Influence of endogenous cytokinins on reserve mobilization in cotyledons of Cicer arietinum L. Artificial restoration of endogenous levels of isopentenyl adenine riboside and isopentenyl adenine

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~mm~m

~ B:olog~ V e g ~

(Fis~log~ Veg~al), Fa~ltad ~ BiologY, Unive~idad ~ S a l m o n , ~3~, S a ~ m a ~ a (Spa~)

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

P l a ~ ~ ~ Merged s/n,

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 ~ t h ~ s ~ to the m O a b ~ 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 'Catalina'; c ~ o ~ n s ; m~abd~m; e n z y m ~ activity; endogenous ~ e ~

Introduction In d ~ o t plant~ according to the most of the research carried out on those seeds in wh~h the axis controls enzymat~ activity and reserve mobil~afion, the regulation by the axis can be accounted for Other by hormonal control or by ~nk effect [1]. Most of the ~udies concerning hormonal con~ol have been carried out by application of growth regulating substances [2-4]. In recent years, the cytokinins have been asfigned an impo~ant regulatory role in the processes ~ading to germination by stimulating different enzymatic activities [2,5-8]. However there are no ~udies which conclufivdy explain the r o ~ of naturally existing cytokinins in seeds concerning the mobil~ation processes, Eight cytokinins were detected in germinated

C°rresp°ndenvCeeget Facd~°~eL~SBi ~, a~og~M,artinu'~ver~daC d~dracom~mend ~ e ~fi~°~da e e Madri& Ciudad U~v~fi~ri~ E-28040 Madrid, Spain.

Abbr~v~t~n~ CKs, c ~ o ~ n s ;

i6Ad~ isopentyl adenine; irAd~ ~open~l adenofin~ Z, zeafin,

~ed ~rm~ation

chick-pea seeds [9]. The cytokinins detected in the coty~dons ~eem to originate from lhe e m b r y o n ~ axes [10,111. Each type of cytokinin seemed to exert a specific action on reserve metabol~m. In a previous work [11] we have studied the regulatory r o ~ of zeatin, zeafin ribo~de and their corresponding glucofides on the processes of mobil~ation reserves. In the present work we show the results obtained, concerning the action of two other isolated cytokinins from chick-pea cotyledons 06Ado, i6Ade), on the above mentioned processes. We have carried out a comparative study of the principal processes taking place during nutrient mobihzation in ch~k-pea seeds under normal conditions and in seeds from which the embryon~ axis had been removed. Our aim, after artificial restoration of normal endogenous levds of i6Ado and i6Ade in exdsed cotyledons (u~ng the substances themsdves extracted from these seeds) was to see whether there is any recovery of the above-mentioned processes in spite of the absence 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

Plant ma~r~l Two kind of seeds were used: Cicer ariet~um L. cv C a ~ d h n a (Ch~k-pea) seeds for the evMuafion of cytokinins and Cucum~ saHvus L. cv Calahor~a ~ucumbe0 seeds for the bioassa~ The ch~k-pea seeds were germinated and grown on a ~ass plate covered with filter papeL in darkness at 25°C with 80% rdafive humidity. The cucumber seeds were germina~d in the same way at 28°C. Extra,ion, purification and evaluation ofcytok~ins Extraction, purification and identification of the different cytokinins was carried out as described by Mufioz et al. [11]. The ex~ac~ duted ~om the Dowex 50W × 8 column were dried and redi~olved in 1 ml of 80% ethanol and the sample spored onto ~hcagel 60 G plates. The plates were developedwithisopropanoFammonia/water(10:l:l), The Chromatograms were later di~ded into 11 band~ Once each band had been duted in 80% ethanol, ill,red and dried, it was used for the bioassay which was performed ufing the cucumber cot~edons. The bands exhibiting cytokinin acridty were used for anMyfis by HPLC. For the HPLC anMyfi~ columns of 290 × 4 mm packed with M~ro -Park (MCH-5) were employed for quantification of cytokining The mobile phase confined of methanoFwater (40:60 v/v to 54:46 v/v), u~ng a linear grad~nt of 12 min. Conditions were as follows: flow ra~, 0.5 ml/min; pH of so~ vent, 5.~ ~mperature, 30°C; pressure, 148 atm; detector beam wave~ngth, 254 nm. The retention times were de~rmined ufing i6Ado and i6Ade ~andards obt~ned ~om Sigma under the condb dons described above, A~ificial restoration of endogenous &ve~ ofi6Ado and ~Ade ~ exceed coty&dons The reproduction of endogenous ~vds of these cytokinins were carried out as described by Mufioz et at. I11]. In order to reproduce the endogenous ~vds of the above -mentioned cytokinin~ the seeds were germinated as usu~. After 6 h of germination the embryon~ a~s was exdsed and substituted by an agar block 0 % agaO into which the tot~ amount of i6Ado extracted ~om the same number ofcot~edons of 12-h imbibed seeds

was injected (di~olved in wate0 with microsyringe. There is ~ways a period of 6 h before the cyto~nins i~ected into the agar have passed into the cotyledons [11]. Endogenous levds of i6Ade were artifidally restored according to the abovementioned m~hod. Endogenous cytokinins were ex~acted after 12, 18, 2~ 36, 48, 72, 96 and 120 h of incubation or germination.

