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BLot~idmtcctet Bloph)'sica Ac¢e, l 137 (1992)338-348 © 1992ElsevierScience Publishers B.V. All lighLsreserved Ol6%4889/92/$05.()0
Phytohormonal regulation of S-adenosylmethionine synthetase by gibberellic acid in wheat aleurones Mukul Mathur,
Minati Satpathy
and R.C. Sachar
Oi~het~£gtz?/ ~tld Mole~ttlat B&dok$'Ltdmrrm~D; Depuet~nenrof Botmly, U~drer~i[yt~f DelJli, Delhi f lndia I (Received 6 May ITS2)
Key words: Wheat alcuroae: S-Adenosylmelhionine synthet~-s¢: Regulation of AdnMct synlhetasc: Oibhercllic acid: Enzyme synthesis, de n~rct):Enzyme suhunit, in yitr(~ hyhritlizannn or Gibhcrcl[ic acid (GA~) brought about a 3-fold stimulation of AdoMcl synthcta.sc activity in wheat aleurones. At the qualitative level, Ihrce isozymes of AdoMet s'ymhetase were observed by E)E-52 chromatography in GA~-Irealed wheat aleuroncs. In contrast, the control wheal aleumacs showed a single isozymc. Thus the phytohnrmone IGA.~, 1 g M ) induced lw~l additional isozymc,s of AdoMet ~nthetas¢ in wheat aleurones. The activity of all the three isozymes in GA3-trcatcd aleurones was considerably decreased by the simultaneous presence of ah~isic acid (ABA. I0 ,u MI. Cs,cloheximidc (20 ~,g/ml) also significantly lowered Ihe levels of the three isozymcs of AdoMet synthetase in GA j-treated aleuron~s, thereby suggesting thc requirement of de-nt~o protein synthesis for ;.~[ecomplete induction of isozymes. FI~wever, wheat aleur~mes excised from embworlated wheat sucds, did not re:quire the application of GA~ ['or the induction of two additional isozymes of AdoMet synthctagc. Appart:nfly. the transport of G A 3 from the embryo to aleuroncs induced two new L,ozymcs of AduMcl synthetase. T h e e i,,~ozymes of AdoMel symhela~ were ;~lso obse~ed in wheat embryos cxci~O |ram germinated wheat grains, '~ithnul e'~ogenous application of GA~,. The molecular weight of nit the three isozymcs of AdoMct synihela~ in wheat system is 181000. The molecular weight of the subunil of the enzyme is 84000. The dimeric nature nf AdoMet synlhetase ~,as established by SDS-PAGE analysis of the purified cnz~tme. In-vitro hybridization of two flanking isoz~mi¢ peaks I and III by IMaCI-fre~z~.thaw method resulted in the appearance of an additional middle activity peak (is~zymc Ill. However. no additional isozymie peaks were generated when isozymic peaks 1 and Ill wcrc individually given a freeze-thaw treatment. Thus the flanking isozymic peaks 1 and Ill n:prc.sent il 9modirners that differed in their net charge. In contrast, life middle isozymic activity peak It, wben subjected to NaCI-frecze-t haw truatmcnts yielded twl~ additional isozymic peaks, [ and Ill. thereby suggesting its heterodimerie nature. Wc envisage that the three isozymeg in GA.wtreatcd wheat aleut(me layers ara formed by the random dimerlzation of two classes of enzyme s~hunils. The two enzyme snbunits which differ in their net charge could be the product of two genes of AdoMcl symhctasc (SAM1 and SAM2). Based on this assumption, wc propos~ that a ,single isozymc I in water imbibed control wheat aleuroncs is the product of 5AAII genc of AdoMct symhctasc. The occurrence of three isozymes in GA,-trcated alcurones could be ascribed to the cxpresskm of an alternate gene of AdoMet synthetas~ (SAM2 gear). Thus the raedom dimerization of two ¢tas~es of ~ubunils, derived from SAMI and SAM2 genes, resulted in the appearance of three isoz~mes in hormone-lrcaled aleuroncs.
Introductlen T h e biosynthesis of S-adenosylmethionine from sub-
strates L-methionine and A T P is catalysed by A d o M e t
Correspondence Io: R.C Sachar, Biochcmi:~tq~and Mi)lccula~"Biology Laboratory. Department of Botany. University of Delhi. Delhi I In ¢d07, labia. Abbreviations: AdoMet synthelase, S-adenosytnnethionine synthetase: AIde or SAM. S-adenosylmethlonlne: A'rP, adenosine txiphosphate: ABA, abscisic acid: BSA, bovine serum albumin; CHI, cycloheximide: GA 3, eihbercllic acid; PUG. ~3otyelhylene glYCol: PAGE. polyacrslamidc gel ~lcctruphuresis: PVP, polyvinyl polypyrrolidoae; PPO. 2,5-diphenyl~xazole; POPOP. L4-bis-2-(5phenylosazolyl~benzen¢; gDS, sodium d~Jcc'jl ,~lphal¢.
synthetase (EC. 2.5.1.6) bolh in prokaryotes vnd cukaryotcs [1]. A d o M e t synthetase has been purified and characterized from ,~iacchuromyces cere~'isae [2], Escherictda coil [3], animal cells [4-7] and plant cells [8]. G e n e r a t e clones of A d o M e t synthetase were isolated from E. co(i [9], Saccharomyce~ ceret'isae [10] and A r a bidopsis dtalimta [I 1], while e D N A clones of this enzyme were constructed from rat liver [12|, Arabidopsis thafimta 13], Carnation [14] and human liver [15]. In Yeast, A d o M e t synthetase is encoded by two genes, S A M ! and S A M 2 [10]. Northern blot analysis showed that vOW low transcripts of S A M 2 gene in Yeast strains containing S A M I disrupted gene in a minimal medium were increased after growth in a medium c o n t a i n i n g methioninc. In c~ontrast, transcripts of S A M I gene ill
339 strains with SAM2-disrupted gone were decreased after growth in presence of methionine. Thus the expression of SAMI and SAM2 genes in yeast is segulated differently under the same growth enid/tings [16]. The two forms (~ and B) of AdcJMet synthetase in rat liver were regulated differently in rats maintained on high methlonine diet. Moth/on/no brought about a 4,5-fold stimulation of a form, while the /3 form showed only 1.5-fold increase in the activity of AdoMet symhetnsc [17]. Regulation of AdoMet synthetase activity, by Lmoth/paine is also reported in wheat embryos [g] and dwarf pea epicotyls [18]. In E. colt, conditional mutants of AdoMet synthetase have been raised by in-vitro mutagenesis of MetK gone. These temperature-sensitive mutants showed extremely low levels of S-adenosylmethionine and decreased activity of AdoMet synthetase when grown in minimal medlum at non permissive temperalure (42~C). However, the mutants grew normally at 42°C in Luria broth medium containing yeast extract and showed normal levels or" S-adenosylmethiunine and AdoMel synthetase activity. Further studies revealed that the expression of an alternate gone of AdoMet synthetase (MetX gone) restored the normal levels of AdoMet and AdoMct synthetase activity [19]. Thus, a second MetX gone of AdoMet synthetase was identified that shares antigenic similarity with MetK gone encoded enzyme. Clearly, the MetX gone is expressed under different growth conditions. The existence of two genes of AdoMet synthetase is now well established in E. cot/[19]. The SAM1 gone in yeast is considered to be the counterpart of MetK gone in E. colt [20] AdoMet synthetase i.~ also encoded by two genes in Arabidopsis thai/ann [13]. Regulation of AdoMet synthetase activity by phytohemagglutinin is reported in cultured human Iympbocytes. Immunological studies suggested that this lecdn-mediated stimulation (2.5-fold) of AdoMet synthetase activity, could be ascribed to enzyme activation and probably accounts for the increased levels of cellular AdoMet [21]. More ~ccently, phytohormonal regulation of AdoMet synthetase was reported from our laboratory in dwarf pea eplcotyls. About 2.S-fold stimulation of AdoMet synlhetase was reported in GAj-rreated epicotyls. Abscisic acid or c~cloheximide inhibited the G:~3-mediated increased activity of AdoMet synthetase. Three isozymes of AdoMgt ~ynthetase were observed in GA ~-treated epicotyls in contrast to a ~ingle isozyme in controls. Clearly, GA 3 regulates the induction of two additional isozymes of AdoMet synthetase in pea epicotyls [18]. With a view to determine whether GA~ also plays a crucial role in the regulation of AdoMet synthetase in other GA 3 responsive tissues, we selected wheat aleurune system for the present investigation. Gibberel[ic
