Gibberellic Acid-Enhanced Synthesis and Release of ...release of a-amylase and ribonuclease by isolated aleurone layers of barley. Some of the earlier experiments (9, 10) were repeated

Planlt Physiol. (1967) 42, 398-406

Gibberellic Acid-Enhanced Synthesis and Release of a-Amylaseand Ribonuclease by Isolated Barley Aleurone Layers

Maarten J. Chrispeels and J. E. VarnerMSU/AEC Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48823

Received December 16, 1966.

Summnary. Gibberellic acid enhances the synthesis of a-amylase in isolated aleuronelayers of barley-seeds (Hordeumn vulgare var. Himalaya). In the presence of 20 mmcalcium chloride the amount of enzyme obtained from isolated aleurone layers isquantitatively comparable to that of the half-seeds used in earlier studies. After alag period of 6 to 8 hours enzyme is produced at a linear rajte. Gibberelflic acid doesnot merely trigger a-amylase synthesis, but it is continuously required during theperiod of enzyme formation. Enzyme synlthesis is inhibited by inhibitors of proteinand RNA synthesis. Small amounts of actinomycin D differentially inhibit enzymerelease and enzyme synithesis suggesting 2 distinct processes. Gibberellic acid simi-larly enhances the formation of ribonuclease which increases linearly over a 48 hoturperiod. During the first 24 hours the enzyme is retained by the aleurone cells andthis is followed by a rapid release of ribonuclease during the next 24 hour period.The capacity to release the enzyme is generated between 20 and 28 hours after theaddition of the hormone. Ribonuclease formation is inhibited by inhibitors o-f proteinand RNA synthesis. These inhibitors also prevent the formation of the releasemechanism if added at the appropriate moment.

Haberlandt observed in 1890 (3) that the aleu-rone layers of rye produce a substance (or sub-stances) which causes liquefaction of the starchyportion of the endosperm and dissoluition of thestarch grains. This observation was confirmed byBrow-n and Escombe (2) who used barley seeds astheir experimental material. Mlany years laterYomo (13) and Paleg (7) showed independentlythat when gibberellic acid is added to endospermportions of barley seeds it initates the productionof amylolytic enzymes and the release of stugars.Briggs (1) extended this observation to includeseveral other hydrolytic enzymes (protease, phos-phatase and t-glucanase).

The only- living ce'lls in the endosperm portionof the seed, judged by presence of respiration andlamino acid incorporation are those of the aleturonelavers and it has been shown that all the a-amylaseis prodtuced by the aleurone cells (6, 9). In severalvarieties of barley it is possible to separate thealeuroine cells (with the seed coat) from thestarchy portion of the endosperm after the halfseeds have been imbibed for 3 days. In this wayone can sttudv the effect of the hormone on anisolated tisstle only 3 cell layers thick and consist-ing only of target cells. The effect can be meas-tired in terms of the resulting increase of severalh-drol -tic enzymes.

In the earlier work of this laboratory (10, 11)isolated aleturonie layers were used occasionally for

studies with inhibitors. No extensive use washowever made of this potentially elegant systembecause half-seeds and isolated aleurone layers werenot quantitatively comparable; the latter produicedonly a quiarter to a third as much a-amylase asthe former and we were therefore not sure thatthey were qualitatively similar either. This prob-lem has now been remedied and we have made amore detailed study of the production and therelease of a-amylase and ribonuclease by isolatedaleurone layers of barley. Some of the earlierexperiments (9, 10) were repeated because we feltit was necessary to define as carefuilly as possiblethe isolated aleurone system in order that it maybe used to investigate the biochemical mode ofaction of the hormone. WN hen the experimentalresults were similar for aleutrone layers and half-seeds, they are mentioned in the text, but the resutltsare not given in detail.

Materials and Methods

Seeds of barley (Hordeutmi vulgare var. Hima-laya) were cutt transversely and the embryo con-taining halves were discarded. The extreme tipsof the distal halves were cut off and the pieces ofendosperm obtained in this way (referred to ashalf seeds) were stored uintil ready for utse. Storagetime never exceeded 2 weeks. Half seeds weresterilized for 20 minutes in 1 % sodium hypochlo-

