P~nt Scienc~ 82 (1992) 161-166 161 Elsefier Sdenfific Publishers Ireland Ltd.

Influence of endogenous cytokinins on reserve mobi zafion in cotyledons of Cicer ar tinum L. Arfifidal restoration of

endogenous levels of opentenyl adenine riboside and isopentenyl adenine

Jose Luis Mu~oz, Luisa Martin, Gregofio Nicolas and Nieves Villalobos

D ~ m m ~ m ~ B:olog~ V e g ~ (Fis~log~ Veg~al), Fa~ltad ~ BiologY, Unive~idad ~ S a l m o n , P la~ ~ ~ Merged s/n, ~ 3 ~ , Sa~ma~a (Spa~)

(Recoved Ju~ 2~h, 1991; ~fifion rec~ved D~emb~ ~h, 1991; ~ e d Decemb~ ~h, 1991)

Of~e Og~ ~ d o ~ n o ~ c ~ o ~ d~e~ed ~ cotyledo~ ~ Citer ~ u m ~ ~ i ~ n ~ n ~ adenine fibo~de execs i~ m~n effect on the me.bolero of ~ e lipids and is less effioem ~th ~ s ~ to the mOab~m of ~ o h y d r m ~ and W o ~ . l ~ n ~ n : ade~ne only h~ a ~ g ~ o ~ effect on c a r b o h y d ~ m ~ a b ~ m .

Key wor~: Cic~ a r ~ u m L cv 'Catal ina ' ; c ~ o ~ n s ; m~abd~m; e n z y m ~ activity; endogenous ~ e ~ ~ed ~rm~ation

Introduction chick-pea seeds [9]. The cytokinins detected in the coty~dons ~eem to originate from lhe embryon~

In d~ot plant~ according to the most of the axes [10,111. Each type of cytokinin seemed to ex- research carried out on those seeds in wh~h the ert a specific action on reserve metabol~m. In a axis controls enzymat~ activity and reserve previous work [11] we have studied the regulatory mobil~afion, the regulation by the axis can be ac- ro~ of zeatin, zeafin ribo~de and their correspon- counted for Other by hormonal control or by ~nk ding glucofides on the processes of mobil~ation effect [1]. Most of the ~udies concerning hor- reserves. In the present work we show the results monal con~ol have been carried out by applica- obtained, concerning the action of two other tion of growth regulating substances [2-4]. isolated cytokinins from chick-pea cotyledons

In recent years, the cytokinins have been asfign- 06Ado, i6Ade), on the above mentioned pro- ed an impo~ant regulatory role in the processes cesses. ~ading to germination by stimulating different en- We have carried out a comparative study of the zymatic activities [2,5-8]. However there are no principal processes taking place during nutrient ~udies which conclufivdy explain the ro~ of mobihzation in ch~k-pea seeds under normal con- naturally existing cytokinins in seeds concerning ditions and in seeds from which the embryon~ axis the mobil~ation processes, had been removed. Our aim, after artificial

Eight cytokinins were detected in germinated restoration of normal endogenous levds of i6Ado and i6Ade in exdsed cotyledons (u~ng the

C°rresp°ndenvCeeget~, Facd~°~eL~SBi a~og~M, artinu '~ver~daC d~dracom~mend ~ e e ~fi~°~da e substances themsdves extracted from these seeds)

Madri& Ciudad U~v~fi~ri~ E-28040 Madrid, Spain. was to see whether there is any recovery of the Abbr~v~t~n~ CKs, c ~ o ~ n s ; i6Ad~ isopentyl adenine; above-mentioned processes in spite of the absence irAd~ ~open~l adenofin~ Z, zeafin, of the embryon~ axis.

