A family of β-galactosidase cDNAs related to development of vegetative tissue in Cicer arietinum

A amily o b-galac osidase cDNAs ela ed o de elopmen o ege a i e issue in Cice a ie inum Rocı ´o Es eban, Emilia Lab ado , Be a Dopico* Dp o. de Fisiologı ´a Vege al, Facul ad de Biologı ´a, Uni e sidad de Salamanca, Cen o Hispano-Luso de In es igaciones Ag a ias, Pza Doc o es de la Reina s/n, Salamanca 37007, Spain Recei ed 26 Ap il 2004; ecei ed in e ised o m 6 Sep embe 2004; accep ed 7 Sep embe 2004 A ailable online 30 Sep embe 2004 Abs ac In he cell wall o Cice a ie inum epico yls, he e a e a amily o b-galac osidases, one o hem named bIII-Gal is a b-galac osidase able o deg ade cell wall pec ins. The ole o he emainde b-galac osidases has no been es ablished. In his pape , we desc ibe he cloning and exp ession pa e n o a amily o h ee C. a ie inum b-galac osidase cDNAs (named CanBGal-1, CanBGal-4 and CanBGal-5) and we compa e hese esul s wi h he p e iously cha ac e ized CanBGal-3 cDNA clone, which encode he bIII-Gal. The sha ed amino acid sequence iden i y among he ou b-galac osidase deduced p o eins (named b-Gal, bIII-Gal, bIV-Gal and bV-Gal) anged om 63% o 81%. All display he pu a i e ac i e si e o amily 35 o he glycosyl hyd olases. An unusual cha ac e is ic o one o he chickpea b-galac osidases (bI-Gal) is he p esence a he C- e minus o he enzyme o a galac ose binding lec in domain. The CanBGals gene exp ession along seedlings and adul plan could sugges s di e en oles o hei co esponding p o ein h oughou he chickpea plan . The exp ession o CanBGal-5 is ela ed o young and me is ema ic s ages wi h high cell di ision a e, such as he me is ema ic hook, e y young epico yls, and apical in e nodes. By con as , CanBGal-1 and -4 seem o be mo e s ongly ela ed o ad anced s ages o epico yl g ow h, inc easing hei exp ession along epico yl age, and also in basal non-elonga ing s em in e nodes. In adul plan s, CanBGal-1 shows i s highes exp ession le els in lea es, while CanBGal-4 seems o be be e ep esen ed in adul oo s. This is he i s epo abou se e al membe s o he genomic amily o b-galac osidases ac ing du ing de elopmen o ege a i e o gans. #2004 Else ie I eland L d. All igh s ese ed. Keywo ds: Cell wall; Cice a ie inum; De elopmen ; Epico yl; b-Galac osidase 1. In oduc ion b-Galac osidases (EC 3.2.1.23), a widesp ead amily o glycosyl hyd olases, a e cha ac e ized by hei abili y o hyd olyze e minal, non- educing b-D-galac osyl esidues om b-D-galac osides. Se e al s udies ha e shown ha b-galac osidases ca alyze he hyd olysis o e minal gala- c osyl esidues om ca bohyd a es, glycop o eins and galac olipids. b-Galac osidase ac ion has been epo ed o elease ee galac ose du ing no mal me abolic ecycling o galac olipids, glycop o eins and cell wall componen s, and o deg ade cell wall componen s du ing cell wall expansion, cell senescence and ui ipening [1–5]. Fu he mo e, unde a o able he modynamic in i o condi ions many b-galac osidases ha e speci ic biosyn he ic ac i i ies h ough bo h ansglycosyla ion and e e se hyd olysis [6,7]. In highe plan s, so ening- ela ed b-galac osidases ha e been pu i ied om oma o [8,9], apple [3], mango [10] and papaya [11] among o he ui s, and hei biochemical p ope ies ha e been cha ac e ized. Mo e ecen ly, hese physiological and biochemical s udies ha e been suppo ed by he cha ac e iza ion o b-galac osidase genes, such as hose desc ibed