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CIIRISPEELS AND VARNER-GA-ENHANCED SYNTHESIS OF HE DROLASES

rite (commiiiiercial liquid bleach, (liluted 1:4), rinsedseveral times in sterile water anid preincubated inpetri dishes on moist sterile sand. (Autoclave10 cm petri (lishes containing 100 g of purified sandand moistened with 20 to 21 ml of sterile distilledwater)- Petri dcishes containing 100 to 160 half-seeds were wrapped in altuminum foil anid allowedto inctubiate at room temperatuire for 3 days. Afterthis time the seed coalts with attached aleturonelayers were easily removed from the starchy por-

tioni of the endosperm with the aid of 2 smallspattulias. This dissection was (lonie in a transferroom titler- aseptic conditionis. Ten such aleturonelayers were puit in a 25 ml Erlenimeyer flask coIn-taiining the incubation meditunm. The latter coIn-sisted of 2 ,umoles of acetate buffer (pH 4.8),200 Lm(-oles of CaCL. and 2 mrumoles of gibberellicacid in a fincal volume of 2.0 ml. These flaskswvere sterilized by autoclaving. Practical grade

gibberellic acid (Sigma Chemical Company) was

used tlhrotughouLt the experiment;s. This prepara-tioIn contaitns miore thail 90 % Gd.\- anid is referredlto in the text as GA. (\Ve cli(d niot stuidy the effectof autoclaving on GA, btut for experiments involvingGA as a critical v-ariable the soluitionls of gibberellicacid were sterilizedl by filtrationi anid added to thesterile flasks). In order to inisuire against anymicrobial growth, 1 drop (30 ,1A') of a soluitionconitainiing 0.5 ,g/ml of streptomnycin, peiiicillinanld mystecliin or 0.5 mg/ml of chlorampheniicol was

added to each flask. The aleuiroine layers were

incubated at 250 on a DuLbnoff metabolic shaker(40 oscillations per minlulte). ANt the end of theincubation period 1 ml of water w-as addedl to theflasks and the meedia were decanted. The aleturoinelayers wer-e rinsed with 2.5 ml of wvater and(l thiswas combined w-ith the media. The aleuirone layerswere ground to a thick paste in a porcelain mortarwith a little sandcI and 0.8 ml of 0.2 m sodlitimchloride. The homogenate \-as diluted with 4.0nil of the samiie solution, pouiredI into a centrifulgetlil)e anicd cenitrifuiged at 2000 X qJ for 10 mintutes.The resulting supernatant fractioni (referre(d to as

extract) anid the meditim were assayed for a-am-

ylase activit\ by the methodl of Shuster and(l Gifford(8)

The a-anm\-lase assay -was calibrated with puri-fiedl malt a-anivlase and a conversion factor of2.7 ug of a--amVlase per unit of optical densitychainge was uisedl. This conversion factor is de-pendent o(i the starch used for the assav. A freshstarch soluttionl was made every day ulsinlg 150 mg

of native (nioni-soluibilized) potato starch (Nuttri-tionial Biochemi-ical Corp.), 600 mg of KH.PO.,and(! 20) Lmoles of calcitum chloride in a final voltumeof 104) mll. The starch suspension was boiled for1 milltlte, coole(l and centrifuiged at 2000 X g for10 minuttes. The clear stiperniataint wvhich was utsedfor the assay w\as carefully separated from thesuispetndled 1u1scoluibilized starch an the bottom of the

tube. The a-amylase assay was carried out in a16 X 150 m-m test ttube with a suitable aliattot ofenzyme (0.02-0.2 ml) and enough water to make1.0 ml. The reaction was statted by the additionof 1 ml of starch soluition, allowed to proceed atroom temperature for a suitable time (1-5 min)and stopped by adding 1.0 ml of iodine reagent.The iodine reagent was made by diluting 1.0 ml ofiodine stock soltution (6 g of KI and 600 mg ofiodine dissolved in 100 ml of water) to 100 ml with0.05 N hydrochloric acid. Five ml of distilled waterwere added to each tuLbe and after thorough mixingthe optical density was read at 620 m/% using aColeman colorimeter. The initial OD of the starchsolution is usually abotut 1.35. The decrease inOD at 620 m/i caused by the action of the enzymeis proportional to the quianitity of a-amylase presentand the length of the reaction time over the rangeof 30 % to 75 % decrease in OD.

Ribonuiclease was determined according to themethod of Wilson (12). One unit of enzymeactivity represeints 0.1 OD uinit of soluible nticleo-tides genlerated duirilng a 40 minutte incubationperio(l.

Results

a-Awmavse Produictiont bv Isolated AleuronteLaoers. A comparison of the a-amylase productionby half seeds and isolated aleturone layers is shownin table 1. After their separation from the starchyportion of the endosperm the aleuirone lavers pro-duce at most half as mulch a-amylase as the intacthalf seeds, and frequently less than a quiarter ofthe total amotunt. It was shownl earlier (9) thatthe starch portion of the endosperm does not con-tribute to the a-amylase prodtuction and the dis-crepancy was therefore ascribed to the existenceof another factor (or factors) in the starch endo-sperm which interacts with the aleturone layers toenhance a-amvlase synthesis (11). \VNe observeda small stimtulation of a-amylase prodtuction whena mixtulre of 16 amillo acids, each at 1 mNi, was

Table I. Production of a-Amnvlase by Half-se-eds andIsolated Aleurone Loae,rs as Affected by

GA and CaCl,Ten half-seeds or 10 aleurone layers were incubated

for 24 hours with buffer, w-ith or without 1 /Am GAand with or without 20 mM CaCl,. Activity of thea-amylase w as measured in the medium and in an ex-tract of the tissue. The data indicated the suim of these2 values.