acid is known to regulate the activity of several hydrolytic [22] and biosynthetic ¢:nzymes [23] in wheat aleutones. Presently we report the phytohormonal regulation of AdoMet synthetase by (3ibberellic acid (GA~) and Abscisie acid (ABA) in wheat aleut"~ne~. The GA3-mediated stimulation of AdoMct synthctase activity was retarded by ABA in wheat aleurones. At the qualitative level, OA 3 induced two additional /spumes of AdoMct ~ymhetase. Thus the three isoz,ym¢~ of AdoMet synthetase were observed in GA.~-treated aleurones as against a single isozyme in control aleutones. In-vltro molecular hybridization of physically separated isazymes of AdoMet synthetase has clearly revealed that the two flanking iso~mie peaks (I and lID are homodimers, while the middle isozYmie peak (II) is a hetarodimer. The d/merle nature of AdoMet synthetase was established by SDS-PAGE analysis of purified enzyme in wheat system [8]. We suggest that the presence of a single isozyme l in control aleurones is formed by the dimgrization of one class of enzyrae subunit derived from SAMI gone of AdoMet synthetase. The three isozymcs in GA3-trcuted aleurones are formed by the random dimerizatlon of two classes of enzyme suhunits that are possibly the products of SAMI and SAM2 genes of AdoMet synthetase, We postulate ~hat GA.~ controls the expression of an alternate gone (SAM2) of AdoMet synthetase in wheat system. Materials and Methods Rea::ents. ['~H]Methylmethionine (spcc. act, 85 Ci/mmol) was purchased from Amersham, UK. Adenosine triphosphate, polyvinyt polypyrrolidone, polyethylene glycol (molecular weight 20000) and SDS protein markers were products of Sigma. Sepahrose CL-6B w~s l:rodnet of Pharraacia. Molecular-wgightealihratit~., kit of marker proteins was purchased from Boehringer, Germany. Phosphoceilose paper(P-el)and DE-52 were obtained from Whatman, UK. Gibberellic acid (GAa ), ahseisie acid and cycloheximide were produets of Sigma. L-moth/paine was purified by reerystallization before use fo~ the enzyme assay. All other reagents were of analytical grade. Materials. Wheat seeds (ear. HD-2009) were supplied by Indian Agricultural Research Institute, Kar. nal, India. Isolatio~I of wheat aleuroaes. Wheat seeds were d¢¢mbryonated by cui{ing them transversely, The embryo.less half-seeds were surface-sterilized with HgCI2 solution (0.02%, w / v for 10 mln.) and rinsed thoroughly with ~teHle distilled water. The half-seeds were washed (6 h) in sterile distilled water at 25°C (I g haIf-~eds/100 ml of distilled water) and then imbibed (48 h) in presence and absence nfGA~ (1 p.M) in dark at 25°C under asceptie conditions. The embwo-less half-seeds were also imbibed (,18 h) in presence of
340 Abseisic acid (ABA, 10 ~.M), Cycloheximide (CHI, 20 ~ g / m l ) and L-methionine (2 raM). Chloramphenicol (50 # g / m l ) was routinely added as a bactericidal agent. Wheat aleurones were ¢.xeised from de-emhryonated half-:seeds of wheat after different treatmLnts and employed for the preparation of enzyme fractions. 1solution and culture of excised wheat embryos. Wheat embryos were manually excised from dry grains as described earlier [24]. The excised embryos were germinated (48 h) asceptieally in sucrose solution (2%) in the presence and absence of abscisie acid (ABA, 10 /zM) and GA~ (1 ,ttM). Chloramphenicol (50 p g / m l ) was added as a bactericidal agent. Germination of wheat seeds. Wheat seeds were surface sterilized with HgCI~ solution (0.02 %, w / v for 10 rain) and rinsed thoroughly in sterile distilled water. The seeds were germinated in dark at 25°C. The embryonic tissue was clipped manually from two-day-old seedlings, frozen in liquid nitrogen and stored at -70°C. Wheat aleurones were also isolated from embryonated seeds and stored at -7&C. Preparation of enzyme fraction. Isolated wheat aleuroues and excised wheat embryos were homogenized in Tris-HCl buffer (50 raM, pH 7.6; 1:3, w/v) containing #-mercaptoethanol (5 mM), MgCI 2 (10 raM, Na-EDTA (0.1 raM), PVP (2%) and acid-washed sand. Triton X-100 (0.1%)was added to the buffer during homogenization of alearones. The crude homogenate was centrifuged at 1 0 0 0 0 x g for i5 rain. at 4°C. The supernatant fraction was preci0itated with ammonium solphate (0-60e/b saturation) and collected by centrifugation. The pellet fraction was suspended in Tris-HCI buffer (20 raM, pH 7.6) cootaining /~-mercaptoethanol (5 raM), MgC[ z (10 mIvi), Na-EDTA (0.l raM) and glycerol (10%). The enriched ammmlium sulphate fraction precipitate was exhaustively dialysed against the above buffer. The desalted ammonium sulphate fraction precipitate was further fractionated on DE-52 ion-exchange column chromatography for the .separation of isozymas of AdoMet synthetase. Physical sel~ration of isozymes of AdoMet synthetase. The desalted ammonium sulphate fraction precipitate (250 mg protein at a concentration of 5 mg/ml) was applied to DE-52 column (50 ml bed volume), equili. brated with Tris-HCl buffer (50 raM, pH %6) containing j[kmercaptoethano! (5 raM), MgCh~ (10 mM), NaEDTA (0.1 raM} and glycerol (10%). The unbound protein fraction was removed with two bed volumes of equilibrating buffer. A linear gradi~,nt of ammonium sulphate (0-.0.4 M), prepared in equilibrating buffer, was employed for the separation of isl~zymesof AdoMet ~nthetase. 211 fractions (I0 ml each) were collected at a flow rate of 2 min/ml. Each fra:tion was concentrated against PEG (molecular weight 20000) and desalted by exhaustive dialysis. The concentration of prorein in each fraction was moasored by the procedure of