in oma o [12,13] o in Japanese pea [14]. Besides he ole o pec ins in ui ipening, he e a e also se e al epo s indica ing ha he loss o pec ic galac ose seems o play an impo an ole in cell wall ex ensibili y and hence pec ic polyme s, which a e highly complex com- pounds, mus be in ol ed in he me abolism o expanding cell walls [15,16]. Fujino and I oh [17] desc ibed a clea www.else ie .com/loca e/plan sci Plan Science 168 (2005) 457–466 * Co esponding au ho . Tel.: +34 9 2324500x1951; ax: +34 9 23294682. E-mail add ess: [email p o ec ed] (B. Dopico). 0168-9452/$ – see on ma e #2004 Else ie I eland L d. All igh s ese ed. doi:10.1016/j.plan sci.2004.09.009 di e ence in cell wall a chi ec u e be ween elonga ing and non-elonga ing egions, sugges ing a modi ica ion in he molecula o m o pec ic polysaccha ides du ing he elonga ion o epide mal cells. Those au ho s sugges ed ha neu al suga side-chains could be in ol ed in he in e ac ion wi h o he cell wall componen s. b-Galac osidase is one o he enzymes ha seems o play a ole in he p ocess o modi ica ion o he pec in s uc u e, emo ing b-galac osyl linkages on he neu al suga side-chains [18–20]. Se e al s udies ha e ocused on he biochemis y o b-galac osi- dases du ing cell wall modi ica ion du ing g ow h [5,21–24], bu as a as we know, ew s udies ha e been ca ied ou on he molecula cha ac e iza ion o b-galac osidase genes in ege a i e elonga ing o gans. The pu i ica ion o a amily o chickpea b-galac osidases in Cice a ie inum epico yls was i s epo ed by Dopico e al. [25]. One o hem, bIII-Gal, was cha ac e ized as a p o ein in ol ed in he cell wall au oly ic p ocess [18,23,26]. I was p oposed ha bIII-Gal migh be impo an in he cell wall changes aking place du ing elonga ion. The cDNA co esponding o bIII-Gal, named CanBGal-3, was subse- quen ly cloned [27] and i s unc ion in pec in deg ada ion was suppo ed by i s exp ession in a po a o sys em. The ole o he emainde b-galac osidases has no been ye es ablished. In his pape , we desc ibed he cloning and exp ession pa e n o h ee C. a ie inum b-galac osidase cDNAs (named CanBGal-1, CanBGal-4 and CanBGal-5) and we compa ed hese esul s wi h he p e iously cha ac e ized CanBGal-3 cDNA clone, which encode he bIII-Gal. The membe s o his amily o b-galac osidase genes, may be in ol ed in galac ose me abolism du ing cell wall u no e , elonga ion p ocesses, o a chi ec u al pec in changes in C. a ie inum plan s. 2. Ma e ials and me hods 2.1. Plan ma e ial Chickpea seeds (C. a ie inum L. c . Ped osillano) 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 8C 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 days, a e which he epico yls we e collec ed. Di e en plan issues we e collec ed o s udies o gene exp ession: hooks, epico yls, mesoco yls ( oo –epico yl junc ion zone), co yledons and oo s om 4-day-old e iola ed seedlings. S ems o 11-day-old plan s we e di ided in o 5 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 ully expanded and la ge han 4 mm. Imma u e pods (P1) we e 1 cm 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. 2.2. RNA ex ac ion Fi e g ams o ozen ma e ial we e 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 o- pyl naph halene sulphonic 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 Mun ˜oz e al. [28]. 