Totdl ,ug of a-amy-lase producedno CaCl, 20 mM Cacl9

Hal f-seeds Control 16 191 /M GA 238 420

Aleurolne lasers Control 47 801 u-\ GA 121 520

399

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PLANT PHYSIOLOGY

Table II. Effect of Calcistni and Otlher Dizalent Ionson the Production of a-,4m-lasc by, .1!curone LaversTen half aleurone lavers were incubated with buiffer

and 1 fXNI GA and the appropriate concentration of thesalt. a-Amylase was assayed in the medium and thetissue extract after 24 hours incubation.

Treatmenrt ,ug of a-amylase per 10aleurone layers

Control 690.1 n1m[ 741 nm-m 198

10 mnm 29320 nmw 328100 nml 33510 mM 22310 mat 4210 mm 7010 mm v

added to the incubation mediuim of the aleuironelayers (J. E. Varner and M. J. Chrispeels, tunpuib-lished), but this addition did not bring the pro(dtuc-tion of the aleuirone layers uip to the level of halfseedls. This however, can easily be achieved bythe simple expedient of adding 20 m.m CaCl., to theincuibation mediuim. Calcium chloride cauises a2-fold increase in the amouint of enzyme obtainedfrom the half seeds and a 4-fold increase whenadded to aletirone layers. The effect of calciuimchloride and chlorides of other divalent ions onaleuirone layers is shown in table II. The requiire-ment for calciuim is satuirated at 20 mm. SrCl.,can be suibstituted for CaCl. but MgCl.2 and BaCl,are withouit effect, while CdCl., is inhibitory.

Concerning the basis of the calciuim effect wefirst explored the possibility that CaCl., affectsthe synthesis of the enzyme, but no evidence to thiseffect coulld be obtained. Next, because a-amvlasefrom malt is known to bind calciuim we investigatedthe effect on the activity of the enzyme. Aleuironelayers were incuibated with variotus concentrationsof calc:'um for 10 houirs and at this time 500 ug ofpuirified malt a-a!mylase were added. At the endof the 24 houlr rutn the a-amylase activity hadalmost completely disappearedl in the absence of

calciuim while it remained in the presence of calcitum(table III). The data show that the same con-centration of CaCl., which gives maximal activityof the enzyme produced uinder the influence of GAis necessary for maximal preservation of addeda-amylase. Again SrCl., is a fairly good suibstituitefor CaCl., while i\IgCl2 is withouit effect. If CaCl.,was added to the medium at the end of the experi-ment (24 hrs) instead of at the beginning, no in-crease in a-amylase activity was observed. Thisstuggests that we are dealing here with an irrever-sible inactivation of the enzyme.

300

E200 /

TOTAL

100 ,[ IN ALEURONE CELLS

0~~~~~~~~

5 8 12 24HOURS OF INCUBATION

FIG. 1. Timecourse of a-amylase synthesis by 10aleurone layers incubated with 1 ,uM GA. Enzyme activityw-as measured in the medium surrounding the aleuronelayers and in the supernatant of a 0.2 -m NaCl extract ofthe aleurone layers. The term total refers to the sumof these 2 activities.

Table III. Protection of a-Amnvlase Against Inactivation by CaCl., lgCI., and SrCI.,Ten half aleurone layers were incubated with buffer and 1 !,LM GA. After 10 hours 500 jug of -pure a-amylase

isolate(d from brewers malt were added. After 14 more hours the media anid tissue extracts were assayed for a-amy-lase. Expected production after 24 hours was determined as a separate experiment, without added malt a-amylase.The figures show the amount w hich was produced in the presence of the same aniounts of the different salts.

a-Amylase (,ug per 10 aleurone layers)Added to themedium after

10 hrs

5Q0500500

300500

Assayed after24 hrs

145610810630121

% Remainiing after 24Expected production hrs of the 500 juI

after 24 hrs added at 10 hrs

55 18202 82320 98230 8081 8

Treatment

Conitrol1 mM CaC12

20 mM CaCl210 mM SrCI210 mmr MgCI2

400



CHRISPEEI-S AND VARNER-GA-ENHANCED SYNTHESIS OF H YDROLASES

Table IN7. Effect oj GA Concentration on a-ArnvlasePr-oduiction

Teni half-seeds or 10 aleurone lavers -,vere incubatedfor 24 hours with buffer, 20 nmi CaCl, and differentconcentrations of GA. Activitv of the a-amylase wa'smeasured in the medium and in an extract of the tissue.The data indicate the sum of these 2 v-alues.