016~945~9~$05.00 © 1992 ~ f i e r Sc~nfific Pubfishers Ireland Ltd. Printed and Published ~ Ireland

1~

MaStiffs and Methods was injected (di~olved in wate0 with micro- syringe. There is ~ways a period of 6 h before the

Plant ma~r~l cyto~nins i~ected into the agar have passed into Two kind of seeds were used: Cicer ariet~um L. the cotyledons [11]. Endogenous levds of i6Ade

cv Ca~dhna (Ch~k-pea) seeds for the evMuafion were artifidally restored according to the above- of cytokinins and Cucum~ saHvus L. cv Calahor~a mentioned m~hod. Endogenous cytokinins were ~ucumbe0 seeds for the bioassa~ The ch~k-pea ex~acted after 12, 18, 2~ 36, 48, 72, 96 and 120 h seeds were germinated and grown on a ~ass plate of incubation or germination. covered with filter papeL in darkness at 25°C with 80% rdafive humidity. The cucumber seeds were Mobilization of reserves ~ ch~k-pea germina~d in the same way at 28°C. For this ~udy both the cot~edons collected

during germination of intact seeds and the Extra,ion, purification and evaluation ofcytok~ins cotyledons from growing seeds wilhout thor axes

Extraction, purification and identification of the (with and w~hout arfifid~ reproduction of endog- different cytokinins was carried out as described enous ~vds of i6Ado and i6AdO were used. Tot~ by Mufioz et al. [11]. The ex~ac~ duted ~om the carbohydrates were determined by the method Dowex 50W × 8 column were dried and redi~olv- described by Dubois at ~. [12]. The hpids were ex- ed in 1 ml of 80% ethanol and the sample spored ~acted according to the method of B~gh and Dyer onto ~hcagel 60 G plates. The plates were de- [13]. Determination of soluble sugars was per- velopedwithisopropanoFammonia/water(10:l:l), formed by the Somog~ [14] and Ndson [15] The Chromatograms were later di~ded into 11 m~hod. Protein extracted after di~olving the refi- band~ Once each band had been duted in 80% due obt~ned with 0.05 N NaOH at 37 ° C for 4 h ethanol, i l l , red and dried, it was used for the [1~ was measured by using the method described bioassay which was performed ufing the cucumber by Bradford [1 ~. For the ~udy of tot~ am~ase cot~edons. The bands exhibiting cytokinin acrid- and pro~ase actifitie~ the enzymatic ex~a~s were ty were used for anMyfis by HPLC. prepared according to the m~hod described by

For the HPLC anMyfi~ columns of 290 × 4 mm Metifier and Paulflo 1~. For protease acti~ty packed with M~ro -Park (MCH-5) were employed both amino add [18] and pepfide [1~ rdease were for quantification of cytokining The mobile phase measured ~ . confined of methanoFwater (40:60 v/v to 54:46 v/v), u~ng a linear grad~nt of 12 min. Conditions Resuhs were as follows: flow ra~, 0.5 ml/min; pH of so~ vent, 5.~ ~mperature, 30°C; pressure, 148 atm; A~ificial re~orat~n of endogenous &veb of~Ado detector beam wave~ngth, 254 nm. The retention and ~Ade ~ exceed coty&dons times were de~rmined ufing i6Ado and i6Ade To re,ore the ~vd of both i6Ado and i6Ade ~andards obt~ned ~om Sigma under the condb (Fig. l) only a fin~e injection was needed, at 6 h dons described above, of incubation, of the respective sub~ances

pre~oufly extracted. So, after the i~ection, an en- A~ificial restoration of endogenous &ve~ ofi6Ado dogenous ~vd was obt~ned of i6Ado and i6Ade and ~Ade ~ exceed coty&dons in exdsed cot~edons fimilar to the level obt~ned

The reproduction of endogenous ~vds of these in cot~edons of germinated seeds. However, the cytokinins were carried out as described by Mufioz ~vds of cytokinins were somewhat lower under et at. I11]. In order to reproduce the endogenous these ~eatments due to a slight retention of both ~vds of the above -mentioned cytokinin~ the sub~ances in the agar. seeds were germinated as usu~. After 6 h of germi- nation the embryon~ a~s was exdsed and Influence of ~Ado and ~Ade on reserve mobH~a- substituted by an agar block 0% agaO into which Hon ~ Cicer ariet~um L. coty&dons the tot~ amount of i6Ado extracted ~om the The treatmentofdetachedch~k-peacot~edons same number ofcot~edons of 12-h imbibed seeds with i6Ado produces confiderable variations in