2.3. Sc eening o b-galac osidases in a chickpea cDNA lib a y The cDNA lib a y used was cons uc ed 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 Mun ˜oz e al. [28]. The sc eening o he lib a y was pe o med as desc ibed in [27]. A he e ologous sc eening o he lib a y was pe o med using a Malus domes ica b-galac osidase clone (pABG1, accession no. L29451, kindly p o ided by D . Ross, New Zealand) as p obe. The he e ologous sc eening a o ded wo di e en chickpea b-galac osidase cDNAs. A mix u e o hese wo chickpea b-galac osidase clones was used as p obe in u he homologous sc eenings in o de o isola e as many chickpea b-galac osidase cDNAs as possible. In hese sc eenings wo new b-galac osidase cDNAs we e isola ed. Thus, we inally collec ed ou di e en chickpea b-galac osidase cDNAs, named CanB- Gal-1, CanBGal-3, CanBGal-4 and CanBGal-5. 2.4. DNA 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 [29]. Sequence analysis was pe o med au oma ically using he dRhodamine e mina o cycle sequencing 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 abases, espec i ely, using he FASTA algo i hm [30]. Alignmen and he phylogene ic ee o he chickpea and o he plan b-galac osidases was done 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 [31] and pu a i e N-glycosyla ion si es and he b-galac osidase ac i e si e [32] we e analyzed a he P osi e web page (h p://www.expasy.ch/p osi e). Conse ed domains o he p o eins we e analyzed using he NCBI R. Es eban e al. / Plan Science 168 (2005) 457–466458 Conse ed Domain Sea ch (h p://www.ncbi.nlm.nuh.go /) [33]. 2.5. No he n analysis No he n expe imen s we e pe o med as desc ibed in Mun ˜oz e al. [28]. cDNA agmen s om he 30non-coding end o he clones we e used as speci ic p obes o minimize any po en ial c oss-hyb idiza ion. cDNA speci ic p obes we e ampli iedbyPCRinaGeneAmpPCRsys em9700 he mocycle (PE Biosys ems, No walk, CT, USA), cleaned by he High Pu e PCR p oduc pu i ica ion ki (Roche, Ge many), and labelled wi h 32 Pusing he Random p imed ki (Roche, Ge many). To al RNA (10 mg/lane) was elec opho esed, ans e ed on o Hybond N nylon memb anes (Ame sham Pha macia Bio ech, UK), and hyb idized using he adiolabelled speci ic p obes. A e hyb idiza ion, memb anes we e washed wice wi h 2SSC, 0.1% SDS a 42 8C o 5min each and wice wi h 0.1SSC, 0.1% SDS a 42 8C 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 4.6 K 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. No he n expe imen s we e pe o med a leas wo imes each. 2.6. Genomic DNA ex ac ion and Sou he n analysis Genomic DNA was isola ed using he Plan DNA isola ion ki (Roche, Ge many) om 14-day-old chickpea plan olioles. DNA (10 mg) was diges ed using HindIII and EcoRI es ic ion enzymes, sepa a ed on aga ose gel, and ans e ed on o a Hybond N nylon memb ane (Ame sham Pha macia Bio ech, UK) ollowing he manu ac u e ’s ins uc ions. Blo s we e hyb idized a 65 8C o 12 h in 1% (w/ ) SDS, 5SSPE, 5Denha ds’solu ion using he [ 32 P]-labelled speci ic p obe, cons uc ed as desc ibed abo e. Memb anes we e washed wice wi h 2SSC, 0.1% SDS a 65 8C o 5 min each, wice wi h 0.5SSC, 0.1% SDS a 65 8C o 20 min each and wice wi h 0.1 SSC, 0.1% SDS a 65 8C o 20 min each. 3. Resul s 3.1. Chickpea b-galac osidase clones Sc eening o he C. a ie inum cDNA lib a y e ealed he p esence o a leas ou di e en cDNAs encoding b- galac osidases: CanBGal-1, CanBGal-3, CanBGal-4 and CanBGal-5. CanBGal-3, CanBGal-4 and CanBGal-5 a e ull-leng h clones whose sizes we e 2600, 2900 and 2700 bp, espec i ely. CanBGal-1, whose size was 