Bradferd [25]. An aliquot of each fraction containing 250 /~g protein was added to the reaction mixture for the a.~say of AdoMet symhetase activity. The enzyme aCliC,ty was expressed as pmol AMe synthesized per ~ng protein. A~rny of AdoMet synthetase. The incubation mixture (125 /tl) contained Tris-HCl buffer (100 raM, pH 7.6), MgClz (20 raM), KCI (70 raM), neutralized glutathione (8 raM), ATP f,lO raM), [3H]metbylmethionine (0.5 i, Ci~, L-mgthioninc (1 mM) and enzyme fraction (250 g g tl~)teiu). The reaction mixture was incubated at 30 ~C for 10 rain. An aliquot (4-0 el) of the reaction mixture was spotted on phosphoccllulose paper (H + form) and dried under infra-red lamp [26]. The filters were washed with chilled phosphate buffer (5 raM, pH 7.0) several times to remove unincorporated [3H]mcthylmethionine. The filter discs were then transferred to scintillation vials. The filters were soaked in ammonium hydroxide solmion (1 ml, 1.5 M) for 5 rain. An aliquot (5 ml each) of scintillation fluid (PPO, 4.0 g; POPOP, 0,05 g; toluene, 667 ml and Triton X-100, 333 ml) was added to each vial for measuring the radioactivity of the reaction product. Determination of molecular weight of AdoMet synthctas¢. The molecular weight of native AdeMet synthetase was determined by subjecting the DE-52 fraction (eluted with 0.05--0.3 M ammonium sulphate) to molecular-sieve chromatography on Sepharose CL-6B (2.24 cm × 50 eml. Fractions (3 ml each) were collected after void volume (V~}ffi75 ml), AdoMet synthetase activity was assayed in each fraction by taking an aliquot containing 100 ttg protein. Sepharose CL-6B column was calibrated with molecular-weight-marker proteins (molecular weigttts: forritin, 440600; eatalase, 240000; aldolasc, 158000; bovine serum albumin, 67000). Snbunit rt~uetuee of AdoMet synthetase. AdoMet synthetase was purified from wheat embryos by the method described by us in Ref. [8]. The subunit structure of purified AdoMct syuthetas¢ was determined by ~abjecting the purified enzyme to SDS-PAGE (7.5%) according to the method of Laemmli [27]. The purified enzyme (5 #.g protein) in Tris-HCI buffer (6fl raM, pH 7.0) was treated with SDS (2%) and p-mercaptocthanol (0.66 M) (overnight at 25"C) end fractionated on SDSPAGE (7.5%) at constant current (20 mA/gel slab). The gel was stained with silver nitrate for developing protein bands according to the method given in Ref. 28. h~-t'itro hybridization of isozymes of AdoMet synthetase. The three isozymes of AdoMet synthctase were physically separated by DE-52 ion.exchange column chromatography. In order to understand the subanit composition of each isozyme, the isolated -isozymes were subjected to in-vitro hybrtdizatic, n in different combinations by NaCI-fraeze-thaw method as de-
34t scribed previously [29]. T h i s treatment brings about complete dissociation followed by random recorabination of the subunits o f isozymes. Initially, the i ~ l a t e d iso~mie fractions were dialyzed against phosphate buffer (0.1 M, p H 7.0) containing #-mereaptocthano[ (5 raM), MgCl~ (10 mM) and N a - E D T A (0.1 mM) for replacing Tris-HCI buffer (20 mM, p H 7.6) and glycerol (10%). T h e isozymes I and Ill were mixed with NaCI (1 M) and subjected to slow freezing treatment at - 20°C. The frozen sample was th¢n thawed rapidly at 25°C and dialyscd against Tris-HCI buffer (20 mM, p H 7.6) containing /~-mercaptoethanol (5 mM), MgCl~ (10 raM), N a - E D T A (0.1 raM) and glycerol 0 0 % ) for removing NaCI. T h e dialysed fraction was refractionated by DE-52 ion-exchange chromatography for determining the isozymie pattern after in-vitro hybridization. Similarly, isozymes I, It and !!I were individually subjeered to the a l i v e treatment t b r studying the isozymic pattern after in-vitro hybridization, T h e controls of these experimental sets were not subjected to NaCIfreeze-thaw treatment, but were refractionated on D E 52 after mixing them in various combinations. T h e hound protein fraction on D E - 5 2 was etuted with a salt gradient ( 0 - 0 . 4 M ammonium sulphate). Each fraction was concentrated against P E G (molecular weight 20000) at 4°C and dialysed against the dialysis buffer to remove salt. An aliquot, containing 2.50 ~zg protein, from each fraction was used for determining the activity o f A d o M e t synthetase. T h e enzyme activity was represented as pmol A M e synthesized p e r mg 9rotein.
,~ ~ o ~O ~ Tt= ~ m 35 ~.~'~ ~ = ~ ": 3C 2~. ~ e "~ ~" ~,. 2 (
"6 ~ w t~
.~_
15
0.1
10 ~~ ~ "~ ~ -6 ~
u
-&
o /~0
80 120 160 200 Elurion volume [ml)
Fig. 1. Regulatory role of GA~ in the induction of t~o isozymes of AdoMet syzithetase in wheat alcttmnes. Embl3~-Iess halFseeds of ~hcat were imbibed in the presence altd absence of GA~ (1 pMh "the DE-52 ion~xchenge chromatogrargy s~owed a slnsl¢ activity peak ot AdoMeI synthetase in control aleurones, whereas the GA3o treated aleurones exhibited three isor/mcs. Application of CH1120 ~g/mll along v~ith GA~ (I saM) to cmh~o-less halt-seeds repressed the a¢ tivity O[ all the three isozymesin isolated wheat aleutoaes~ ( • ) Control tel GA 3, (Q) GA3 +CHL
Results o
Regulatio~t o f .4doMet s~wtherase in wheat aleurones Embryo-less half-~eeds of wheat were imbibed in sterile distilled water (eo,~trol) and also in a solution of G A 3 (1 /aM) for 4~, h. A d o M e t synthetase activity was measured in DE-52 fraction p r e p a r e d from isolated wheat aleurones. A significant stimulation (3-fold) of A d o M e t synthetase activity was observed in G A 3treated ateurones. This stimulatory response of O A a
was nugliiicd by the simultaneous application of A B A (10 #.M) or CHI (20 ~ g / m l ) (Table 1), A t the qualitative level, the control alearones showed a single activity peak of A d o M e t synlhetasc, whereas three i~:oz~aes were observed in" ~ A 3 - t r e a t e d alearones (trig. I). Clearly, the phytohormone induced two addiEonal isoz'ymas of A d o M e t synthetasc in wheat aleurones. T h e GA3-evoked actlviW peaks of two isozyn':es o f
TABLE I Regldutian of AdoMet ~nrheta.~e actil'(ty by Bib~vrclfic acid CGA.)), t.-methfocliel¢ and absci~ic acid in W]lCal~l~¢roHes Embwo-less half-seeds of wheat were imbibed in GA 3 tl/xM). GA 3 (l /~M)+ CHI (20 #g/ml), GA 3 (I gM)+ABA (10 ~M) and t+-melhionirie (2 raM) for 4g h. Aleurones 'were manually isolated after these treatlmcnts and frozen in liquid nitrogen. The DE-S2 fraction (0.12-0.3 M) was prepared fmra =xci~edwheat aleumnes for the assay of AdoMet synlhetase activity. Trcalment
Control
Adomet synthetas¢ activity (rmol of SAM syath¢siz¢d/ nab prot¢inl
RelaLh,e a¢liviW
556
t.00
(3A:.tl ~¢M)
1734
3.10
GAj (I gtM)+ CHI tea/zg/ml) GA~ (I/~M)+ ABA ( tO gM) L-Methionine (2 raM)
71g 4hi 1332
2,40
1,29 0.83
342
~', ~o
3c
wheat aleurones , e x e i ~ d from water imbibed cmblyonated wheat seeds, is regulated by the internal pool of GA1 which is transported from the embryo to the aleurnue layers. Thus thc induction o f two additional i s o ~ m e s o f A d o M e t syntheta~se in wheat aleuronos by the exogenous application of G A 3 truly represents a physiological system present in nature.
g~ e'~
2:
Regulation of AdoMel .~ynttherase in wheat embryus
"g~e' ~
z~
~
~-
0.~
&O
80 120 150 200 Elution volume (ml} Fig. 2. Regulation orAdoMet synthetase isoz~mesby GA 3 and ABA
ia wheat aleuron~. Erobryo-less half-~eds of wheat were imbibed in f~lullOrlof GA 3 (l /xM) and GA 3 (l p.M}+ ABA (IO FM) for 4S h. Ammonium sulphate fraction plx~cipitatt: (0-~'~% saturation} was prepared from excised wheal aleurones and was desalled by exhauslive dialysis. This en.-,y~e preparation Was fraellonated By L)E-52 column chromatography for the separation of isozymes of AdnMet syntheta-se. Three is0mymic peaks of AdoMet ~ynthetase were observed in GAa-treatcd a!eulones. Al~¢isic acid repressed the activity Of all the thlee isozymc~ uf AdoMcl synthclas~ in GA~-trcaled alearoaes. (o) GA~. (0) OA~ +ABA.