163

" ' "I 1200 1000 ~

600 ~ 10 ~ ~oo! ~ ~ 7- ~_~____~__~__:~__~_~ 200- ~ ,,~.-.--,- ,-- ,-- -] - ] : ~ - - - ~ ~ - - ~ - ~ - - ~

0 6 ~ 24 36 48 ~ 96 120 ~ ~ __~

5 I~OO~ B 1200~ lOOO~

1 ~ ~ t~o ~ ~1 / ~ ~ ~ ~ ~a a ~ ~ o'~ ~ 'OO] // ~ ~ INCUOATION TI"E ~,ur~ •

~- - ...... - ~ ~ ~ - - Fig. 2. Va~ationinlipidcontcnt~cotylcdon of Cic~r ar~t~ ~ . . . . . . . • , , ~ ~

0 6 ~ 2~ s6 ~e T2 e6 120 L. ~eds. CoW,dons of ~tact seeds (~ untreated detached cotyledons (O~ de~chcd c~:edons after ap~ication of i~Ado

I~ATI~ ~H[ ~0~ (~) and a~er appl~a~on of i~Ade (~. Each p~ sho~s the

~ I. ArS~ ~cproduction of endo~nous ~v~s of i~Ado average of 3 ~plications; expe~m~ ~er¢ re~cd 3 ~me~ and i~Ade in ex~scd coW,dons of ch~a seed~ (A) Con- Ve~c~ bars ~c~e S.E. ~: endo~nous i~Ado ~ norm~ ~eds (~ aphelion ~ 6 h of ~cuba~on of the i~Ado ~xtracted ~om cotyledons of seeds ~inat~ ~r 12 h (O). (B) Co~r~: endogenous i~Ade ~ norm~ seeds (~ injection at 6 h of incuba~on of the i~Ad~ extracted ~om cotyledons of seed~ ~rm~a~d ~r 12 h (@).

EaChwere reN~edPNm sh°ws3 times'the VerficNaVera~ °fbars3 ~cme~N~m~nmS'E'expefimenu ~so~

-

lipid contents as may be seen in Fig. 2. Of particu- ~ ~,oo ~ ~ lar interest is the fact that recovery is much greater = ~ ~ ~ ~ ~ / : ~ at the beginning than at the end of the process, ~ ~ / with a value at 48 h of incubation, for example, be- ~ ~ ~ _ ~ _ ~ ~ ing approximately 74.6% whereas at 120 h it is ~ ~9 only 38%. Nevertheless, this cytokinin seems to ~ ~ ~ ~ ~ _ ~ ~ - ~ have little effect on the metabolism of total car- ~ bohydrates during g e ~ n a t i o n (Fig. 3), since there ~ ~ is an approximation to the values obtained in nor- ~ m~ conditions, recovery ~ so low that at 120 h of * ~ ~ ~ n s~ ~ ~ ~ ~,

incubafiOnresul~ obtNnedit is withonly soluble34%. Incarbohydra~sSpi~ of thiS,(Fig.the ~ ~ ~,~,~

4) are rather spectacular ~nce there is a graduM re- N~ ~ V a ~ n ~ mtN carbohydra~ co~ents/c~edon of covery ~om 48 h of incubation onwards such that Cicer ar~et~um ~ seed~ CoW,dons of ~tact seeds (~); u~- at 120 h the vMues obtNned are very ~ose to those N~m~ntreated detached cotyledons i~Ado (~) and after application of i~Ade (~. Each detached coW,dens a ~ a~ d e ~ i n e d in the cot~edons of seeds geminated pN~ shows the av~a~ of 3 ~N~ati~n~ exNfi~enU we~ under n o r m conditions, repe~ed 3 fime~ VerficN bars ~ c m e S.E.

