Expression of cysteine proteinase mRNA in chickpea (Cicer arietinum L.) is localized to provascular...

8/10/2019 Expression of cysteine proteinase mRNA in chickpea (Cicer arietinum L.) is localized to provascular cells in the dev

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J. Plant Physiol. 158. 1463 1469 (2001)Urban & Fischer Verlaghttp: / /www.urbanf ischer.de/ journals/ jpp

Expression of cysteine proteinase mRNA in chickpea ( Cicer ariet inumL.)is localized t o p rovascular cells in the deve loping root

Emilio Cervantes1 * , Juan a G. De Diego 2 , M ara Dolo res Gm ez3 , Javier De Las Rivas1, Jos M ariano Igual 1,Encarna Velzquez4 , Phil ippe Grappin5 , M anu el Cercs3 , Juan Carbonell3

1 IRNA-CSIC, Apartado 257, 37079, Salamanca, Spain2 Departam ento de Bioqum ica y Biologa M olecular, Edific io Departamen tal, Campu s M iguel de U nam uno, Universidad d e Salamanca, 37007,Salamanca, Spain3 Instituto de Biologa M olecular y Celular de las Plantas (IBM CP), CSIC-Universidad Politcnica de Valencia, Camino de Vera s/n, 46022, Valencia,Spain4 Departam ento de M icrobiologa, Universidad de Salamanca, Edific io Departam ental, Campu s M iguel de Unam uno, 37007, Salamanca, Spain5

Laboratoire de Biologie de Semences, INRA, 78026, Versailles Cedex, France

Received Apri l 5, 20 01 A ccepted July 2, 20 01

Summary

A full-len g th cD N A clon e en co d ing a ch ickp ea (C icer arietinum L.) cysteine p roteina se, ho m o log ou s

to rd 21gene ofA rab idop sis as w ell as to cysteine p roteina se g en es from leg um e s, w as isolated .

E xp ression of the m R N A in g row ing rad icles ha s b ee n loc ated in a very p rec isely d efine d lon g itu-

d ina l zone in the stele, co rresp on d ing to the area of d ifferen tiation of vascular tissues. Th is resultsug g ests tha t the en co d ed p roteinase m ay b e involved in p rog ram m ed ce ll d ea th even ts tha t p re-

ce d e vascu lar d evelop m ent.

Key words: C icer a rietinum cysteine p roteina se g erm ina tion roo t d eve lop m e nt va scu lar

b und le

Abbreviations:aa am ino ac id s.P C D P rog ram m e d C ellD eath

Introduction

G e rm ina tion an d ea rly se ed ling d eve lop m en ta re hig hly c om -

p lex p roc esses, d irec ted b y the co ord ina te ac tion of m ultip le

reg ulatory p athw ays. V ery ea rly in roo t d eve lop m e nt, P C D

* E -m ailco rresp on d ing au tho r:e ce rvan t@ u sal.es

The nu cleotide seq uen ce rep orted in this p ap er has b een sub m itted

to the EM B L N ucleotide Se qu ence da tab ase und er accession num -

berX82011.

m ust oc cu r in ord er to allow the co -ord ina te form ation of ne w

vascu lar tissue (Fuku d a 1997, Ye a nd Va rner 1996 ).T hu s,in a

you ng see d ling , a lim ited nu m b er of c ell d ivision s sep arate

the q uiesce nt ce nter from the ce lls tha t are prec ursors to the

vascular b und le,an d in a shortspa ce occu p ied by a red uced

nu m b er of ce lls, the p roteo lytic even ts tha t lea d to ce lld ea th

m u stb e a cc urately p rog ram m e d .

P roteina ses ha ve b ee n p urified an d the ir corresp on d ing

cD N A s clone d from leg um e seed s and seed ling s (B ecker et

al. 1994, Jones et al.199 6, C ercs et al.199 9, Fischer et al.