Treatmeiwt

Control0.001 ,ui GA0.01 mI GA0.1 A-m GA1.0 ;4N GA

,ug of a-amylase per10 aleurone layers

54157410454451

At the end of the 3 day pre-incuibation periodthe half-see(ds contain a small amouint of a-amylase,uisually less than 1 ,ug per half aleuirone layer.WVhen GA is added the pro(Iduction of the enzyme islinear for 16 to 24 houirs after an initial lag-periodof 6 to 8 hoturs (fig 1). Most of the enzyme issecreted into the mediuim by the aleturone lavers,while the cells retain only a small amouint whichremains constanit from 12 to 24 hours. Only asmall amouint of a-amylase is sy-nthesized in theabsence of exogenotus GA. These data are similarto those obtained with half-seedIs (9, 10,11).

In some experiments 10 half aleuirone layerspro(duice as muich as 600 yg a-amvlase in 24 hours,

-GA

100p

'l 12 18 24HOURS OF INCUBATION

FIG. 2. Effect of the removal of GA after a-amvlasesvnuthesis has been initiated. Aleurone layers Nvere incu-bated in 10 mjAc GA for 11 hours. GA s-as theni re-

moved by frequent rinsing and at 12 hours GA was eitheradded or withheld subse(quently.

in other as little as 2175 ug. HowN ever, enzymeprodtuction is always at a constant linear rate re-gardless of the amouint which is prodticed in 24houtrs. The requirement for GA is satisfied at aconcentration between 0.01 pt and 0.1 /AMi (tableIV).

Figtire 2 shows that there is a continious re-quirement for GA duiring the periodI of enzymesynthesis and that it is not stufficient to have GApresent only duiring the inductioni period. Aletironelayers were rinsed, to remove the GA, 11 houirsafter the start of the incubation. After the removalof GA a-amylase synthesis continued at a slightlyreduiced rate for approximately 6 houirs. Duiringthe next 6-hoiur period it contilniued at 50 % of therate present in the presence of GA. That substan-tial enzyme synthesis continuies after removal ofGA may be dIule to GA remaininig in the tissiie.

0200

a) Totl /

0 /

0)

-Do XMedium

0 100 /

:D/

Hours of incubationFIG. 3. Time course of I)roduction of ribonuiclease

by 10 aleurone layers incubated with 5 mrum GA.

Ribonutcleasc Production bv Isolated Ale-roneLayers. Ribonuiclease is synthesized by the aleui-rone layers in response to added GA at a linearrate, which contintues for at least 48 houirs afterthe start of the experiment (fig 3). Almost noribontlclease is released into the mediuim in the first24 hoLurs, cauising it to accimullate in the aleuironecells. This is followedI by a rapi(l release of theacculmtulated enzyme in the next 24 houirs, and 48houirs after the start of the incuibationi no ribo-nuiclease remains in the aleulrone layers. Thissuiggests that the aleiurone layers do not have thecapacity to secrete the enzyme iulltil 24 to 28 houirsafter the additioni of GA.

Isolated aleuronie layers produice as muich ribo-nuclease as half-seeds indicatinig that the aleuironecells are the souirce of the enzyme. Dry half-seedscontain a small amouint of ribonuclease (abouit 15uinits per 10 half-seeds). In 1 experiment seeds

400h

300Fa

Ea

I%I-a

2001

AA I a A

401



PLANT PHYSIOLOGY

Table V. Increase itn Ribonuclease during Pre-incubationof Half-seeds

Sterile half-seeds were incubated on moist sterile sandfor the number of days indicated in the left column. Atthe end of the incubation they were homogenized in 0.2 NNaCl and ribonuclease was assayed in this extract of thetissue.

Units of ribonucleaseDays of pre-incubation per 10 half-seeds

0 (Dry half-seeds) 14.21 17.12 23.23 33.84 45.13 Writh 10 ug/ml cycloheximide 17.4

were handled with gloves to make sure that noenzyme was transferred from the hands to theseeds. There is a steady rise in the endogenouslevel of ribonuclease during pre-incubation of thehalf-seeds on moist sand (table Vr). This ribo-nuclease is also located in the aleurone cells andnot in the starchy portion of the half-seed endo-sperm (tunpublished results).

The requirement for GA is satisfied at a con-centration of 1 mjum (fig 4), which is well belowthat required for maximal a-amylase synthesis.Ribonticlease synthesis also occturs in the absenceof GA and the addition of GA cautses only a 2-foldincrease in the amount of ribonuclease produced in48 hours. Most of the ribonuclease syinthesized inthe absence of GA and in the presence of lowconcentrations of GA (0.01 and 0.1 muM) remains

0D200H

4'

a'U

0.M

0

0)

c

1001

0 1011 10- 10 10o9

GA concentration [M]l0o-

FIG. 4. Dose response curve of the production ofribonuclease by aleurone layers at different concentrationsof GA. Ribonuclease was measured in the medium andan extract of the tissue after 48 hours of incubation.

in the aleurone layers after 48 hours. The rapidrise in the amount of ribonuclease released into themedium over the concentration range 0.3 to 3 m,Msuggests that both synthesis and release of theenzyme are independently controlled by GA.