2656, is no a ull-leng h clone. The mRNA leng h es ima ed o his clone by No he n blo was 3200 n . The p o eins deduced om he chickpea CanBGal clones, named bI-Gal, bIII-Gal, bIV-Gal and bV-Gal, had a signi ican le el o sha ed amino acid sequence iden i y wi h one ano he and also wi h o he published plan b-galac osidases. bIII-Gal and bIV-Gal a e he closes a amino acid sequence le el (81% iden i y). bI-Gal and bV-Gal show a lowe deg ee o iden i y: abou 63% be ween each o he and wi h espec o he o he wo b- galac osidases. The p o eins bIII-Gal, bIV-Gal and bV-Gal p esen a p edic ed N- e minal hyd ophobic signal pep ide, whose pu a i e clea age si e is indica ed by an a ow in Fig. 1,as de e mined by Signal P [31]. The hyd ophobic signal pep ides a e 29, 23 and 26 amino acids long o bIII-Gal, bIV-Gal and bV-Gal, espec i ely. A e he elease o he signal pep ide, he ma u e p o eins ha e a molecula mass o 77.9, 78 and 80 kDa o he bIII-Gal, bIV-Gal and bV-Gal, espec i ely. bIII-Gal and bV-Gal show a high p edic ed isoelec ic poin (8.06 and 8.45), whe eas he p edic ed isoelec ic poin obse ed o he bIV-Gal was 5.79. No conclusi e da a could be ob ained o bI-Gal, owing o he lack o app oxima ely 140 amino acids a he N e minus o his p o ein. The ou chickpea b-galac osidase p o eins display he ac i e si e o amily 35 o he glycosyl hyd olases desc ibed by Hen issa [32] (ma ked by as e isks in Fig. 1). Posi ion 6 o his ac i e si e was di e en in he ou p o eins, bIII-Gal and bIV-Gal ha e Me whe eas bI-Gal and bV-Gal ha e Leu. Acco ding o Hen issa [32], Me and Leu a e possible conse ed susbs i u ions. A cha ac e is ic o he chickpea b-galac osidases is ha hey ha e di e en leng h. In pa icula , bI-Gal is 115 amino acids longe a he C- e minal end han he o he chickpea b-galac osidases (Fig. 1). I is in e es ing o no e ha his ex a pep ide o he bI-Gal comp ises an unusual galac ose binding lec in domain, as poin ou he analysis o he con- se ed domains o he p o eins using he NCBI Conse ed Domain Sea ch [33]. No o he chickpea b-galac osidases p esen his domain. All chickpea b-galac osidases show a leas one pu a i e N-glycosyla ion si e. bI-Gal and bIII-Gal ha e only one, whe eas bIV-Gal and bV-Gal ha e ou and wo, espec- i ely. The pu a i e N-glycosyla ion si es a e highligh ed in Fig. 1. 3.2. Phylogene ic ee The di e ences among chickpea b-galac osidases a e clea ly seen in he phylogene ic ee, compiled using he ou deduced chickpea p o eins and o he plan b-galac osidases, based on p o ein sequence alignmen (Fig. 2). The dis- ibu ion o chickpea b-galac osidases in his ee sugges s ha he e a e subg oups o b-galac osidases dis inguishable by hei sequence. C. a ie inum b-galac osidases appea R. Es eban e al. / Plan Science 168 (2005) 457–466 459 sepa a ed in h ee di e en phylogene ic b anches. bI-Gal appea s in g oup I, oge he wi h p o eins wi h a molecula mass anging om 92.1 o 93.6 kDa and 832 o 847 aminoacid esidues. The la ge size o hose p o eins was p ima ily due o an addi ion o app oxima ely 100 amino acids a hei ca boxyl e mini, as we ha e desc ibed abo e in bI-Gal (Fig. 1). All he p o eins included in his g oup I p esen a galac ose binding lec in domain. The bV-Gal p o ein appea s in b anch II, oge he wi h b-galac osidases om Phaseolus ulga is,Vi is ini e a and Lycope sicon esculen um TBG6. This g oup is he e ogeneous in hei molecula mass, bu shows se e al conse ed egions