0176-1617/01/15 8/1114 63 $ 15.00 /0


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Figure1. M u ltip le a lig nm en to fthe C acG 1 seq ue nc e (364 aa ) from C ice rarietinum tog ethe r w ith 6 cysteine p roteina ses from p lan ts:3 sim ilar p roteinPha seolus vulgaris364 aa,and Pisum sativum 367 aa);2 of the closest p roteina se seq uen ces from the A rab idop sis tha lian a g enom e (A R ATH 2,46 2

b ase a nd A R AT H 1,376 aa, w ith ac cess-num b er 02318 5);an d c aricain from C arica p ap aya (348 aa).The last90 aa ofthe A R ATH 2 seq uenc e is not s

rest of the alig nm en t.S eve rald om ains p red icted in the seq ue nc e o fC ac G 1 are ind ica ted :a signalp ep tide from aa 1 to 3 3; an activation pep tide fro

aa 110 to 36 3, w hich inc lud es an initial variab le reg ion of 20 a a. M o reo ver, tw o d ifferen t d om ains insid e C acG 1 p rotein are rec og nised : an alp ha h

b eta d om ain from 131 to 14 1 a nd 23 6 to 36 4. Th e sec on d ary struc ture p red iction (H for alp ha he lix,E for b eta stran d s,a nd L for loo p s),tog ethe r w i

an d e for externa lreg ion ),are a lso p resen ted in the lasttw o line s. B elow the alig nm en t,a d raw ing w ith the m ain seco nd ary struc ture fea tures g ives

In the C acG 1 seq ue nc e, the fully co nse rved ch arg ed aa as w e llas six cysteine s tha tform sulp hid e b rid g es (SH 1, S H 2 a nd S H 3),are m arked w ith a


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1465E xp ression of a c ysteine p roteina se

20 00 an d referen ce s the rein); ho w eve r, the ir exp ression p at-

terns an d reg ulatory m ech an ism s ha ve no t be en clarified .

Th e sp ec ific p hysiolog ica lrole for ea ch d escrib ed p roteina se

is also a m atter of c on troversy, an d the ir su b strate p roteins

rem ain to b e id entified .

In a nim a ls,the P C D p rog ram s co nsisto fa casc ad e o fp ro-

teolytic even ts, ac curately reg ulated ,in w hich a p articular set

of cysteine p roteina ses, the casp ases, ha ve a p red om ina nt

role (C ryns an d Yua n 1998 ). In p lan ts, cysteine p roteina ses

have be en associated w ith PC D p roce sses (Fukud a 1997),

and the existence of caspases h as b een sugg ested , based

m ainly on exp erim en ts tha t use d inh ib itors (D el P ozo an d

Lam 1998), bu t so far, no sequ enc es w ith hom olog y to cas-

p ase s h ave b ee n id en tified .

In this w ork, w e d escrib e the clon ing , exp ression p attern,

an d struc turala na lysis ofa c ysteine p roteina se g en e, cacG 1,

tha t is ind uc ed d uring ch ickp ea g erm ina tion . In situ hyb rid i-

zation exp erim e nts show m R N A exp ression in a p rec ise zon e

co rresp on d ing to vascular elem en t differen tiation in the d e-

velop ing roo t. Th at the cacG 1 g en e is e xp resse d in ce lls a s-sociated w ith the m a turation of vascular tissu e, b oth in xylem

an d p hloe m p recu rsor cells, sug g ests involvem ent in the

co ntrolo fp rotein d eg rad ation d uring this p roc ess.

M ate rials and M ethods

Plant m aterial

Seeds ofchickpea (C ice r arietinu m , L.) w ere im b ib ed on m o isten ed

filter p ap er a nd g erm ina ted in the d ark at 25 C . Fo r the trea tm e nts

w ith ethylen e, the excised co tyled on s w ere im b ib ed on filter p ap er in

close d , 2-L tup p er-w a retm co ntaine rs (20 see d s p er co ntaine r). E thyl-

en e w as injec ted throu g h a rub b er stop p er ad ap ted to the lid .

RNA isolation and northern blot analysis

S ee d s (or exc ised co tyled on s w h ere ind ica ted ) w ere g rou nd in liq uid

nitrog en and R N A isolated b y the m e tho d of W ad sw orth et al. (1988 ).