Effect of Metabolic Inhibitors on a-A mylaseSyvnthesis. The production of a-amylase by aleu-rone layers is inhibited by the tuncouplers 2,4-dini-trophenol and carbonylcyanide-mii-chlorophenyl hy-drazone (CCP) and by the protein synthesisinhibitors cycloheximide, puromycin, and p-fluoro-phenylalanine. These data are not reported herebecause they are similar to those obtained with half-seeds (9, 10, 11). The formation of the enzymecan also be arrested by the addition of the inhibi-tors in mid-course during enzyme production (e.g.12 or 15 hrs after the addition of GA). Table VIshows the effect of cycloheximide and CCP added15 hoturs after GA. Both inhibitors stop a-amylasesynthesis immediately. This eliminates the possi-bility that any part of the a-amylase exists as anenzymatically inactive precursor which is beingconverted into an active enzyme under the infltuenceof GA.

Table VI. Mlid-coturse Inhibition of a-A inylase Forma-tion by Cyclohcxintide and CCP

Ten aleurone layers were incubated with buffer, 1,/zM GA and 20 mmi CaCl,. Activity of the a-a-n-lase was measured in the medium and in an extract ofthe tissue. The data indicate the sum of these 2 values.Inhibitors (CH -- cycloheximide 2 ,ug/ml; CCP =carbon-lcvanide-ini-chlorophen-lhydrazone 20 ANi) areadded at 15 hours and the samples are assayed 4 hourslater.

IAtg of a-amylase perTreatment 10 aleurone layers

GA for 15 hrs 196GA for 19 hrs 370Add CH at 15 hrs 220Add CCP at 15 hrs 189

Synthesis of a-amvlase is inhibited by actino-mycin D, an inhibitor of RNA synthesis, and bysome base analogues (8-azaguanine and 5-azacy-tidine) btut not by others (5-bromouracil and5-fluoroturacil). When the antibiotic is added atthe same time as GA a high concentration is neededto prevent a-amylase formation (table VII). Even100 4g/ml of actinomycin D inhibit,s a-amylasesynthesis by only 60 %. In a parallel experimentwe measuired the inhibition of 14C-uridine incor-poration by actinomycin D in the 4 to 8 houir periodafter the addition of the antibiotic. In this experi-ment no GAk was added to avoid the complicatingfactor of a possible increase in the pool size ofuridine dute to the presence of GA-enhanced ribo-nuclease. The inhibitory action of actinomycin Don the incorporation of 14C-tiridine increases with

402



CIIRISPEELS AND VARNER-GA-ENHANCED SYNTHESIS OF HYDROLASES

Table VII. Inhiibition of a-Amylase Production by Actinomvcin DSamiiples of 10 aleurone layers were incubated with buffer, 1 Am GA and 20 mar CaCI2 Appropriate amounts of

actiMomycin D were added at the same time as GA. The enzyme was assayed after 24 hours incubation. In a parallelexp)eriment aleurone layers were inicubated witlhout GA and allowed to incorporate 14C-uridine (1 Ac per flask specificactivity 26 mc per Mmllole) between 4 and 8 hours after the addition of actinomycin D. RNA -vas extracted by themetlho(d of Kirbh (5).)

a-Amylase,ug Per 10

Treatment aleuronie layers % Inhliibition

% Inhibition ofinicorporationof 14C-tiridine

Conitrol2O tg/iii Act. D50 jig/mll Act. D100 ,ug ml Act. D

increasiing conceintrations of the antibiotic. Thelowest coinceintrationi of actinomycil 1) (25 ug/ml)which har(lly affects a-amylase synthesis inhibitsdridine incorporation by only 40 % The (lata indi-

cate that the failuire of actinomyvcin D, especiallyat lower concentrations, to inhibit a-amylase syn-thesis may be relate(d to its ineffectiveness in block-ing RNA synthesis in this system.

The lower concentrations of actinomycin I)which have relatively little influlence oII enzymeformation have a profound effect on the releaseof a-amylase 1h the aleuirone cells (table VIII).II the presence of 25 ,ug/ml of actinomycin D moreth(an half the a-amilxvlase is retaine(d 1)v the aleuironecells, instead of the utstial 1i to 20 %. Enzvmerelease is proportioinally more affecte(d than enzymesyilthesis inidicating that the process maybe af-fecte(l in(lependlently.

Table VIfI. Inhibition of a-Anvliase Seciretion b}\Actinow vein D

Details tlle same as in table VII.