Ammonium sulphate fraction precipitate ( 0 - 6 0 % saturation) from germinated wheat embryos (48 h) was fractionated on DE-52 ion-exchange chromatography for determining the isoz'ymic pattern of A d o M e t synthetas¢. T h r e e iso~mic activity peaks o f AdoMgt synthetase were observed in excised germinated wheat e m b e d s without eJtogenou5 application of GA~ (Fig. 4). Furthermore, G A 3 failed to alter the isozvmie oatturn of A d 0 M e t synthetase in wheat embryos. It is quite likely that the induction of two additional isozymes of A d o M e t synthetase in wheat embryos is regulated by the endogenous high levels o f G A 3. This was in contrast to wheat aleurones, excised Dora deembryonated half-seeds where exogenous addition of GA_~ was mandatory for the induction of two addi-
~o A d o M e t synthetase were virtually abolished by A B A (10 ~ M ) (Fig. 2). Cyclohexlmlde (20 ~ g / m ] ) re,pressed the activity of all the three isozymes of A d o M e t synthetase in G A s - t r e a t e d aleurones (Fig. D. T h u s it appears that de-nova protein synthesis is necessary fur complete induction of GA~-regulated A d o M e t synthetase isozymes. Both C H i (20 g g / m l ) and A B A (10 ~ M ) were also effective in inhibiting ( 5 0 - 0 0 % ) the single activity peak of A d o M e t s~nthetase in control wheat aleurones (data not presented). The true significance of the phytohormonal regulation of A d o M e t synthetase became explicit when we eampared the isozymie pattern in aleurones excised from embryonated and de-embryonated seeds of when L Although a singte isozyme was observed in aleurones excised from de-embryonated half-seeds, there were three isozymes in aleurones dissected from emblyonated seeds (Fig. 3). Thus the pattern o f isozymes in aleurones dissected from emhryonated seeds was identical to that observed in GA~-treated alcurones dissect,~d front the de-embryonated wheat half-seeds. Apparently, the induction of two additional isozymes in
/
l
"d-
zs
30
15
'~ "-~ ~
10 !
-
o.t,
} A1
0
Eluti0n v0tume (mi). Fig. 3. comparison of bo~mlc panern of AdnMet synthelase from alcurones isolated tram germinate0 ¢mbl~onaled s~2ds and dc-cmbryonated half-seeds of wheal. Aleuroncs excised tidal water imbibed de-embrvonalcd hall-seeds of wheal showed a stogie JsDx~llle of AdoMet syrahetase by DE-52 ion-exchange chrornatngr,iph]~, In contrast, aleurones exci~ed from water imbued embr~mul~d s~cds of wheat ~howed t h ~ isozyrrles of AdoMel wrahetas¢. (o) A]eutone5 excised from de-cmbr~tmatcd scuds; ( o ) ateumnes exci~ed from ¢lBbl~olmlcdm:t:ds.
343
Molecular welghl and subunh structure ,of AdoMet synthetase Batch elated DE-$2 fraction (0.05-0.3 M ame,:-> nium sulphate) containing three iso~mes of AdoMet synthetase was p r e p a r e d f r o m w h e a t embryos a n d subj e c t e d to molecular sieve chromatography on Sepharose
~'_.
,= ='zc
02
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'~.
so 120 1so Elution votume(
2UO mt )
CL-6B. A single molecular form was observed after gel permeation with a molecular weight of l g l 0 0 0 (Fig. 6A, B). Molecular-sieve chromatography of physically separated individual isozymcs alsO showe,d a single activity peak on Scpharosc CL-6B that corresponded to the molecular weight of 1810(KLThis suggested that the three isozymes of AdeMot symhetase have an identical molecular weight, lsozyme II wa~ purified to clectrophoretic homogeneity_ as previousl~ described [8]. Analysis of the purified AdoMeI aynt'.mtase on SDS-PAGE showed a single silver-stained pruiein band. The molecular weif, ht of the subunit is 84000 as determined from the relative mobility of standard protein markers (Fig. 6C). Thus wheat AdoMet syutheta~ is dimeric in nature.
o
Fig. 4. Negativercgulalionof AdoMe! synthetm~¢ by abscisicacid in gemtinated e~¢ised wheat emb~os. Esciscd wh~al gmbryo~ were Senilinalcdin the presence anti absence of ABA (10 ~.Mk Fraeti,nalien of d~aly~d ammonium sulphate fraction precipitate (O-6Oe~ satut'atlon) on DE-gZ revealed three isogymic poohs of AdoM¢I synthetas¢ in control crab,s. Administratior~of ABA (1O #M) to wheat emblg'Osduring germinationsubstantially inhibited the ~.clivity of two iso,~mes uf AdoMe! synthetase. (o ) Control,t~) ABA.
m ,°4o
g=
~
3o
/
//
/
/
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with ABA (10 a M ) was quite effective in lowering the levels of two i~zYmes of AdoMet synthetas~ (Fig. 4). Earlier, we have shown that CHI (20 # g / m l ) completely abolished the activity of three isozymes of wheat embryos, thereby suggesting the requirement of denero prolein synthesis for the induction of AdoMet synthetase [8]. Conclusive proof for the de-nero synthesis of AdoMet s~nthetase in wheat embryos was also shown by labelling the enzyme in vivo with [~804] 2and eventually recovering the 3aS label in methionine residue of the ~gS-labclled purified AdoMet synthetase
g >~.~ Q
01 ~ .
18]. 120
Regulation of AdoMet synthetase by t..methionine Imbibition of d¢-cmbtyonated wheat half-seeds in solution of L-methionine (2 m M ) stimulated ( 2 . 4 f f o l d ) AdoMet synthetase activity in wheat aleurones (Table 1). Fraetionation of ammonium sulphate fraction precipitate on D E - 5 2 s h o w e d three i ~ z y m e s in methionine-treated aleurones (Fig. 5). Thus the induction of two additional isozym~:s by t.-methionine was comparable to that observed in GA3-treated wheat aleurones.
flution
volume
I~0
2'00
(m:)
Fig, 5, |rldurtio~ of is~lRrs of AduM~:t ~ynthctas¢ by i.-m©tMor(in¢ in ~vhe;nale~ ~he!;. E~llh,~/o-le~.half+seedsof wheal were imbibed in Ihe ,d~cnce and presence of L-metbionine (2 raM) for 48 h. Al~a. tones wcze mc-n~all~ excL~edf~om ¢mbr,o-less half seeds. The de,salted ammonium sulphate fraction precipitate, prepared from ts'hcat 'aleumnes, wa~ subjected ru DE-52 rhroraatogr~phy with a linear salt gra.dielU. ;~ single isozynx~ o; ..'~dtJMet syLithrtase was observed in comrol alruron¢,~, t:Metbionin¢ Ireatmenl of ~lletlmn¢~ indug~J two additional isozymes of AdoMet synlhetas¢. (~.) Control, (e) 1.methionine.
3,14
In-vitro hybridization of subunits of AdoMet synthetase With a view to determine the number of A d o M e t s~nthetase genes that contribute to the indttetion of three isozymes ill GA3-treated aleurones and wheat embryos, we resorted to in-vitro hybridization o f physica:ly separated isozymes in different combinations. Characterization of the purified A d o M e t synthetase in wheat system indicated that all the three isoz-./mes ~re dimerie with identical molecular weight but differ in their net charge. W e considered the possibiliW of the cMstcnCc o f two discrete classes o f enzyme subunits
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|
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obtained by molecular sieving. (B) Calibration cui'v¢ of standard protein markers. The standard protein markers of known molecular weight were used for calibrating the S¢-phatose CL-CiBcolumn. The logla molecular weight of each protein marker was plotted as a funelion of ils g~. value. The molecular weight of native AgoMet s3,nthctase (tat000)was calculated from its K,~ value. (C) Subunit strucltur¢ of purified AdoMet syarhctasc in wheat embryos. The purified preparation of AdoMet synthetase (5 Jag protein) from germinated excised wheat embryos was subjected to YD,-PAGE (7.5%). Lane a: Silver-stained protein band of subunit of AdoMet synthet~e; lane b: SDS protein markers of known molecular weight. stained with silver nitrate.