N orthern b lots w ere prep ared as d escrib ed b y Jo hn et al.(1995 ).T he

g els w e re staine d w ith ethid ium b rom id e an d the rR N A b an d s visua l-

ized to verify e q ua lsa m p le loa d ing .

cDNA library construction and screening

P oly (A )+ R N A w as p urified b y affinity ch rom atog rap hy using an oli-

g o-d T cellulose colum n (B oeh ring er). D oub le strand ed cD N A w as

synthe sized using po ly (A )+ R N A isolated from g erm inated seed s

using a Z ap -cD N A synthe sis kit (S tratag en e). Th e cD N A lib rary w as

con structed in Lam bd a Z ap II vector (Stratag ene ) ac cord ing to the

m a nufacturers instructions. To clone cacG 1 c D N A , the lib rary w as

screen ed w ith the P C R frag m e nt enc od ing the p artialse q uen ce for a

p utative cysteine p roteina se (C e rvantes et al.19 94 ).

Radio labeling of probes

Fo r the no rthe rn b lots, the co m p lete inse rt of clon e cacG 1 w as p uri-

fied from ag arose g els w ith G ene clea n (B IO 101 inc .). D N A p rob es

w ere rad io lab eled w ith [32 P ]dC TP using ran do m p rim ers a cco rd ing

to F einb erg a nd Vo g elstein (1983 ).

Nucleotide sequencing

N ucleo tid e se q ue nc es w ere o b taine d using d ou b le stran d ed p lasm id

tem p late D N A an d the T 7 nu cleo tid e se q ue nc ing kitfrom P ha rm a cia.

Protein structure a nalysis

P roteins w ith a sig nifica nt sim ilarity to the C acG 1 d ed uc ed am ino

acid sequence w ere found in the EM B L/SW ISS PR O T databases,

using the m ethods FAS TA (Pearson and Lipm an 1988) and B LA ST

(b asic loc al alig nm en t search too l) (A ltsch ul et al. 1990 ) as sea rch

tools. A m u ltip le align m e nt of 16 seq uen ces of hom olog ou s p roteins

from p lan ts w as con structed using the C LU STA L-X p rog ram (Th om p-

son et al. 1997). Th e alig nm e nt allow s the id en tifica tion of fully c on -served resid ue s and the rec og nition of the am ino ac id s involved in d i-

sulp hid e b rid g es an d in the ac tive site. A tree of sim ilarity, d ed uc ed

from p air-w ise co m pa rison an d cluster a na lysis of the seq ue nc es,

w as also co nstruc ted to b etter evalua te the sim ilarities of the se-

qu ence s.T he align m ent and the consen sus seq uenc e d erived w ere

use d for sec on d ary struc ture an d solven t ac ce ssib ility p red iction s b y

the PH D m ethod (R ost et al. 1994). The com p arison w ith the 3D -

struc ture o fp roc arica in (1p ciA in P D B d atab ase ) an d p ap ain (1p p n in

P D B da tab ase) allow ed the reco gn ition of the d om ains of the C acG 1

p rotein.

In situ hybridizatio n

In situ hyb rid ization exp erim e nts w e re d on e, essentially a s d escrib ed

b y D avies (1993 ), using d ig oxig enin lab eled R N A p rob es. For tissue

p rep aration , rad icles of g erm ina ted ch ickp ea see d ling s (48 ho urs

p ost im b ib ition , 2 cm in len g th) w ere excised an d d ivid ed into five

eq ua l po rtion s (see Fig . 3). P araffin section s (7m ) corresp on ding

Figure 2.N orthe rn b lot sho w ing cacG 1 trans crip t exp ression in the

exc ised co tyled on s (im b ib ed after ab lation of the em b ryon ic axis)

un d er d ifferen t e thylen e co nc en tration s. A ll the sam p les w ere co l-

lec ted at 48 ho urs after im b ib ition .C o ntrolc orres p on d s to n orm a lco ty-

led on s from intac tse ed ling s.


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1466 Em ilio C ervantes etal.

to p ortions 1 and 2 w ere em p loyed in the exp erim en ts. S ense and

antisense R N A p rob es w ere ge nerated using a stand ard p rotoco lfor

in vitrotran scrip tion ,w ith the D ig R N A lab eling m ix (B oe hring er M an n-

heim ) as source for the nuc leotid es. Tem p lates w ere ob tained by d i-

g estion of the p lasm id s w ith E co R 1 o r B sp 120 , resp ec tively, for the

sense an d antisense R N A prob es.