,ug Of a-anmxlaseper 10 aleuronie laversT reatnient

Coiitrol25 ,Lg/ml of Act. D50 g,/nlm of Act. D

Medium324176108

Extract79197194

Total403373302

Effect of Metabolic Inhibitors on RibonuicleaiseProducticont (aid Rcle(ase. Inhibitors of protein syin-

thesis inhibit the formation of ribonuclease byaleuronie layers. Cycloheximide (5 pug/ml), p-fluo-rophenylalaniine (2.5 mNt) and puromycin (250p,g/mI) inhibit enzyme synthesis by 98 %, 52 %and 22 % respectively. The increase in ribonu-

clease which is observed during the 3 (lay pre-incu-bation periodl is also inhibited bv cycloheximide(table V). \When the inhibitor is added in mid-course (e.g. 24 hrs after GA) it inhibits ribonu-clease formation very rapidly, without inhibitingthe release of the enzyme from the aleurone layers

Table IX. Cyclohexim ide Inhzibits Ribonuiclease Synthesiswhen Added 20 Houirs After GA

Ten aleuronie layers were incubated -,vith buffer, 20m-m CaCI., anid 20 n11,UmM GA. After 24 lhours 5 pkg/ml ofcvcloheximide m-vas a(lded to 1 set.

Units of RNAaseper 10 aleuironie layers

0 Hrs24 Hrs48 Hrs48 Hrs, add cycloheximide

at 24 lhrs

Medium Extract28.0

8.5 98.1219. 5 11.5

Total28.0106.6231.0

100.6 17.4 118.0

in the following 24 houLr period (table IX). This

indicates that cycloheximide (loes not inhibit the

release process per se.

An1 experimelnt w'vas (lesignie(I to test the possi-bilitv that the formation of the release capacityrequiires protein synthesis. Time couirse stui(lies on

the appearance of ribonuiclease in the mediuim had

shown that this capacity develops between 20 anid(28 hoturs after the a(ldition of GA (fig 3). Cyclo-

heximide was a(ldedl to the aleuirone layers 20, 24

an(I 28 houlrs after GA and(I the release of ribo-

nutclease from the aleuirone layers was measture(l in

2 4-hour perio(ds followving the ad(litionl of cyclo-heximide (table N). In this xvay it is possible tostuidy enzyme release in the absence of enzyme

svnthesis.The restults show that if cycloheximide is added

20 houlrs after GA, release of the enzyme is slow,even betweeni 24 and( 28 houirs, indicatinig that therelease capacity is not (leveloping in these aleuironelayers. The enzyme is released more rapidlv if theinhibitor is adde(d after 24 or 28 houirs. Most oIr

all of the release capacity has alrea(ly (evelopedl at

this stage.Ribonuiclease synthesis is also inhibited by

actiniomycin D (50 and 100 yg/ml). A time cotursestudly indicates that there is no inhibition in thefirst 24 hours,, while the inhibition is almost com-

plete in the next 24 houirs (table XI). Actinomycin

359332241152

. .

7%33 %58%

40 %63 %75 %

403



Table X. Release of Ribonuclease from Aleurone Layers after Inhibition with CyclolecxiwiideAleurone layers were incubated for 20, 24 or 28 hours with 5 m,"m GA. At these times they were rinsed 3 times

for 5 minutes with sterile medium containing GA and 10 ,ug/ml of cycloheximide to remove the ribonuclease whichhad already been released in the medium. The aleurone layers were then further incubated with medium containing5 mtum of GA and 10 ,ug/ml of cycloheximide for 4 and 8 hours. The aleurone layers were collected and the ribo-nuclease remaining in the aleurone cells was determined. Units of ribonuclease released were calculated by difference.

Time or additionof cycloheximide

Time period aftercycloheximide

Units of ribonucleaseper 10 aleurone layers

Units of ribonucleasereleased in 4 hrs

20 hrs Initial 75.6After 4 hrs 59.1After 8 hrs 47.7



Table XI. Inhibition of Ribonuclcase Synthesis and Secrction by Actinoinycin DAleurone layers were incubated in the presence of 5 mum GA and actinomycin D.

16.511.4

47.721.9

73.417.9

Treatment Time Units of ribonuclease per 10 aleurone layers

Medium Extract TcytalControl 24 hrs 8.7 94.2 106.9Act. D 50 ,g/ml 9.3 82.4 91.7Act. D 100 jug/ml 5.7 86.7 92.4Control 48 hrs 191.5 3.7 195.2Act. D 50 ,ug/ml 86.0 24.4 110.4Act. D 100 ,ug/ml 18.2 78.9 97.1

Table XII. Inhibition of Ribonuclease Release by Actinomycin DDetails as in table X. To 1 set actinomycin D (100 ug/ml) was added from the beginning. After 26 hours the

aleurone layers were rinsed and further incubated in a similar meditum conitainiing 10 /.g/ml of cycloheximide. Theribonutclease remaining in the aleurone layers was measured 4 and 8 hours later.