Mobilization of reserves ~ ch~k-pea For this ~udy both the cot~edons collected during germination of intact seeds and the cotyledons from growing seeds wilhout thor axes (with and w~hout arfifid~ reproduction of endogenous ~vds of i6Ado and i6AdO were used. Tot~ carbohydrates were determined by the method described by Dubois at ~. [12]. The hpids were ex~acted according to the method of B~gh and Dyer [13]. Determination of soluble sugars was performed by the Somog~ [14] and Ndson [15] m~hod. Protein extracted after di~olving the refidue obt~ned with 0.05 N NaOH at 37° C for 4 h [1~ was measured by using the method described by Bradford [1 ~. For the ~udy of tot~ am~ase and pro~ase actifitie~ the enzymatic ex~a~s were prepared according to the m~hod described by Metifier and Paulflo 1~. For protease acti~ty both amino add [18] and pepfide [1~ rdease were measured ~ . Resuhs

A~ificial re~orat~n of endogenous &veb of~Ado and ~Ade ~ exceed coty&dons To re,ore the ~ v d of both i6Ado and i6Ade (Fig. l) only a fin~e injection was needed, at 6 h of incubation, of the respective sub~ances pre~oufly extracted. So, after the i~ection, an endogenous ~ v d was obt~ned of i6Ado and i6Ade in exdsed cot~edons fimilar to the level obt~ned in cot~edons of germinated seeds. However, the ~vds of cytokinins were somewhat lower under these ~eatments due to a slight retention of both sub~ances in the agar. Influence of ~Ado and ~Ade on reserve mobH~aHon ~ Cicer ariet~um L. coty&dons The treatmentofdetachedch~k-peacot~edons with i6Ado produces confiderable variations in

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I ~ A T I ~ ~H[ ~ 0 ~ ~ I. A r S ~ ~cproductionof endo~nous ~v~s of i~Ado and i~Ade in ex~scd coW,dons of c h ~ a seed~ (A) Con~ : endo~nous i~Ado ~ norm~ ~eds ( ~ aphelion ~ 6 h of ~cuba~on of the i~Ado ~xtracted~ o m cotyledonsof seeds ~inat~ ~r 12 h (O). (B) Co~r~: endogenous i~Ade ~ norm~ seeds ( ~ injectionat 6 h of incuba~on of the i~Ad~ extracted ~ o m cotyledons of seed~ ~ r m ~ a ~ d ~r 12 h (@).

Fig. 2. Va~ationinlipidcontcnt~cotylcdonof Cic~r a r ~ t ~ L. ~eds. CoW,dons of ~tact seeds ( ~ untreated detached cotyledons (O~ de~chcd c~:edons afterap~ication of i~Ado (~) and a~er appl~a~on of i~Ade (~. Each p ~ sho~s the average of 3 ~plications;e x p e ~ m ~ ~er¢ r e ~ c d 3 ~me~ V e ~ c ~ bars ~ c ~ e S.E.

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lipid contents as may be seen in Fig. 2. Of particular 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, being approximately 74.6% whereas at 120 h it is only 38%. Nevertheless, this cytokinin seems to have little effect on the metabolism of total carbohydrates during g e ~ n a t i o n (Fig. 3), since there is an approximation to the values obtained in norm ~ conditions, recovery ~ so low that at 120 h of

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N~ ~ V a ~ n ~ mtN carbohydra~ co~ents/c~edon of Cicer ar~et~um ~ seed~ CoW,dons of ~tact seeds (~); u~N~m~ntreatdetached ed i~Ado cotyledons (~) and after application detached coW,dens of i~Ade(~. a ~ Ehca a~ pN~ shows the av~a~ of 3 ~N~ati~n~ exNfi~enU we~ repe~ed 3 fime~ VerficN bars ~ c m e S.E.

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~co~don ~ V~°nof~ c e r a°rf ~ uc°ntentsL, m seed~ °f s~UNec~edonsCa~°~dra~o~tact f seeds ~); unheard detached c~edons ~ detached cotyledons~ ~ c a t i o n ~ i ~ (~ and a~er aphelion of i ~ ~ . Each p~m ~ows ~e avera~ of 3 ~ o n ~ ex~fimen~ were ~pe~ed 3 times. Vertic~ ba~ ind~a~ S . E . The ~ud~s concerning am~ase a ~ N i ~ (~g. 5) ag~n c o ~ a recove~ ~ n o ~ ~vds after ap~c~on ~ i~. For i ~ d o , at the momem of ma~mum a ~ 0 6 h ~ ~ c u b ~ o n ) , a recovery

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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 content (Fig. 6) or protease activity measured as peptide 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). When the e ~ c t of i6Ade on nutrient mobilization was studied, an absence of any e ~ c t on the variation ~ ~ d ~v~s in coW,dons ~ i ~ d ~ a c ~ ed axe~ ( H ~ ~ was observed, ~ o u ~ there was 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 ~ t h ~ s ~ c t to tot~ (~g. 3) and to s o ~ e carbohydra~s (~g. ~. T~s recovery became more pronounced towards the end of the period ~u~ed, SO that ~ r ~ t ~ carbohydr~es (~g. 3) it was 43% at 48 h and 62% at 120 h and ~ r s ~ u ~ e carbohydrates ( ~ ~ ~ was 56% at 48 h and 67% at 120 h of ~cubafion. The v~ues of am~ase acfifi~ observed wi~ ~is treatment (~g. 5) show a recove ~ of 52% at the momem of ma~mum a ~ y . U n i t e the resets obt~ned concer~ng carboh~

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