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derived from two genes of A d o M e t synthetase that could generate three isozymes by random dimerization o f the snbunits. T h i s hypothesis was tested by the in-vitro hybridization of subunits o f physically separated isoxymes of A d o M e t synthetase. The two flanking activity peaks (isozymes I and I l l ) of A d o M e t synthetase were isolated by D E - 5 2 chromatography and then subjected to NaCl-freese-thaw treatment. This treatment dissociates the dimerie isozymes and then permits the random association o f enzyme subunits into directs. T h e in-vitro-hybridized isozyates were analysed by D E - 5 2 chromatography. T h e untreated control samples ,showed the original two activity peaks comparable to isozymes I and i11. T h e treated samples, however, showed an additional middle activity peak in
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BLot~idmtcctet Bloph)'sica Ac¢e, l 137 (1992)338-348 © 1992ElsevierScience Publishers B.V. All lighLsreserved Ol6%4889/92/$05.()0
Phytohormonal regulation of S-adenosylmethionine synthetase by gibberellic acid in wheat aleurones Mukul Mathur,
Minati Satpathy
and R.C. Sachar
Oi~het~£gtz?/ ~tld Mole~ttlat B&dok$'Ltdmrrm~D; Depuet~nenrof Botmly, U~drer~i[yt~f DelJli, Delhi f lndia I (Received 6 May ITS2)
Key words: Wheat alcuroae: S-Adenosylmelhionine synthet~-s¢: Regulation of AdnMct synlhetasc: Oibhercllic acid: Enzyme synthesis, de n~rct):Enzyme suhunit, in yitr(~ hyhritlizannn or Gibhcrcl[ic acid (GA~) brought about a 3-fold stimulation of AdoMcl synthcta.sc activity in wheat aleurones. At the qualitative level, Ihrce isozymes of AdoMet s'ymhetase were observed by E)E-52 chromatography in GA~-Irealed wheat aleuroncs. In contrast, the control wheal aleumacs showed a single isozymc. Thus the phytohnrmone IGA.~, 1 g M ) induced lw~l additional isozymc,s of AdoMet ~nthetas¢ in wheat aleurones. The activity of all the three isozymes in GA3-trcatcd aleurones was considerably decreased by the simultaneous presence of ah~isic acid (ABA. I0 ,u MI. Cs,cloheximidc (20 ~,g/ml) also significantly lowered Ihe levels of the three isozymcs of AdoMet synthetase in GA j-treated aleuron~s, thereby suggesting thc requirement of de-nt~o protein synthesis for ;.~[ecomplete induction of isozymes. FI~wever, wheat aleur~mes excised from embworlated wheat sucds, did not re:quire the application of GA~ ['or the induction of two additional isozymes of AdoMet synthctagc. Appart:nfly. the transport of G A 3 from the embryo to aleuroncs induced two new L,ozymcs of AduMcl synthetase. T h e e i,,~ozymes of AdoMel symhela~ were ;~lso obse~ed in wheat embryos cxci~O |ram germinated wheat grains, '~ithnul e'~ogenous application of GA~,. The molecular weight of nit the three isozymcs of AdoMct synihela~ in wheat system is 181000. The molecular weight of the subunil of the enzyme is 84000. The dimeric nature nf AdoMet synlhetase ~,as established by SDS-PAGE analysis of the purified cnz~tme. In-vitro hybridization of two flanking isoz~mi¢ peaks I and III by IMaCI-fre~z~.thaw method resulted in the appearance of an additional middle activity peak (is~zymc Ill. However. no additional isozymie peaks were generated when isozymic peaks 1 and Ill wcrc individually given a freeze-thaw treatment. Thus the flanking isozymic peaks 1 and Ill n:prc.sent il 9modirners that differed in their net charge. In contrast, life middle isozymic activity peak It, wben subjected to NaCI-frecze-t haw truatmcnts yielded twl~ additional isozymic peaks, [ and Ill. thereby suggesting its heterodimerie nature. Wc envisage that the three isozymeg in GA.wtreatcd wheat aleut(me layers ara formed by the random dimerlzation of two classes of enzyme s~hunils. The two enzyme snbunits which differ in their net charge could be the product of two genes of AdoMcl symhctasc (SAM1 and SAM2). Based on this assumption, wc propos~ that a ,single isozymc I in water imbibed control wheat aleuroncs is the product of 5AAII genc of AdoMct symhctasc. The occurrence of three isozymes in GA,-trcated alcurones could be ascribed to the cxpresskm of an alternate gene of AdoMet synthetas~ (SAM2 gear). Thus the raedom dimerization of two ¢tas~es of ~ubunils, derived from SAMI and SAM2 genes, resulted in the appearance of three isoz~mes in hormone-lrcaled aleuroncs.
Introductlen T h e biosynthesis of S-adenosylmethionine from sub-
strates L-methionine and A T P is catalysed by A d o M e t
Correspondence Io: R.C Sachar, Biochcmi:~tq~and Mi)lccula~"Biology Laboratory. Department of Botany. University of Delhi. Delhi I In ¢d07, labia. Abbreviations: AdoMet synthelase, S-adenosytnnethionine synthetase: AIde or SAM. S-adenosylmethlonlne: A'rP, adenosine txiphosphate: ABA, abscisic acid: BSA, bovine serum albumin; CHI, cycloheximide: GA 3, eihbercllic acid; PUG. ~3otyelhylene glYCol: PAGE. polyacrslamidc gel ~lcctruphuresis: PVP, polyvinyl polypyrrolidoae; PPO. 2,5-diphenyl~xazole; POPOP. L4-bis-2-(5phenylosazolyl~benzen¢; gDS, sodium d~Jcc'jl ,~lphal¢.
synthetase (EC. 2.5.1.6) bolh in prokaryotes vnd cukaryotcs [1]. A d o M e t synthetase has been purified and characterized from ,~iacchuromyces cere~'isae [2], Escherictda coil [3], animal cells [4-7] and plant cells [8]. G e n e r a t e clones of A d o M e t synthetase were isolated from E. co(i [9], Saccharomyce~ ceret'isae [10] and A r a bidopsis dtalimta [I 1], while e D N A clones of this enzyme were constructed from rat liver [12|, Arabidopsis thafimta 13], Carnation [14] and human liver [15]. In Yeast, A d o M e t synthetase is encoded by two genes, S A M ! and S A M 2 [10]. Northern blot analysis showed that vOW low transcripts of S A M 2 gene in Yeast strains containing S A M I disrupted gene in a minimal medium were increased after growth in a medium c o n t a i n i n g methioninc. In c~ontrast, transcripts of S A M I gene ill
339 strains with SAM2-disrupted gone were decreased after growth in presence of methionine. Thus the expression of SAMI and SAM2 genes in yeast is segulated differently under the same growth enid/tings [16]. The two forms (~ and B) of AdcJMet synthetase in rat liver were regulated differently in rats maintained on high methlonine diet. Moth/on/no brought about a 4,5-fold stimulation of a form, while the /3 form showed only 1.5-fold increase in the activity of AdoMet symhetnsc [17]. Regulation of AdoMet synthetase activity, by Lmoth/paine is also reported in wheat embryos [g] and dwarf pea epicotyls [18]. In E. colt, conditional mutants of AdoMet synthetase have been raised by in-vitro mutagenesis of MetK gone. These temperature-sensitive mutants showed extremely low levels of S-adenosylmethionine and decreased activity of AdoMet synthetase when grown in minimal medlum at non permissive temperalure (42~C). However, the mutants grew normally at 42°C in Luria broth medium containing yeast extract and showed normal levels or" S-adenosylmethiunine and AdoMel synthetase activity. Further studies revealed that the expression of an alternate gone of AdoMet synthetase (MetX gone) restored the normal levels of AdoMet and AdoMct synthetase activity [19]. Thus, a second MetX gone of AdoMet synthetase was identified that shares antigenic similarity with MetK gone encoded enzyme. Clearly, the MetX gone is expressed under different growth conditions. The existence of two genes of AdoMet synthetase is now well established in E. cot/[19]. The SAM1 gone in yeast is considered to be the counterpart of MetK gone in E. colt [20] AdoMet synthetase i.