Time period after Units of riboniuclease Units of ribomucleasecycloheximide, per 10 aleurone lavers released in 4 hrs

Actinomycin D Initial 72.6After 4 hrs 73.2 0After 8 hrs 62.0 9.4

Control Initial 70.6After 4 hrs 47.6 23.0Afiter 8 hrs 22.8 24.6

D also prevents the release of the enzyme from thealeurone layers in the second 24 hour period, sug-gesting that RNA synthesis may be required forthe formation of the release capacity.

The effect of actinomycin D on the developmentof the release capacity was measured in the follow-ing, more direct way. Aleurone layers were in-cubated for 26 hours with GA and 100 ,ug/ml ofactinomycin D and then treated with cycloheximideto stop any further ribonuclease synthesis. By thistime (26 hrs) most of the release capacity will havedeveloped in the controls and cycloheximide will nothave a profound infltuence on the release of the

enzyme (see data in table X). This allows us tomeasure the effect of actinomycin D on the de-velopment of the release capacity in the absenceof further ribonuclease synthesis and in the absenceof the effect which cycloheximide itself has on thedevelopment of the release capacity. Release ofenzyme into the medium was then measured overthe next 2 4-houir periods. The data in table XIIshow that release is almost completely inhibited ifthe aleurone layers have been incubated with actino-mycin D for 26 hours. Addition of actinomycin Dto the controls, 26 hours after GA (the beginningof the actual release experiment) had n?o effect on

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the rate of release indicating that the antibioticdoes niot interfere with the release process itself.

Discussion

Usiiig isolatedl aleurone layers we have con-firmed ouir earlier results on gibberellic acid in-dtice(I a-amn-lase synthesis obtained with half-seeds.Previouislv work carried out with isolated aleuronelavers ( 9, 10, 11) was hampered by the fact thatthey seemed able to produice only smaller amountsoft a-atniylase than half-seeds. This problem hasbeen remedied by showing that calciuim ions areneeded for obtaining maximal enzyme activity andwve canl noxv l)e assuired that aleuirone layers arecompar-al)1e to half-seeds quantitatively as well asqualitatixely. This is also truie for the GA-trig-gere(l sy-nthesis of ribonutclease.

One problem remain s: detaching the aleturonelavers froml the starchy endosperm initiates theformationi of a small amouint of a-amvlase andl alarge anmotunit of ribonticlease anid in;creases thebackgrotunid agaiinst which one has to work. It isnotexworthy that the enzymes have dlifferent optimain their G X requiiiremenit. A concentrationi of 2mpurt GA is elnoLlgh to obtaiin maximal ribonucleasesN-nthesis. but this constituites only a 2-fold increaseover the conitrol valtue. Is it possible that enzymesvnthesis in the absence of added GA is duie toen(logenotis gib)berellins ? To test this possibilitx350 aleurI-onie layers xx erc extractedI according tothe metllo(d of Kende and Labag (4), anid the p,,li-fied extract x-as bioassayed wxith the d -dwarf corntest. The resuilts indlicated that aleurone lavers doconltaini oibl)berellinl-like substainces (Chrispeels aindJonies, iunipuiblislhed data). \Whether these en(log-enois gil)lherellin-like sulbstanCces caIn trigger off thesvnthesi' of a-amNlase or ril)onticlease remainsunl;kox- . It is, however, conceival)le that processesw hich ar-e triggered off by small amouniits of GA(e.g. riboul-clease svnthesis) caii be iuitate(l moreeasilx- xxithotit added GA than processes xx'hich re-qillire larger amotints of hormone (e.g. ce-amylasesyntthesis). This coullcl accollnt for the olservationthat in the absence of GA relatively more ribo-iiclease is synthesized than a-amy-lase.

Eiuzyme synthesis can be blocked bl the additionof uincotiplers of phosphorylation or inhibitors ofprotein synthesis added either at the same time asGA or in mid-couirse xvhen enz\me svynthesis is inprogress. Both kindIs of inhibitors stop enzymesv-ithesis alImost immediately. This eliminates thepossibilitv that there is an eilzvmatically inactiveprecuirsor wxxhich is converted into active enzymes,unlless it is asslinledI that this conversion is in tuirndependlenit on1 coIntinuiouis proteiin synthesis, becauseit is brouight about by an uinstable enz_yme. Enzymesynthesis is also blocked by inhibitors of RNA syn-thesis stiggesting that a nexw RNA is requiirecl forthe s-ntmlesis of the enzymes.