I~

ao was obt~ned of a p p r o ~ m a ~ 4 6 ~ A ~udy of the e ~ c t of i ~ d o a p ~ e d to exosed c ~ e d o ~ on protein metabolism, shows a lack of any e ~ c t

~ . ~ ~ ~ of this substance on the variation in protein con- -

= ~ tent (Fig. 6) or protease activity measured as pep- ~ 2u tide release (Fig. 7). There is only a slight e ~ c t ~ ~ which pewits a small recovery of the n o d a l ~ ~ ~ ~ levels ofprotease activity as measured as the ~ ~ ~ release of amino acids (fig. 8). ~ ~B~ ; ~ ~ ~ ~ ~ When the e ~ c t of i6Ade on nutrient mobiliza-

. / ~ tion was studied, an absence of any e ~ c t on the variation ~ ~ d ~v~s in coW,dons ~ i ~ d~ac~ ed axe~ (H~ ~ was observed, ~ o u ~ there was a

~0~ ~ ~ ~ ~ ~ ~ ~ ~ ~a recove~ of the ~vds obt~ned in c o ~ o n s of seeds ~ a t e d under n o ~ f l con~fions both

I~UATI~ ~ME ~eu~ ~ t h ~ s ~ c t to tot~ (~g. 3) and to s o ~ e car-

~co~don ~ V~°nof ~cer a r ~ u m ° f c°ntentsL, seed~ °f s~UNec~edonsCa~°~dra~of ~tact bohydra~s (~g. ~. T~s recovery became more seeds ~); unheard detached c~edons ~ detached pronounced towards the end of the period ~u~ed, cotyledons ~ ~ca t ion ~ i ~ (~ and a~er aphelion SO that ~ r ~ t ~ carbohydr~es (~g. 3) it was 43% of i ~ ~ . Each p~m ~ows ~e avera~ of 3 ~ o n ~ at 48 h and 62% at 120 h and ~ r s~u~e car- ex~fimen~ were ~pe~ed 3 times. Vertic~ ba~ ind~a~ S.E. bohydrates ( ~ ~ ~ was 56% at 48 h and 67% at

120 h of ~cubafion. The v~ues of am~ase acfifi~ The ~ud~s concerning am~ase a~Ni~ (~g. 5) observed wi~ ~is treatment (~g. 5) show a recov-

ag~n c o ~ a recove~ ~ n o ~ ~vds after ap- e ~ of 52% at the momem of ma~mum a ~ y . ~ c ~ o n ~ i ~ . For i ~ d o , at the momem of Uni t e the resets obt~ned concer~ng carboh~ ma~mum a ~ 06 h ~ ~cub~on) , a recovery

'°1 -1 ~ ; | ~ ~ ~

~" ~ ~0- '

~ ~ = ~- ~ ° ~

: ~ 20.

~ " ~ ~ ' ~ , - , ~ ,

~ 0 ~ I~ a 2~ ~6 ~ T~ 9~ I~0 0, i ~ ~ ~ ~ ~ ~0

~ B ~ N Tilt ~euf~ INCUBATION Ti l t ~euf~

Fig. ~ V ~ o n ~ ~ amylase act~ty/c~edon ~ Cicer ~ ~ Vadation of p r ~ n c o n ~ ~ o n of Ocer a r ~ L. seed~ C~edons of ~tact ~eds ~ untreated a r ~ L. seeds. CoW,dons of ~ seeds (~; u~re~ed ~tac~d co~ons (~; de~ched c~y~dons a~r ~ca t i on ~tac~d co~ons (~; ~ c ~ o n s a~r a p ~ n of i6Ado (~ and a~r ~ o n of i6Ade (~. Each p ~ i~do (~ and a~er ~ o n of i~de ( ~ Each p ~ ~OWS ~e avera~ of 3 r e ~ a t i o n s ; e x ~ m e n t s weft r e ~ shows the avera~ of 3 f f ~ a t i o n ~ ex~dmen~ weft repe~ed 3 time~ Ve~c~ bars ~ c ~ e S.E. 3 dme~ Vertic~ ba~ ind~a~ S.E.