~ also encoded by two genes in Arabidopsis thai/ann [13]. Regulation of AdoMet synthetase activity by phytohemagglutinin is reported in cultured human Iympbocytes. Immunological studies suggested that this lecdn-mediated stimulation (2.5-fold) of AdoMet synthetase activity, could be ascribed to enzyme activation and probably accounts for the increased levels of cellular AdoMet [21]. More ~ccently, phytohormonal regulation of AdoMet synthetase was reported from our laboratory in dwarf pea eplcotyls. About 2.S-fold stimulation of AdoMet synlhetase was reported in GAj-rreated epicotyls. Abscisic acid or c~cloheximide inhibited the G:~3-mediated increased activity of AdoMet synthetase. Three isozymes of AdoMgt ~ynthetase were observed in GA ~-treated epicotyls in contrast to a ~ingle isozyme in controls. Clearly, GA 3 regulates the induction of two additional isozymes of AdoMet synthetase in pea epicotyls [18]. With a view to determine whether GA~ also plays a crucial role in the regulation of AdoMet synthetase in other GA 3 responsive tissues, we selected wheat aleurune system for the present investigation. Gibberel[ic
acid is known to regulate the activity of several hydrolytic [22] and biosynthetic ¢:nzymes [23] in wheat aleutones. Presently we report the phytohormonal regulation of AdoMet synthetase by (3ibberellic acid (GA~) and Abscisie acid (ABA) in wheat aleut"~ne~. The GA3-mediated stimulation of AdoMct synthctase activity was retarded by ABA in wheat aleurones. At the qualitative level, OA 3 induced two additional /spumes of AdoMct ~ymhetase. Thus the three isoz,ym¢~ of AdoMet synthetase were observed in GA.~-treated aleurones as against a single isozyme in control aleutones. In-vltro molecular hybridization of physically separated isazymes of AdoMet synthetase has clearly revealed that the two flanking iso~mie peaks (I and lID are homodimers, while the middle isozYmie peak (II) is a hetarodimer. The d/merle nature of AdoMet synthetase was established by SDS-PAGE analysis of purified enzyme in wheat system [8]. We suggest that the presence of a single isozyme l in control aleurones is formed by the dimgrization of one class of enzyrae subunit derived from SAMI gone of AdoMet synthetase. The three isozymcs in GA3-trcuted aleurones are formed by the random dimerizatlon of two classes of enzyme suhunits that are possibly the products of SAMI and SAM2 genes of AdoMet synthetase, We postulate ~hat GA.~ controls the expression of an alternate gone (SAM2) of AdoMet synthetase in wheat system. Materials and Methods Rea::ents. ['~H]Methylmethionine (spcc. act, 85 Ci/mmol) was purchased from Amersham, UK. Adenosine triphosphate, polyvinyt polypyrrolidone, polyethylene glycol (molecular weight 20000) and SDS protein markers were products of Sigma. Sepahrose CL-6B w~s l:rodnet of Pharraacia. Molecular-wgightealihratit~., kit of marker proteins was purchased from Boehringer, Germany. Phosphoceilose paper(P-el)and DE-52 were obtained from Whatman, UK. Gibberellic acid (GAa ), ahseisie acid and cycloheximide were produets of Sigma. L-moth/paine was purified by reerystallization before use fo~ the enzyme assay. All other reagents were of analytical grade. Materials. Wheat seeds (ear. HD-2009) were supplied by Indian Agricultural Research Institute, Kar. nal, India. Isolatio~I of wheat aleuroaes. Wheat seeds were d¢¢mbryonated by cui{ing them transversely, The embryo.less half-seeds were surface-sterilized with HgCI2 solution (0.02%, w / v for 10 mln.) and rinsed thoroughly with ~teHle distilled water. The half-seeds were washed (6 h) in sterile distilled water at 25°C (I g haIf-~eds/100 ml of distilled water) and then imbibed (48 h) in presence and absence nfGA~ (1 p.M) in dark at 25°C under asceptie conditions. The embwo-less half-seeds were also imbibed (,18 h) in presence of
340 Abseisic acid (ABA, 10 ~.M), Cycloheximide (CHI, 20 ~ g / m l ) and L-methionine (2 raM). Chloramphenicol (50 # g / m l ) was routinely added as a bactericidal agent. Wheat aleurones were ¢.xeised from de-emhryonated half-:seeds of wheat after different treatmLnts and employed for the preparation of enzyme fractions. 1solution and culture of excised wheat embryos. Wheat embryos were manually excised from dry grains as described earlier [24]. The excised embryos were germinated (48 h) asceptieally in sucrose solution (2%) in the presence and absence of abscisie acid (ABA, 10 /zM) and GA~ (1 ,ttM). Chloramphenicol (50 p g / m l ) was added as a bactericidal agent. Germination of wheat seeds. Wheat seeds were surface sterilized with HgCI~ solution (0.02 %, w / v for 10 rain) and rinsed thoroughly in sterile distilled water. The seeds were germinated in dark at 25°C. The embryonic tissue was clipped manually from two-day-old seedlings, frozen in liquid nitrogen and stored at -70°C. Wheat aleurones were also isolated from embryonated seeds and stored at -7&C. Preparation of enzyme fraction. Isolated wheat aleuroues and excised wheat embryos were homogenized in Tris-HCl buffer (50 raM, pH 7.6; 1:3, w/v) containing #-mercaptoethanol (5 mM), MgCI 2 (10 raM, Na-EDTA (0.1 raM), PVP (2%) and acid-washed sand. Triton X-100 (0.1%)was added to the buffer during homogenization of alearones. The crude homogenate was centrifuged at 1 0 0 0 0 x g for i5 rain. at 4°C. The supernatant fraction was preci0itated with ammonium solphate (0-60e/b saturation) and collected by centrifugation. The pellet fraction was suspended in Tris-HCI buffer (20 raM, pH 7.6) cootaining /~-mercaptoethanol (5 raM), MgC[ z (10 mIvi), Na-EDTA (0.l raM) and glycerol (10%). The enriched ammmlium sulphate fraction precipitate was exhaustively dialysed against the above buffer. The desalted ammonium sulphate fraction precipitate was further fractionated on DE-52 ion-exchange column chromatography for the .separation of isozymas of AdoMet synthetase. Physical sel~ration of isozymes of AdoMet synthetase. The desalted ammonium sulphate fraction precipitate (250 mg protein at a concentration of 5 mg/ml) was applied to DE-52 column (50 ml bed volume), equili. brated with Tris-HCl buffer (50 raM, pH %6) containing j[kmercaptoethano! (5 raM), MgCh~ (10 mM), NaEDTA (0.1 raM} and glycerol (10%). The unbound protein fraction was removed with two bed volumes of equilibrating buffer. A linear gradi~,nt of ammonium sulphate (0-.0.4 M), prepared in equilibrating buffer, was employed for the separation of isl~zymesof AdoMet ~nthetase. 211 fractions (I0 ml each) were collected at a flow rate of 2 min/ml. Each fra:tion was concentrated against PEG (molecular weight 20000) and desalted by exhaustive dialysis. The concentration of prorein in each fraction was moasored by the procedure of