R. Es eban e al. / Plan Science 168 (2005) 457–466460 Fig. 1. Alignmen o he ou deduced b-galac osidase amino acid sequences o he CanBGal 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). Boxes indica e 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. The ac i e si e o amily 35 o glycosyl hyd olases is ma ked by as e isks. Pu a i e N-glycosyla ion si es a e highligh ed. wi hin he g oup (da a no shown), such as Gln a he posi ions 272 and 361, whe eas a e Glu in he o he g oups. The p o eins analyzed in his g oup ha e been ela ed o ea ly de elopmen p ocesses in g ape [34,35] and oma o [13] ui s. The bIII-Gal and bIV-Gal, oge he wi h b-galac osi- dases om o he leguminosae such as Vigna adia a and Lupinus angus i olius a e clus e ed in g oup IV. All he galac osidases in his g oup IV a e be ween 721 and 731 amino acids long and ha e molecula weigh s anging om 79 o 82.1. Mos o hem ha e been ela ed o cell wall pec in deg ada ion. Al hough sha ing a molecula weigh simila o g oup IV, he p o eins clus e ed in g oup V p esen a clea di e ence in he ac i e si e o amily 35 o he glycosyl hyd olases desc ibed by Hen issa [32]. All o hem ha e Me and Ala a posi ions 6 and 7, espec i ely, ins ead o Leu and Se , p esen in mos o he plan b-galac osidases desc ibed o da e. Some b-galac osidases ha e Me o Ala a hose posi ions bu ne e bo h oge he as in g oup V. The b-galac osidase om Dian hus ca yophyllus, de ec - able in senescing ca na ion pe als and unde s ic egula ion by e hylene [1], appea s on a sepa a e b anch. 3.3. Exp ession pa e n du ing g ow h The empo al exp ession pa e n in epico yls o he ou CanBGal was examined using RNA ex ac ed om epico yls anging in age om 2 days (when he epico yl eme gence s a s) o 8 days (when he a e o g ow h s a s o all) (Fig. 3). (Da a p e iously published e e ed o CanBGal-3 [27] we e included o a be e discussion o he esul s). Speci ic p obes we e used o each cDNA in o de o minimize any po en ial c oss-hyb idiza ion. CanBGal-1 and CanBGal-4 mRNAs we e de ec ed h oughou he g ow h o R. Es eban e al. / Plan Science 168 (2005) 457–466 461 Fig. 2. Phylogene ic ee ep esen ing he e olu iona y ela ionship among he ou deduced p o eins om he CanBGal cDNAs o Cice a ie inum and se e al plan b-galac osidases. epico yls and hei mRNA le els inc eased wi h he leng h o he epico yls. On he con a y, he CanBGal-5 mRNA le els dec eased wi h he age o epico yls. CanBGal-3 s a ed o be exp essed on 4 days and no ansc ip was obse ed in e y young epico yls (2 and 3 days) [27]. Exp ession along he s em in e nodes om 11-day-old plan s (Fig. 4) showed a simila co ela ion o g ow h as ha ound along epico yl age (Fig. 3). The mRNA le els o CanBGal-1 and CanBGal-4 inc eased om he apical young in e node (5 h) o he basal one (1s ), whe eas CanBGal-5 exp ession ollowed he opposi e pa e n, showing he highes mRNA le els in apical in e nodes and dec easing owa ds he basal ones, whe e almos no exp ession was de ec ed. CanBGal-3 showed he highes le el o ansc ip s in he 3 d in e node and almos no exp ession was ound in he younges (5 h) in e node; i.e., he one closes o he me is ema ic issue (Fig. 4). 