Results

Cloning and sequencing of cacG1

S creen ing of the cD N A lib rary m a d e from co tyled on s of g er-

m ina ted ch ickp ea seed ling s,w ith the rad io lab eled P C R frag -

m ent co rresp on d ing to a p utative cysteine p roteina se (C er-

vantes et al. 1994; acce ssion num be r X70375), yielde d sev-

eral p ositive clon es. T he co rresp on d ing p hag em id s in vivo

w ere excised an d the co m p lete nuc leo tid e seq uen ce of on e

of the clone s w as d eterm ined .T he clon e w as nam ed cacG 1:

it is 1217 nu cleo tid e p airs in len g th, an d it co ntains on e op en

rea d ing fram e (109 2 nu cleo tid es) flan ked b y 5 and 3 non-co d ing seq ue nc es a nd en d s in a p oly-A tail. Th is seq uen ce

co ntains a reg ion w ith >95 % id en tity to the P C R frag m en t

that w as used a s a prob e.

Structural features of cacG1

cacG 1 enc od es a p rotein of 364 am ino ac id s. C om p arative

an alysis ofthis se q ue nc e sho w ed ch arac teristics of an en d o-

p ep tid ase of the p ap ain sup erfam ily. A co m p lete sea rch in

the p rotein d atab ases w as d on e in ord er to find ou t the m ost

sim ilar p rotein seq ue nce s of other p lan tp rotea ses. B ased on

the sim ilarity,16 seq ue nc es of cysteine p roteina ses w ere se-lec ted an d a m u ltip le seq ue nc e alig nm en tw as d on e. Th is al-

low e d the id entification of three d ifferen t reg ions in the se-

que nce of C acG 1 protein: a sig nal pep tide from aa 1 to 33,

w ith ch arac teristics of a lea d er seq ue nc e; a prop ep tide from

aa 34 to 110, w hich m ig ht b loc k the ac tive site; an d the m a-

ture p rotein, from aa 110 to 36 4, w hich is the ac tive en zym e

an d inc lud es an initial20 aa variab le reg ion ,w he re the clea v-

ag e for ac tivation occ urs. Fig ure 1 show s the seq uen ce of

C acG 1 w ith the structural features d escrib ed ab ove. Th e

strong hom olog y w ith carica in, a p roteinase of kn ow n 3 D

structure,allow s a reco g nition of tw o d ifferentd om ains insid e

C acG 1 p rotein: an alp ha he lix d om a in from aa 142 to 23 5,

an d a m ainly b eta d om ain from 131 to 141 an d 23 6 to 3 64 .

Th e reco g nition of the se d om ains is also ob taine d b y an

ind ep en d en t p red iction of se co nd ary struc ture of the se-

q ue nc e (R ost et al. 199 4). Fig ure 1 illustrates this sec on d ary

structure p red iction, tog ethe r w ith the solvent accessib ility of

the d ifferen t p arts of the seq ue nc e. In the fig ure, the se-

q uenc e of C acG 1 is ca lled as C acG 1 364 (as b eing a C icer

arietinum p rotein w ith 36 4 aa ), and it is alig ne d to 6 se-

q ue nc es: three of ve ry sim ilar c ysteine p roteina ses of othe r

legu m es (Vicia sativa, P ha seolus vulg aris an d Pisum sa-

tivu m ,); tw o of the closest p roteina se seq ue nce s from the

A rabidopsis thalian a g en om e ; and the m en tion ed C aricain

from C arica p ap aya. Fina lly,F ig ure 1 also show s othe r struc -

tural fea tures tha t ca n b e d isting uishe d in C acG 1. Th e three

am ino acid s that co nstitute the ac tive site of this p ap ain-typ e

of cysteine p roteina ses are fully co nserved in the alig nm en t,

illustrated b y a g rey b ac kg rou nd in the fig ure. Th ese resid ue s

in C acG 1 sequ enc e are: C ys155, H is311, and A sn331. Six

fully co nserved cysteine resid ue s tha t form three sulp hid e

b rid g es in the p roteina se struc ture are also rec og nized an d

m a rked in Fig ure 1. A llthe se fea tures allow a g oo d struc tural

assigm e nt of the C acG 1 sequenc e, and supp ort a cysteine

p roteinase activity for C acG 1.