Bradferd [25]. An aliquot of each fraction containing 250 /~g protein was added to the reaction mixture for the a.~say of AdoMet symhetase activity. The enzyme aCliC,ty was expressed as pmol AMe synthesized per ~ng protein. A~rny of AdoMet synthetase. The incubation mixture (125 /tl) contained Tris-HCl buffer (100 raM, pH 7.6), MgClz (20 raM), KCI (70 raM), neutralized glutathione (8 raM), ATP f,lO raM), [3H]metbylmethionine (0.5 i, Ci~, L-mgthioninc (1 mM) and enzyme fraction (250 g g tl~)teiu). The reaction mixture was incubated at 30 ~C for 10 rain. An aliquot (4-0 el) of the reaction mixture was spotted on phosphoccllulose paper (H + form) and dried under infra-red lamp [26]. The filters were washed with chilled phosphate buffer (5 raM, pH 7.0) several times to remove unincorporated [3H]mcthylmethionine. The filter discs were then transferred to scintillation vials. The filters were soaked in ammonium hydroxide solmion (1 ml, 1.5 M) for 5 rain. An aliquot (5 ml each) of scintillation fluid (PPO, 4.0 g; POPOP, 0,05 g; toluene, 667 ml and Triton X-100, 333 ml) was added to each vial for measuring the radioactivity of the reaction product. Determination of molecular weight of AdoMet synthctas¢. The molecular weight of native AdeMet synthetase was determined by subjecting the DE-52 fraction (eluted with 0.05--0.3 M ammonium sulphate) to molecular-sieve chromatography on Sepharose CL-6B (2.24 cm × 50 eml. Fractions (3 ml each) were collected after void volume (V~}ffi75 ml), AdoMet synthetase activity was assayed in each fraction by taking an aliquot containing 100 ttg protein. Sepharose CL-6B column was calibrated with molecular-weight-marker proteins (molecular weigttts: forritin, 440600; eatalase, 240000; aldolasc, 158000; bovine serum albumin, 67000). Snbunit rt~uetuee of AdoMet synthetase. AdoMet synthetase was purified from wheat embryos by the method described by us in Ref. [8]. The subunit structure of purified AdoMct syuthetas¢ was determined by ~abjecting the purified enzyme to SDS-PAGE (7.5%) according to the method of Laemmli [27]. The purified enzyme (5 #.g protein) in Tris-HCI buffer (6fl raM, pH 7.0) was treated with SDS (2%) and p-mercaptocthanol (0.66 M) (overnight at 25"C) end fractionated on SDSPAGE (7.5%) at constant current (20 mA/gel slab). The gel was stained with silver nitrate for developing protein bands according to the method given in Ref. 28. h~-t'itro hybridization of isozymes of AdoMet synthetase. The three isozymes of AdoMet synthctase were physically separated by DE-52 ion.exchange column chromatography. In order to understand the subanit composition of each isozyme, the isolated -isozymes were subjected to in-vitro hybrtdizatic, n in different combinations by NaCI-fraeze-thaw method as de-
34t scribed previously [29]. T h i s treatment brings about complete dissociation followed by random recorabination of the subunits o f isozymes. Initially, the i ~ l a t e d iso~mie fractions were dialyzed against phosphate buffer (0.1 M, p H 7.0) containing #-mereaptocthano[ (5 raM), MgCl~ (10 mM) and N a - E D T A (0.1 mM) for replacing Tris-HCI buffer (20 mM, p H 7.6) and glycerol (10%). T h e isozymes I and Ill were mixed with NaCI (1 M) and subjected to slow freezing treatment at - 20°C. The frozen sample was th¢n thawed rapidly at 25°C and dialyscd against Tris-HCI buffer (20 mM, p H 7.6) containing /~-mercaptoethanol (5 mM), MgCl~ (10 raM), N a - E D T A (0.1 raM) and glycerol 0 0 % ) for removing NaCI. T h e dialysed fraction was refractionated by DE-52 ion-exchange chromatography for determining the isozymie pattern after in-vitro hybridization. Similarly, isozymes I, It and !!I were individually subjeered to the a l i v e treatment t b r studying the isozymic pattern after in-vitro hybridization, T h e controls of these experimental sets were not subjected to NaCIfreeze-thaw treatment, but were refractionated on D E 52 after mixing them in various combinations. T h e hound protein fraction on D E - 5 2 was etuted with a salt gradient ( 0 - 0 . 4 M ammonium sulphate). Each fraction was concentrated against P E G (molecular weight 20000) at 4°C and dialysed against the dialysis buffer to remove salt. An aliquot, containing 2.50 ~zg protein, from each fraction was used for determining the activity o f A d o M e t synthetase. T h e enzyme activity was represented as pmol A M e synthesized p e r mg 9rotein.
,~ ~ o ~O ~ Tt= ~ m 35 ~.~'~ ~ = ~ ": 3C 2~. ~ e "~ ~" ~,. 2 (
"6 ~ w t~
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80 120 160 200 Elurion volume [ml)
Fig. 1. Regulatory role of GA~ in the induction of t~o isozymes of AdoMet syzithetase in wheat alcttmnes. Embl3~-Iess halFseeds of ~hcat were imbibed in the presence altd absence of GA~ (1 pMh "the DE-52 ion~xchenge chromatogrargy s~owed a slnsl¢ activity peak ot AdoMeI synthetase in control aleurones, whereas the GA3o treated aleurones exhibited three isor/mcs. Application of CH1120 ~g/mll along v~ith GA~ (I saM) to cmh~o-less halt-seeds repressed the a¢ tivity O[ all the three isozymesin isolated wheat aleutoaes~ ( • ) Control tel GA 3, (Q) GA3 +CHL
Results o
Regulatio~t o f .4doMet s~wtherase in wheat aleurones Embryo-less half-~eeds of wheat were imbibed in sterile distilled water (eo,~trol) and also in a solution of G A 3 (1 /aM) for 4~, h. A d o M e t synthetase activity was measured in DE-52 fraction p r e p a r e d from isolated wheat aleurones. A significant stimulation (3-fold) of A d o M e t synthetase activity was observed in G A 3treated ateurones. This stimulatory response of O A a
was nugliiicd by the simultaneous application of A B A (10 #.M) or CHI (20 ~ g / m l ) (Table 1), A t the qualitative level, the control alearones showed a single activity peak of A d o M e t synlhetasc, whereas three i~:oz~aes were observed in" ~ A 3 - t r e a t e d alearones (trig. I). Clearly, the phytohormone induced two addiEonal isoz'ymas of A d o M e t synthetasc in wheat aleurones. T h e GA3-evoked actlviW peaks of two isozyn':es o f
TABLE I Regldutian of AdoMet ~nrheta.~e actil'(ty by Bib~vrclfic acid CGA.)), t.-methfocliel¢ and absci~ic acid in W]lCal~l~¢roHes Embwo-less half-seeds of wheat were imbibed in GA 3 tl/xM). GA 3 (l /~M)+ CHI (20 #g/ml), GA 3 (I gM)+ABA (10 ~M) and t+-melhionirie (2 raM) for 4g h. Aleurones 'were manually isolated after these treatlmcnts and frozen in liquid nitrogen. The DE-S2 fraction (0.12-0.3 M) was prepared fmra =xci~edwheat aleumnes for the assay of AdoMet synlhetase activity. Trcalment
Control
Adomet synthetas¢ activity (rmol of SAM syath¢siz¢d/ nab prot¢inl
RelaLh,e a¢liviW
556
t.00
(3A:.tl ~¢M)
1734
3.10
GAj (I gtM)+ CHI tea/zg/ml) GA~ (I/~M)+ ABA ( tO gM) L-Methionine (2 raM)
71g 4hi 1332
2,40
1,29 0.83
342
~', ~o
3c
wheat aleurones , e x e i ~ d from water imbibed cmblyonated wheat seeds, is regulated by the internal pool of GA1 which is transported from the embryo to the aleurnue layers. Thus thc induction o f two additional i s o ~ m e s o f A d o M e t syntheta~se in wheat aleuronos by the exogenous application of G A 3 truly represents a physiological system present in nature.
g~ e'~
2:
Regulation of AdoMel .~ynttherase in wheat embryus
"g~e' ~
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80 120 150 200 Elution volume (ml} Fig. 2. Regulation orAdoMet synthetase isoz~mesby GA 3 and ABA
ia wheat aleuron~. Erobryo-less half-~eds of wheat were imbibed in f~lullOrlof GA 3 (l /xM) and GA 3 (l p.M}+ ABA (IO FM) for 4S h. Ammonium sulphate fraction plx~cipitatt: (0-~'~% saturation} was prepared from excised wheal aleurones and was desalled by exhauslive dialysis. This en.-,y~e preparation Was fraellonated By L)E-52 column chromatography for the separation of isozymes of AdnMet syntheta-se. Three is0mymic peaks of AdoMet ~ynthetase were observed in GAa-treatcd a!eulones. Al~¢isic acid repressed the activity Of all the thlee isozymc~ uf AdoMcl synthclas~ in GA~-trcaled alearoaes. (o) GA~. (0) OA~ +ABA.
