion o expan- sin exp ession by IAA seems o be common o mos expan- sins, such as in oma o [3] o cucumbe [30]. By con as , he e is no gene al e ec o GA 3 on plan a-expansins. Thus, he exp ession o speci ic expansin genes in ice is co ela ed wi h he induc ion o apid in e node elonga ion by gibe el- lins [7,23,24]. Howe e , in chickpea, GA 3 has no e ec on epico yl elonga ion [33] and does no a ec Ca-EXPA ans- c ip le els (Fig. 6), showing ha p obably GAs induce expansin gene exp ession only in sys ems whe e GAs induce elonga ion. In summa y, he ansc ip ion pa e n o chickpea expan- sin genes sugges s a speci ic ole o each o he ou expan- sins in di e en phases o de elopmen o in di e en plan o gans. The accumula ion o Ca-EXPA2 mRNA indica ed an impo an in ol emen o his pa icula expansin in epico yl and s em elonga ion. Ca-EXPA3 would be ela ed o adicle de elopmen , while Ca-EXPA4 seems o be in ol ed in pod de elopmen . The conside able inc ease in he le el o all Ca-EXPA ansc ip s ha accompanied he IAA plus BR- induced elonga ion o excised epico yl segmen s sugges s egula ion o chickpea expansins by hese ho mones. 4. Me hods 4.1. Plan ma e ial Chickpea seeds (C. a ie inum L. c . Cas ellana) p e- iously s e ilized in 0.1% (w/ ) sodium hypochlo i e we e ge mina ed in wa e in he da k a 25 °C and 80% ela i e humidi y on glass pla es co e ed wi h il e pape . The g ow h pe iod anged om 2 o 8-d. Epico yls and adicles we e collec ed om 2- o 8-day-old and om 2- o 5-day-old seedlings, espec i ely. Di e en plan issues we e collec ed o s udies o gene ansc ip ion: hooks, epico yls, mesoco- yls ( oo –epico yl junc ion zone), co yledons and oo s om 4-day-old seedlings. S ems o 11-day-old plan s, g own in he ligh , we e di ided in o i e in e nodes, numbe ed 1–5 om base o apex. Roo s om 11-day-old plan s and olioles, pods, lowe s and seeds om adul plan s we e also collec ed. Imma u e olioles (L1) we e hose coiled up and smalle han 4 mm, whe eas ma u e olioles (L2) we e hose who we e ully expanded and la ge han 4 mm. Imma u e pods (P1) we e 1-cm long g een pods whe eas ma u e pods (P2) we e ull-g own g een pods (abou 2.5 cm). Imma u e seeds (S1) we e g een seeds om ma u e pods. Finally, ma u e d y seeds (S2) we e also s udied. 4.2. Plan g ow h egula o ea men s A e 4do g ow hinwa e , subapical 10 mm epico yl- sec ions we e excised. A e insing in dis illed wa e o 1 h, subapical sec ions we e imme sed in a 10 mM po assium- ci a e bu e (pH 6.0) in he p esence o 10 µM IAA, 10 µM GA 3 , 10 µMABA o 0.2 µM BR o 4 o 8 h. Con ol sec ions we e imme sed in 10 mM po assium-ci a e bu e (pH 6.0). Th ee di e en se s o 50 segmen s each we e used in each ea men . 4.3. RNA and genomic DNA ex ac ion Fo RNA ex ac ion,5go ozen ma e ial was g ound in liquid ni ogen and esuspended in 10 ml o ex ac ion bu e [10 mM T is–HCl (pH 8.0), con aining 1% (w/ ) iisop opyl naph halene sul onic acid, 6% (w/ ) 4-amino salicylic acid and 5% ( / ) phenol mix u e (500 g phenol, 70 ml me a- c esol, 0.5 g 8-hyd oxyquinoline, 150 ml dis illed wa e )]. Samples we e ex ac ed as desc ibed in Muñoz e al. [33]. Genomic DNA was isola ed using he Plan DNA isola ion ki (Roche, Ge many) om1go 14-day-old chickpea plan olioles. 