It is usually difficult to separate experimentallythe release of an enzyme from its synthesis. Aleuronelayers contain only a small amount of a-amylaseand this enzyme is therefore not suitable for astudy of release. Time curves of enzyme synthesisindicate that a-amylase is released as soon or almostas soon as it is synthesized. The only way inwhich the 2 processes could be separated is byinhibition with actinomycin D. Small amotunts ofthis antibiotic (25 ,ug/ml) inhibit a-amylase releasemuch more severely than its synthesis. A differentpictture is obtained with ribonuclease. No ribonui-clease is released in the first 24 hours and in theabsence of GA or in the presence of small amountsof GA very little enzyme is released in the next24 hoturs. Larger amounts of GA on the otherhand trigger a very rapid release of the enzymein the second 24 hour period. The release capacitydevelops between 20 and 28 hours after the additionof GA. Addition of cycloheximide after 20 hoturs,but not after 28 houirs, stops the development ofthe release capacity. Addition of actinomycin Dat the same time as GA does not inhibit enzvmesynthesis duiring the first 24 hours, buit iInhibitsboth synthesis and release in the inext 24 hourperiod. It appears therefore that enzyme synthesisand release are activated and inhibited differen-tially, indicating that they are 2 distinct processes.Ho,wever, they may be interdependent and the pos-sibility that an inhibition of release resuilts in aninhibition of synthesis cannot be excltuded.

Whether xre are dealing with an active secretionor with a pzssible release of the enzymes due tonecrosis of the cells is not yet clear. After 24houirs the cells have lost a large portion of theirnitrogen (11) and visual observation (Varner,unpublished) shoxvs them to be completely vacuo-lated. However, respiration remains unchanged for24 hoturs, then declines slowlv whether GA has beenadded or not. This indicates that the biochemicalmachinery of the cells remains intact, at leastduiring the first 24 hours, and there is no reasonwhy the aleuirone cells should not be able to carryouit a controlled release. The release of a-amylasefollows a different pattern from that of ribontu-clease suiggesting again a specific mechanism.

Acknowledgments

The authors gratefully acknowledge the technical as-sistance of Miss Nancx Joseph. We wish to thank R. A.Nilan for supplying the Himalaya variety of barley. Theactinomycin D was a gift from Merck, Inc. This workwxas supported bx the United States Atomic EnergyCommission uinder cointract No. At (11-1) 1338.

Literature Cited

1. BRIGGS, D. E. 1963. Biochemistry of barley ger-mination action of gibberellic acid on barley endo-sperm. J. In.t. Brewing 69: 13-19.

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2. BROWN, H. T. AND F. ESCO'MBE. 1898. On thedepletion of the endosperm of Hordcum vulgareduring germination. Proc. Roy. Soc. 63: 3-25.

3. HABERLANDT, G. 1890. Die Kleberschicht des Gras-Endosperms als Diastase aussclheidendes Driisen-gewebe. Ber. Deut. Botan. Ges. 8: 40-48.

4. KIRBY, K. S. 1965. Isolation and characterizationof ribosomal ribonucleic acid. Biochem. J. 96:266-69.

5. MACLEOD, A. M. AND A. S. MILLAR. 1962. Effectsof gibberellic acid on barley endosperm. J. Inst.Brex-ing 68: 322-32.

6. PALEG, L. 1960. Physiological effects of gibber-ellic acid. I. On carbohy drate metabolism andamn lase activity of barley enidosperm. PlantPhysiol. 35: 293-99.

7. SHUSTER, L. AND R. H. GIFFORD. 1962. Changes in3'-nucleosidase during the germination of xN-heat

embryos. Arch. Biochem. Biophys. 96: 534-40.

8. VARNER, J. E. 1964. Gibberellic acid conitrolledsynthesis of a-amylase in barley enclosperm. PlanitPhysiol. 39: 413-15.

9. V"ARNER, J. E. AND G. RA.1 CHANDRA. 1964. Hor-nional control of enzyme synthesis in barlev enclo-spermii. Proc. Natl. Acad. Sci. U. S. 52: 100-06.

10. VARNER, J. E., G. RAM CHANDRA, AND ,M. J. CHRIS-PEELS. 1965. Gibberellic acid controlled svnthesisof a-amiylase in barley endospermii. J. CelluilarCompii). Physiol. 66. Suppl. 1: 55-68.

11. WVILSON, C. 'M. 1963. Chromatograpliic separationof ribonucleases in corn. Biochem. Biophys. Acta68: 177-84.

12. Yo'Io, H. 1960. Studies on the a-anivlase activat-ilng substance. IV. On the amylase activatingactioIn of gibberellin. Hakko Kyokaishi. 18: 600-02. (cited in) Chemical Abstracts, 55:26145(1961).

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Documents

Gibberellic Acid-Enhanced Synthesis and Release of ...release of a-amylase and ribonuclease by isolated aleurone layers of barley. Some of the earlier experiments (9, 10) were repeated