3.4. Tissue speci ici y in seedlings and adul plan s As expec ed, acco ding o he sc eening o he lib a y, all he CanBGals we e exp essed in epico yls, showing hei R. Es eban e al. / Plan Science 168 (2005) 457–466462 Fig. 3. No he n analysis o CanBGal mRNA exp ession le els in seedling epico yls du ing elonga ion (A). Signals we e quan i ied and no malized acco ding o 18S RNA hyb idiza ion and a e p esen ed as in eg a ed op ical densi y (B). Numbe s e e o days a e sowing. Fig. 4. Di e en ial exp ession o CanBGals in he i e in e nodes o 11-day-old plan s (A). Signals we e quan i ied and no malized acco ding o 18S RNA hyb idiza ion and a e p esen ed as in eg a ed op ical densi y (B). In e nodes a e numbe ed 1–5 om he basal o he apical one. highes le el o exp ession in hese seedling o gans, excep o CanBGal-4 (Fig. 5). CanBGals ansc ip s we e also de ec ed in o he elonga ing seedlings o gans, such as mesoco yls and adicles, bu a lowe le els han in epico yls, excep o CanBGal-4 whe e he highes ansc ip le el was ound in bo h mesoco yls and adicles. Only CanBGal-1 was signi ican ly exp essed in co yledons, al hough a e y low mRNA le el o CanBGal-4 was also de ec ed in hose o gans. T ansc ip s o CanBGal-1, -4 and -5 we e ound in apical hooks (me is ema ic issue) bu always a lowe le els han in epico yls. CanBGal-5 exp ession in hooks ep e- sen ed he second highes ansc ip le el, a e ha o epico yls. No CanBGal-3 ansc ip s we e de ec ed in hooks [27]. The exp ession pa e n o CanBGal clones in adul plan s was b oad. Besides he s em in e nodes, in adul plan s he highes exp ession o CanBGal-1 and CanBGal-3 was ound in ma u e lea es. O no e was he highe ansc ip le el in old lea es (L2) as compa ed wi h young ones (L1). CanBGal-4 and CanBGal-5 showed maximum ansc ip le els in young pods (P1) and in he case o CanBGal-4, also in adul oo s (Fig. 6). All he clones showed e y low o no exp ession in d y seeds and in ma u e pods. I should be no ed ha CanBGal-3 was poo ly exp essed in young lea es and no exp essed in adul oo s. CanBGal-4 had he b oades ange o exp ession and CanBGal-5 was poo ly exp essed in ma u e lea es and lowe s (Fig. 6). 4. Discussion Mos s udies on b-galac osidase genes e e o hei exp ession in ipening ui , whe e he deg ada ion o galac an is associa ed wi h he ipening p ocess ( e iewed by R. Es eban e al. / Plan Science 168 (2005) 457–466 463 Fig. 5. Di e en ial exp ession o CanBGals in di e en pa s o C. a ie inum 4-day-old seedlings (A). Signals we e quan i ied and no malized acco ding o 18S RNA hyb idiza ion and a e p esen ed as in eg a ed op ical densi y (B). H: hook; E: epico yl; M: mesoco yl; C: co yledon; R: adicle. Fig. 6. Di e en ial exp ession o CanBGals in di e en pa s o C. a ie inum adul plan s (A). Signals we e quan i ied and no malized acco ding o 18S RNA hyb idiza ion and a e p esen ed as in eg a ed op ical densi y (B). 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. B ummell and Ha ps e [36]). Howe e , in elonga ing ege a i e o gans, he unc ion o he di e en b-galac o- sidase genes has been s udied o a lesse ex en , al hough changes in he neu al side chains o pec in ha could be media ed by galac osidases du ing g ow h ha e been epo ed [15,23,37–40]. Kikuchi e al. [41] sugges ed ha he neu al side chains o RG-I, such as galac an and a abinan, play an impo an ole in linkages be ween pec ins and o he componen s, such as cell wall hemicelluloses, enhancing he igidi y o cell walls. In C. a ie inum, we ha e p e iously epo ed se e al b-galac osidases pu i ied om epico yl cell walls [25], and one o hem, bIII-Gal was p oposed o be in ol ed in he cell wall changes aking place du ing elonga ion [23]. Du ing