4.4. Isola ion o ␣ -expansins in a chickpea cDNA lib a y RT-PCR and genomic DNA PCR ampli ica ion we e used o he isola ion o se e al chickpea a-expansin agmen s. These agmen s we e used o he isola ion o ull-leng h a-expansins by sc eening a chickpea cDNA lib a y. To al RNA ex ac ed om 5-day-old e iola ed epico yl was used o syn hesize i s -s and cDNAs, using he Supe sc ip II p eampli ica ion sys em o i s -s and cDNA syn hesis (Gi- bco BRL, USA) using oligo dT as p ime , and was subse- quen ly used as a empla e o PCR ampli ica ion. Genomic DNA isola ed om lea es was also used as a empla e o PCR ampli ica ion. Degene a ed PCR p ime s used in PCR ampli ica ion o bo h cDNA and genomic DNA we e desi- gned wi h e e ence o he conse ed amino acid sequences o 34 expansins. The sequence o he ups eam p ime was 5′ 714 M.A. Sánchez e al. / Plan Physiology and Biochemis y 42 (2004) 709–716 TG GG(A/G/T) GG(A/G/T) GCT TGT GG(A/G) TA(C/T)G 3′and ha o he downs eam p ime was 5′(C/G)(A/T) (C/T)TG CCA GTT (C/T)TG (C/G/T)CC CA 3′. The wo oligonucleo ides co esponded o he amino acid sequences o MGGACGYG and NWGQNWQ. The PCR eac ions we e subjec ed o 30 cycles o 94 °C o 1 min, 55 °C o 1 min and 72 °C o 2 min. The ampli ied cDNA agmen s we e cloned in o pGEM-T easy ec o (pGEM Vec o Sys- em om P omega) ollowing he manu ac u e ’s ins uc- ions. The agmen s we e sequenced as desc ibed below. Th ee di e en a-expansin agmen s we e ob ained: Expan 8 ( om genomic DNA PCR ampli ica ion) and Expan 30 and Expan 36 ( om RT-PCR o 5-day-old epico yl o al RNA). The cDNA lib a y used was cons uc ed in he Uni-ZAP XR ec o om poly (A) + RNA ex ac ed om epico yls o 5-day-old e iola ed chickpea seedlings g own in wa e as desc ibed by Muñoz e al. [33]. A mix u e o Expan 8, Expan 30 and Expan 36 agmen s was used as p obe o he sc ee- ning o his cDNA lib a y. The p obe was labeled wi h 32 P using he Random p imed ki (Roche, Ge many). 4.5. DNA sequencing and deduced p o ein sequence analysis Plasmid DNA was p epa ed using he Wiza d plus SV minip ep ki (P omega, Madison, WI, USA) based on he alkaline lysis me hod [41]. Sequence analysis was pe o med au oma ically using he dRhodamine e mina o cycle se- quencing eady eac ion ki on an ABI P ism 377 gene ic analyze (bo h om PE Biosys ems, No walk, CT, USA). The DNA and deduced p o ein sequences we e compa ed wi h o he sequences in he EMBL/GenBank and Swall da a- bases, espec i ely, using he FASTA algo i hm [37]. Align- men and he phylogene ic ee o he ou chickpea and o he plan a-expansins we e ob ained using he Clus al me hod om he MegAlign p og am: pa o he Lase gene sequence analysis so wa e a ailable om DNASTAR. Pu a i e signal pep ides we e analyzed based on he SignalP 2.0 p og am [36] and pu a i e N-glycosyla ion was analyzed a he P osi e web page (h p://www.expasy.ch/p osi e). 4.6. No he n analysis No he n expe imen s we e pe o med as desc ibed in Muñoz e al. [33]. cDNA agmen s om he 3′non-coding end o he clones we e used as speci ic p obes o minimize any po en ial c oss-hyb idiza ion among a-expansin mR- NAs. cDNA speci ic p obes we e ampli ied by PCR in a GeneAmp PCR sys em 9700 he mocycle (PE Biosys ems), cleaned by he High Pu e PCR p oduc pu i ica ion ki (Ro- che, Ge many), and labeled wi h 32 P using he Random p imed ki (Roche, Ge many). To al RNA (10 µg pe lane) was elec opho esed, ans e ed on o Hybond N nylon mem- b anes (Ame sham Pha macia Bio ech, UK), and hyb idized using he adiolabeled speci ic p obes. A e hyb idiza ion, memb anes we e washed wice wi h 2× SSC, 0.1% SDS a 42 °C o 5 min each and wice wi h 0.1× SSC, 0.1% SDS a 42 °C o 20 min each. The au o adiog aphs we e analyzed on a Bioimage 60S Image Analyze (Millipo e, Bed o d, MA, USA,Visage 4.6K So wa e). Subsequen hyb idiza ion o he blo s using a 500 bp agmen om 18S RNA as p obe was used as in e nal con ol o de e mine he ela i e amoun s o RNA pe lane. 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