Expression o f cacG1 in seed s during germ ination

cacG 1 m R N A w as und etected in dry seeds as w ell as in

seed s im b ibe d for 2 and 6 hou rs. The sig nal w as first de -

tec ted at 12 ho urs after im b ib ition an d inc rea sed , rea ch ing a

m axim um at 4 da ys (C ervantes et al. 1994 and unp ub lished

results).

Figure3.O utline of the in situh yb rid ization exp erim en t. G e rm ina ted

chickp ea seed ling s w ere selected at a leng th of 2 cm and d ivid ed

into five eq ua lp ortion s o f 4 m m eac h. P ortions 1 a nd 2 w ere used for

the in situhyb rid iza tion exp erim ents.


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1468 Em ilio C ervantes etal.

Expression of cacG1 mRNAin e xcised cotyledon s isresponsive to ethylene

Fig ure 2 show s the effec tof d ifferen tethylen e co nc en tration s

on the leve ls of cacG 1 R N A in exc ised co tyled on s (co tyle-

d on s im b ib ed after ab lation of the rad icle).Th e co ntrol lan e

sho w s tran scrip t leve ls in co tyled on s of intac t see d s at 4 8

ho urs after im b ib ition .Th e results sh ow tha tethylen e g as at aco nc en tration of 100 L/L w as ab le to inc rea se cacG 1 tran-

scrip t levels in excised co tyled on s. Th is ind uc tion is ind e-

p end ent of the p H be cau se the sam e results w ere ob tained

at p H 3.2 a nd p H 7.0 (no tsh ow n).D ose-resp onse an alysis of

this effec t is co m p arab le to othe r ethylen e-reg ulated g en es

that sho w m axim al ind uction at aroun d 100 pp m ethylene

(C hen and B leecker 1995).

Expression of cacG1associated w ith v ascular t issuedif ferent iat ion in d eveloping radicles

A s ethylene w as ab le to ind uce the expression of cacG 1

m R N A , and since this ph ytoho rm one has b een rep orted to

b e invo lved in the p rog ram m ed ce ll d ea th (P C D ) in p lan ts

(O rzaez an d G ran ell19 97), w e d ec id ed to investig ate cacG 1

cysteine p roteina se m R N A leve ls in the ea rly d eve lop m en t of

vascular tissue in the rad icle. T hu s, 2-cm -len g th rad icles of

ch ickp ea seed ling s a t48 ho urs p ost-im b ib ition w ere selec ted

an d d ivid ed lon g itud ina lly into 5 p ortion s (4 m m ea ch , see

Fig . 3). S ec tion s c orresp on d ing to p ortion s 1 an d 2 in Fig ure

3 w ere p rep ared for the in situ h yb rid ization exp erim e nts.

cacG 1m R N A w as on ly d etec ted throu g ho ut a reg ion loc ated

b etw een 3 an d 4 m m from the roo ta p ex, i.e. alw ays in p ortion

1, and thus co rresp on d ing to ea rly stag es of vascu lar e le-m en td ifferen tiation (Fig . 4 B ,D ). S am p les p rep ared from p or-

tion 1 w ere also hyb rid ized , w ith the sen se p rob e g iving no

sig na l(Fig s.4 A ,C ).E xp ression of cacG 1 m R N A w as notde-

tec ted in the stele of sec tion s co rresp on d ing to p ortion s 2

throu g h 5 in the rad icle. Fig ure 4 E show s an tisense staining

in a section close to w here exp ression oc cu rs, i.e. low er sec-

tion s of p ortion 2 show ing on ly a faint sig na lin the p erip he ry

ofthe stele.