Ammonium sulphate fraction precipitate ( 0 - 6 0 % saturation) from germinated wheat embryos (48 h) was fractionated on DE-52 ion-exchange chromatography for determining the isoz'ymic pattern of A d o M e t synthetas¢. T h r e e iso~mic activity peaks o f AdoMgt synthetase were observed in excised germinated wheat e m b e d s without eJtogenou5 application of GA~ (Fig. 4). Furthermore, G A 3 failed to alter the isozvmie oatturn of A d 0 M e t synthetase in wheat embryos. It is quite likely that the induction of two additional isozymes of A d o M e t synthetase in wheat embryos is regulated by the endogenous high levels o f G A 3. This was in contrast to wheat aleurones, excised Dora deembryonated half-seeds where exogenous addition of GA_~ was mandatory for the induction of two addi-
~o A d o M e t synthetase were virtually abolished by A B A (10 ~ M ) (Fig. 2). Cyclohexlmlde (20 ~ g / m ] ) re,pressed the activity of all the three isozymes of A d o M e t synthetase in G A s - t r e a t e d aleurones (Fig. D. T h u s it appears that de-nova protein synthesis is necessary fur complete induction of GA~-regulated A d o M e t synthetase isozymes. Both C H i (20 g g / m l ) and A B A (10 ~ M ) were also effective in inhibiting ( 5 0 - 0 0 % ) the single activity peak of A d o M e t s~nthetase in control wheat aleurones (data not presented). The true significance of the phytohormonal regulation of A d o M e t synthetase became explicit when we eampared the isozymie pattern in aleurones excised from embryonated and de-embryonated seeds of when L Although a singte isozyme was observed in aleurones excised from de-embryonated half-seeds, there were three isozymes in aleurones dissected from emblyonated seeds (Fig. 3). Thus the pattern o f isozymes in aleurones dissected from emhryonated seeds was identical to that observed in GA~-treated alcurones dissect,~d front the de-embryonated wheat half-seeds. Apparently, the induction of two additional isozymes in
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Eluti0n v0tume (mi). Fig. 3. comparison of bo~mlc panern of AdnMet synthelase from alcurones isolated tram germinate0 ¢mbl~onaled s~2ds and dc-cmbryonated half-seeds of wheal. Aleuroncs excised tidal water imbibed de-embrvonalcd hall-seeds of wheal showed a stogie JsDx~llle of AdoMet syrahetase by DE-52 ion-exchange chrornatngr,iph]~, In contrast, aleurones exci~ed from water imbued embr~mul~d s~cds of wheat ~howed t h ~ isozyrrles of AdoMel wrahetas¢. (o) A]eutone5 excised from de-cmbr~tmatcd scuds; ( o ) ateumnes exci~ed from ¢lBbl~olmlcdm:t:ds.
343
Molecular welghl and subunh structure ,of AdoMet synthetase Batch elated DE-$2 fraction (0.05-0.3 M ame,:-> nium sulphate) containing three iso~mes of AdoMet synthetase was p r e p a r e d f r o m w h e a t embryos a n d subj e c t e d to molecular sieve chromatography on Sepharose
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CL-6B. A single molecular form was observed after gel permeation with a molecular weight of l g l 0 0 0 (Fig. 6A, B). Molecular-sieve chromatography of physically separated individual isozymcs alsO showe,d a single activity peak on Scpharosc CL-6B that corresponded to the molecular weight of 1810(KLThis suggested that the three isozymes of AdeMot symhetase have an identical molecular weight, lsozyme II wa~ purified to clectrophoretic homogeneity_ as previousl~ described [8]. Analysis of the purified AdoMeI aynt'.mtase on SDS-PAGE showed a single silver-stained pruiein band. The molecular weif, ht of the subunit is 84000 as determined from the relative mobility of standard protein markers (Fig. 6C). Thus wheat AdoMet syutheta~ is dimeric in nature.
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Fig. 4. Negativercgulalionof AdoMe! synthetm~¢ by abscisicacid in gemtinated e~¢ised wheat emb~os. Esciscd wh~al gmbryo~ were Senilinalcdin the presence anti absence of ABA (10 ~.Mk Fraeti,nalien of d~aly~d ammonium sulphate fraction precipitate (O-6Oe~ satut'atlon) on DE-gZ revealed three isogymic poohs of AdoM¢I synthetas¢ in control crab,s. Administratior~of ABA (1O #M) to wheat emblg'Osduring germinationsubstantially inhibited the ~.clivity of two iso,~mes uf AdoMe! synthetase. (o ) Control,t~) ABA.
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with ABA (10 a M ) was quite effective in lowering the levels of two i~zYmes of AdoMet synthetas~ (Fig. 4). Earlier, we have shown that CHI (20 # g / m l ) completely abolished the activity of three isozymes of wheat embryos, thereby suggesting the requirement of denero prolein synthesis for the induction of AdoMet synthetase [8]. Conclusive proof for the de-nero synthesis of AdoMet s~nthetase in wheat embryos was also shown by labelling the enzyme in vivo with [~804] 2and eventually recovering the 3aS label in methionine residue of the ~gS-labclled purified AdoMet synthetase
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Regulation of AdoMet synthetase by t..methionine Imbibition of d¢-cmbtyonated wheat half-seeds in solution of L-methionine (2 m M ) stimulated ( 2 . 4 f f o l d ) AdoMet synthetase activity in wheat aleurones (Table 1). Fraetionation of ammonium sulphate fraction precipitate on D E - 5 2 s h o w e d three i ~ z y m e s in methionine-treated aleurones (Fig. 5). Thus the induction of two additional isozym~:s by t.-methionine was comparable to that observed in GA3-treated wheat aleurones.
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Fig, 5, |rldurtio~ of is~lRrs of AduM~:t ~ynthctas¢ by i.-m©tMor(in¢ in ~vhe;nale~ ~he!;. E~llh,~/o-le~.half+seedsof wheal were imbibed in Ihe ,d~cnce and presence of L-metbionine (2 raM) for 48 h. Al~a. tones wcze mc-n~all~ excL~edf~om ¢mbr,o-less half seeds. The de,salted ammonium sulphate fraction precipitate, prepared from ts'hcat 'aleumnes, wa~ subjected ru DE-52 rhroraatogr~phy with a linear salt gra.dielU. ;~ single isozynx~ o; ..'~dtJMet syLithrtase was observed in comrol alruron¢,~, t:Metbionin¢ Ireatmenl of ~lletlmn¢~ indug~J two additional isozymes of AdoMet synlhetas¢. (~.) Control, (e) 1.methionine.
3,14
In-vitro hybridization of subunits of AdoMet synthetase With a view to determine the number of A d o M e t s~nthetase genes that contribute to the indttetion of three isozymes ill GA3-treated aleurones and wheat embryos, we resorted to in-vitro hybridization o f physica:ly separated isozymes in different combinations. Characterization of the purified A d o M e t synthetase in wheat system indicated that all the three isoz-./mes ~re dimerie with identical molecular weight but differ in their net charge. W e considered the possibiliW of the cMstcnCc o f two discrete classes o f enzyme subunits
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Fig. 6. (A) Molecular-sieve chromatography for delermbling the m l e c u l a r weight or AdoMet syntheta.se. The DE-52 fraction, prepared front germinated exci~d wheat embryos, was subjected to l~ol¢¢ular-sigve chromatography on Sepharose CL-fiB column (2.24 ¢mxSO cm), A single molecular form el AdoMet synthelase w~s >, o
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obtained by molecular sieving. (B) Calibration cui'v¢ of standard protein markers. The standard protein markers of known molecular weight were used for calibrating the S¢-phatose CL-CiBcolumn. The logla molecular weight of each protein marker was plotted as a funelion of ils g~. value. The molecular weight of native AgoMet s3,nthctase (tat000)was calculated from its K,~ value. (C) Subunit strucltur¢ of purified AdoMet syarhctasc in wheat embryos. The purified preparation of AdoMet synthetase (5 Jag protein) from germinated excised wheat embryos was subjected to YD,-PAGE (7.5%). Lane a: Silver-stained protein band of subunit of AdoMet synthet~e; lane b: SDS protein markers of known molecular weight. stained with silver nitrate.
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derived from two genes of A d o M e t synthetase that could generate three isozymes by random dimerization o f the snbunits. T h i s hypothesis was tested by the in-vitro hybridization of subunits o f physically separated isoxymes of A d o M e t synthetase. The two flanking activity peaks (isozymes I and I l l ) of A d o M e t synthetase were isolated by D E - 5 2 chromatography and then subjected to NaCl-freese-thaw treatment. This treatment dissociates the dimerie isozymes and then permits the random association o f enzyme subunits into directs. T h e in-vitro-hybridized isozyates were analysed by D E - 5 2 chromatography. T h e untreated control samples ,showed the original two activity peaks comparable to isozymes I and i11. T h e treated samples, however, showed an additional middle activity peak in
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