ou sea ch o cDNAs coding b-galac osidases, a o al o ou cDNA clones, named CanBGal-1, CanBGal-3, CanBGal-4 and CanBGal-5, all wi h a signi ican le el o sequence iden i y, we e ound. These ou genes would be membe s o a small amily o b-galac osidase genes. All o hem appea o be p esen in he chickpea genome as a single copy, as de e mined by Sou he n blo (da a no shown). The high sequence homology o he ou clones wi h one ano he and wi h o he plan b-galac osidases, oge he wi h he p esence o he glycosyl hyd olase amily 35 consensus sequence [32] (Fig. 1), sugges ha hey may all ha e b-galac osidase ac i i y. O he b-galac osidase gene amilies ha e been desc ibed in plan s, such as in L. esculen um wi h se en membe s [13] o Ca ica papaya [11] wi h h ee membe s. In ully sequenced plan genomes such as A abidopsis haliana he e a e 43 da abase en ies co esponding o b-galac osi- dases o he glycosyl hyd olase amily 35, howe e se e al o hem a e jus agmen s. In O yza sa i a,se enb- galac osidases appea in he da abases. The cha ac e iza ion and analysis o he gene exp ession o se e al CanBGals could help o unde s and he pu a i e ole o he co esponding encoded p o eins in chickpea cell wall me abolism. In a ecen epo , he CanBGal-3 was iden i ied as he cDNA co esponding o bIII-Gal and i s unc ion in cell wall pec in deg ada ion was suppo ed by i s exp ession in a po a o sys em [27]. He e, we s udied he gene exp ession a he mRNA le el o he emainde CanBGals in o de o ind he possible ole o each chickpea b-galac osidases. F om he p esen esul s we can obse ed ha he exp ession o CanBGal-5 was ela ed o young and me is ema ic s ages wi h a high cell di ision a e, such as me is ema ic hooks, e y young epico yls and apical in e nodes (Figs. 3–5). I would appea ha he p o ein bV-Gal is in ol ed in he ini ial s ages o plan de elopmen . Few examples o b-galac osidases ela ed o high cell di ision s ages ha e been desc ibed. As a as we know, only Nunan e al. [35] in g ape, and Smi h and G oss [13] in oma o (TBG6), ha e epo ed b-galac osidases whose ansc ip s we e accumula ed speci ically in he ea ly s ages o ui de elopmen ; a s age wi h an in ensi e cell di ision ac i i y ollowed by cell expansion. I is in e es ing o no e ha hese wo p o eins appea ed oge he wi h chickpea bV- Gal in he g oup II o he phylogene ic ee, indica ing a possible ela ionship be ween he s uc u e and unc ion o hese p o eins. In con as wi h CanBGal-5, CanBGal-1 and -4 seem o be mo e exp essed in ad anced s ages o epico yl g ow h, inc easing wi h age and also in basal, non-elonga ing s em in e nodes (Figs. 3 and 4). No b-galac osidase genes ela ed o ad anced s ages o g ow h ha e been desc ibed, al hough he exp ession o se e al b-galac osidases has been epo ed o inc ease du ing senescence, as in ca na ion [1] and in aspa agus [42,43]. Howe e , he elease o galac ose in senescence is accompanied by cell wall disassembly, whe eas he exp ession pa e ns o CanBGal-1 and -4 a e highes when he e is an inc ease in cell wall igidi y. We can, he e o e, p opose ha he ac i i y o hese p o eins could be o hyd olyze an unknown subs a e whose modi ica ion causes cell wall igidi y, p obably by compac - ing pec ins. E en hough CanBGal-1 and CanBGal-4 seem o sha e he same pa e n o exp ession a he mRNA le el in epico yls and s ems, i should be no ed ha he p edic ed isoelec ic poin s o he bI-Gal and bIV-Gal p o eins a e qui e di e en . This sugges s he possibili y ha hese p o eins migh be bound o a di e en subs a e loca ed a a di e en si e in he cell wall. Fu he mo e, in adul plan s CanBGal-1 shows i s highes exp ession in lea es, while CanBGal-4 seems o be mo e impo an in adul oo s. I should also be no ed ha he CanBGal-1 ansc ip s a e he only ones abundan in co yledons, whose cell walls could ha e galac ose- ich polyme s di e en om hose ound in epico yls, indica ing a pu a i e subs a e speci ici y. The p esence o a galac ose binding lec in domain in he p o ein bI-Gal sugges s a pa icula ole o his p o ein. Al hough his domain is a away om he pu a i e ac i e si e, and he e o e migh no a ec he ca aly ic p ope ies o he enzyme, his domain, i p o en o be unc ional could a ec he biochemical cha ac e is ics o he enzyme con aining i . The p esence o his domain is no usual be ween b-galac osidases; acco ding o da abases only abou 42 p o eins among all he plan b-galac osidases desc ibed con ain i [33]. T aino i e al. [44] e e wo b-galac osidases in s awbe y wi h a simila lec in-like domain, sugges ing hei possible suga -binding ac i i y on he basis o he highly conse ed s uc u al iden i y be ween animal lec ins and he C- e minal domain o hese wo s awbe y p o eins. The exp ession pa e n o he CanBGal cDNAs s udied in he p esen epo was qui e di e en om he exp ession o CanBGal-3 encoding he bIII-Gal able o hyd olyze cell wall pec ins [27]. In ha case, he highes le el o ansc ip accompanied he s ages o maximum elonga ion, such as 4-day-old epico yls [27] o s em in e nodes wi h high g ow h abili y (Fig. 4). No ansc ip was ound in he egions wi h high cell di ision a e such as apical in enodes, hooks o e y young epico yls. R. Es eban e al. / Plan Science 168 (2005) 457–466464 The CanBGals gene ansc ip ion in seedlings and adul plan (Figs. 5 and 6) could sugges s speci ic unc ion and/o loca ion o hei co esponding p o ein h oughou he chickpea plan . The idea ha hese ou b-galac osidases om chickpea could be in ol ed in di e en modi ica ions o galac osyl esidues in he cell wall in di e en phases o de elopmen o in di e en o gans is based on he high numbe o speci ic na u al subs a es desc ibed o b-galac osidases. An example indica ing such high speci- ici y is p o ided by he b-galac osidase om Copai e a langsdo ico yledons, wi h conside able speci ici y owa ds xyloglucan oligosaccha ide [45]. Fo hyd olysis, his b-galac osidase ecognises only he galac osyl sub- s i u ion o he xylose adjacen o he non- educing end glucose esidue o xyloglucan oligosaccha ides. In a simila way o C. a ie inum, i is common o ind b-galac osidase iso o ms in he same species; hese do no usually show he same abili y o hyd olyze pec ins o o he cell wall subs a es. In gene al, hese iso o ms p esen di e ences, indica ing ha hey ac in a di e en way o agains di e en subs a es wi h di e en deg ees o b anching [11,25,46]. The cloning o se e al b-galac osi- dase iso o ms could help o cla i y he ole o each one in he same o di e en p ocesses, al hough o da e his poin has only been s udied in ui s such as oma o [13]. Se e al b-galac osidase clones ha e been desc ibed in F aga ia ananassa o Phaseolus au eus, bu o ou knowledge no da a on hese clones ha e been published. As a as we know, his is he i s epo o se e al membe s o he genomic amily o b-galac osidases ac ing in ege a i e o gans. 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