DiscussionIn a p reviou s w ork a p artial cysteine p roteina se cD N A w as

am p lified b y P C R (C ervan tes et al. 199 4). E xp ression an aly-

sis show ed that the corresp ond ing g ene w as up reg ulated

d uring g erm ina tion of ch ickp ea ,a nd ethe p ho n ind uc tion sug -

g ested tha t ethylen e w as invo lved in the p roc ess. In this

w ork, w e u sed tha t cD N A to scree n a cD N A lib rary from g er-

m inated chickpe a seed s and to clone the correspo nd ing

cD N A . A full-len g th cD N A w ith co nse rved fea tures of p lan t

cysteine p roteina ses w as ob tained an d used as a p rob e in

no rthe rn hyb rid ization s. Th e co rresp on d ing tran scrip t is un -

d etec ted in d ry se ed s and d etec ted for the firsttim e b etw ee n

6 an d 12 ho urs after seed im b ib ition . O u r results w ith ethyl-

en e g as ind ica te tha t the rep orted ethe p ho n ind uc tion in ex-

cised cotyled ons is du e to ethylene and not to any other

che m ica l relea sed b y ethe p ho n. Thu s, excised co tyled on s

resp on d to e thylen e a nd cacG 1exp ression is ethylen e sensi-

tive, w ith a d ose-resp onse relationship sim ilar to other ethyl-

ene m ed iated resp on ses (C hen an d B lee cker 1995 ).

R esults on the localization ofcacG 1 transcrip t b y in situ

hyb rid ization in g row ing rad icles after im b ib ition show ed a

very w ell-d efine d exp ression in an area tha t co rresp on d s to

ce lls tha t w ill g ive rise to the vascular b un d le, in b oth tra-

che ary elem e nts as w ell as p hloe m ce lls (M itler an d La m

199 5). D arker ce lls are, in g en eral, sm aller an d m ay co rre-

sp ond to an earlier d ifferen tiation stag e. The transcrip t w as

not d etec ted in the roo t in reg ions loc ated ab ove an d b elow

this area (Fig . 3). B ased on these ob servation s, w e m ay

spe cu late tha t the cysteine p roteina se en cod ed b y cacG 1

co uld b e invo lved in the m e tab olic eve nts co rresp on d ing tothe ea rly step s tha t lea d to ce lld ea th in this reg ion , an d tha t

these ea rly step s m ay b e co m m o n b etw een xylem trac he ary

elem en ts tha t und erg o au tolysis a nd P C D an d p hloem ce lls

that rem a in living after d ifferentiation. O ther cysteine p rotein-

ases ha ve b ee n rep orted to b e assoc iated w ith p roc esses of

PC D in p lants (Ye a nd Varner 1996, B eers 1997,Fukud a 1997,

X u an d C hye 1999).T hese p lant p roteina ses d o no tb elon g to

the fam ily of c asp ases id en tified in an im a l system s (C ryns

and Yu an 1998 ).To d ate, only ind irect evid enc e b ased on in-

hib itor stud ies sug g ests tha tca sp ase s m ay b e ac tive in P C D

p roc esse s in p lan ts (D elP ozo an d La m 199 8);thu s,in p lan ts,

othe r cysteine p roteina ses of the p ap ain sup erfam ily m a y b e

im p ortan t in this p roc ess. M o re b ioc he m ica le vid en ce w illb ereq uired to confirm the p rec ise roles of the d ifferent p rotein-

ases.

O ne interesting asp ec t m ay co nc ern the p ossib le role

p layed b y ethylene in the reg ulation of the exp ression of

cacG 1 m R N A. E thylene has b ee n rep orted to b e involved in

m any p roc esses related to sene scen ce an d ag ing in p lants

(O rzaez and G rane ll 1997, N ood en and Leo po ld 1988), as

w ella s in the P C D p roc esse s tha t lea d to vasc ular form ation

(Ye an d Varner 1996) a nd ae ren ch ym a form ation m ed iated

b y h yp oxia (H e e ta l.19 96 ).O n the othe r ha nd ,e thylen e is in-

volved in the ind uc tion of a -g luc ana se g en e in em b ryonic

axes of g erm ina ted p ea seed ling s after 50 ho urs ofim b ib ition

(P etruze llie t al.199 9). Fu rthe r an alysis of m ec ha nism s reg u-

lating g en e exp ression d uring see d g erm ina tion m ay reve al

new asp ec ts ab ou t the com p lex role of ethylene in p lan t d e-

velop m en t.

Acknowledgements. W e tha nk P ilar C olorado an d A nton io R od rg ue z

for h elp w ith the cD N A lib rary c on struc tion . M a ra d el M a r S n ch ez

he lp ed w ith the nu cleo tid e seq ue nc ing an alysis.


7/7

1469E xp ression of a c ysteine p roteina se

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Expression of cysteine proteinase mRNA in chickpea (Cicer arietinum L.) is localized to provascular...

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