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. This g oup
o b-galac osidases ac s du ing he de elopmen and g ow h
o ege a i e o gans in C. a ie inum and could hus a ec
di e en p ocesses and allow a high egula ion o he
p ocesses in ol ed. Mo e wo k will be necessa y o es ablish
he speci ic subs a e and ac ion o each b-galac osidase.
Acknowledgemen s
The au ho s hank D . Ga in S. Ross o p o iding he
cDNA clone pABG1 om Malus domes ica. This wo k was
suppo ed by a G an om he EC (CT972224) and a G an
om he Di eccio
´n Gene al de Ensen
˜anza Supe io e
In es igacio
´n Cien ı
´ ica, Spain (BOS2002-01900).
Re e ences
[1] K.G. Ragho hama, K.A. Law on, P.B. Goldsb ough, W.R. Woodson,
Cha ac e iza ion o an e hylene- egula ed lowe senescence- ela ed
gene om ca na ion, Plan Mol. Biol. 17 (1991) 61–71.
[2] E.J.I. De Veau, K.C. G oss, D.J. Hube , A.E. Wa ada, Deg ada ion and
solubiliza ion o pec in by b-galac osidases pu i ied om a ocado
mesoca p, Physiol. Plan . 87 (1993) 279–285.
[3] G.S. Ross, T. Weg zyn, E.A. MacRae, R.J. Redgwell, Apple b-
galac osidase. Ac i i y agains cell wall polysaccha ides and cha ac-
e iza ion o a ela ed cDNA clone, Plan Physiol. 106 (1994) 521–528.
[4] M.S. Bucke idge, J.S. Reid, Pu i ica ion and p ope ies o a no el
b-galac osidase o exo-(1,4)-b-D-galac anase om he co yledons
o ge mina ed Lupinus angus i olius L. seeds, Plan a 192 (1994)
502–511.
[5] M.B. Singh, R.B. Knox, b-Galac osidase o Lilium pollen, Phy o-
chemis y 24 (1985) 1639–1643.
[6] E. Bonning, M. Lahaye, J. Vigou oux, J.F. Thibaul , P elimina y
cha ac e iza ion o a new exo-b-(1,4)-galac anase wi h ans e ase
ac i i y, In . J. Biol. Mac omol. 17 (1995) 345–351.
[7] J.H. Yoon, K. Ajisaka, The syn hesis o galac opy anosyl de i a i es
wi h b-galac osidases o di e en o igins, Ca bohyd . Res. 292 (1996)
153–163.
[8] R. P essey, b-Galac osidases in ipening oma oes, Plan Physiol. 71
(1983) 132–135.
[9] A.T. Ca ey, K. Hol , S. Pica d, R. Wilde, G.A. Tucke , C.R. Bi d, W.
Schuch, G.B. Seymou , Toma o exo-b(1-4)-D-galac anase: isola ion,
changes du ing ipening in no mal and mu an oma o ui , and
cha ac e iza ion o a ela ed cDNA clone, Plan Physiol. 108
(1995) 1099–1107.
[10] Z.M. Ali, S. A mugam, H. Lazan, b-Galac osidase and i s signi-
icance in ipening mango ui , Phy ochemis y 38 (1995) 1109–
1114.
[11] Z.M. Ali, S.Y. Ng, R. O hman, L.Y. Goh, H. Lazan, Isola ion,
cha ac e iza ion and signi icance o papaya b-galac anases o cell
wall modi ica ion and ui so ening du ing ipening, Physiol. Plan .
104 (1998) 105–115.
[12] D.L. Smi h, D.A. S a e , K.C. G oss, A gene coding o oma o ui
b-galac osidase II is exp essed du ing ui ipening, Plan Physiol.
117 (1998) 417–423.
[13] L.D. Smi h, K.C. G oss, A amily o a leas se en b-galac osidase
genes is exp essed du ing oma o ui de elopmen , Plan Physiol. 123
(2000) 1173–1183.
[14] A. Ta eishi, H. Inoue, H. Shiba, S. Yamaki, Molecula cloning o
b-galac osidase om Japanese pea (Py us py i olia) and i s gene
exp ession wi h ui ipening, Plan Cell Physiol. 42 (2001) 492–
498.
[15] K. Kakegawa, Y. Edashige, T. Ishii, Me abolism o cell wall poly-
saccha ides in cell suspension cul u es o Populus alba in ela ion o
cell g ow h, Physiol. Plan . 108 (2000) 420–425.
[16] W.G.T. Willa s, C.G. S eele-King, S.E. Ma cus, J.P. Knox, Side chains
o pec ic polysaccha ides a e egula ed in ela ion o cell p oli e a ion
and cell di e en ia ion, Plan J. 20 (1999) 619–628.
[17] T. Fujino, T. I oh, Changes in pec in s uc u e du ing epide mal cell
elonga ion in pea (Pisum sa i um) and i s implica ions o cell wall
a chi ec u e, Plan Cell Physiol. 39 (1998) 1315–1323.
[18] B. Dopico, G. Nicola
´s, E. Lab ado , Cell wall localiza ion o he
na u al subs a e o a b-galac osidase, he main enzyme eponsible o
he au oly ic p ocess o Cice a ie inum epico yl cell walls, Physiol.
Plan . 80 (1990) 636–641.
[19] P. Vale o, E. Lab ado , Inhibi ion o cell au olysis and auxin-induced
elonga ion o Cice a ie inum epico yls by b-galac osidase an ibodies,
Physiol. Plan . 89 (1993) 199–203.
[20] H. Konno, H. Tsumuki, Pu i ica ion o a b-galac osidase om ice
shoo s and i s in ol emen in hyd olysis o he na u al subs a e in cell
walls, Physiol. Plan . 89 (1993) 40–47.
[21] H. Konno, Y. Yamasaki, K. Ka oh, Cha ac e is ics o b-galac osidase
pu i ied om suspension cul u es o ca o , Physiol. Plan . 68 (1986)
46–52.
[22] H. Konno, Y. Yamasaki, K. Ka oh, Enzyma ic deg ada ion o pec ic
subs ances and cell walls pu i ied om ca o cell cul u es, Phy o-
chemis y 25 (1986) 623–627.
[23] B. Dopico, G. Nicola
´s, E. Lab ado , Changes du ing epico yl g ow h
o an au olysis- ela ed b-galac osidase om he cell wall o Cice
a ie inum, Plan Sci. 72 (1990) 45–51.
[24] H. Konno, S. Nakashima, T. Naka o, K. Ka oh, Pec in-bound
b-galac osidase p esen in cell walls o ca o cells unde he di e en
calcium s a us, Physiol. Plan . 114 (2002) 213–222.
R. Es eban e al. / Plan Science 168 (2005) 457–466 465