Plant Physiol. (1967) 42'. 1O08-l0l1

Hormonal Control of Enzyme Synthesis: On the Mode of Actionof Gibberellic Acid and Abscisinin Aleurone Layers of Barley'

Maarten J. Ghrispeels2 and J. E. VarnerMSU/AEC Plant Research Laboratory, Michigan State University

East Lansing, Michigan 48823

Received March 31, 1967.

Summary. Gibberellic acid (GA) enhances the synthesis otf a-amylase andl ribo-nutclease in isolated aleurone layers andl this process is inhibited by abscisiin. Removalof gibberellic acid in mid-course of a-amylase productioni results in a slowing downof a-amylase synthesis, suggesting a continued requirement of GA for eiizymesynthesis. This is paralleled by a continuous requirement for RNA synthesis. Additionof 6-methylpurine or 8-azaguanine in mid-course results in ani inhibition of a-amylasesynthesis within 3 to 4 hours. However, adtinomycin D added in inid-course isalmost without effect. 'rhis is not due to its failuire to enter the cells, because itdoes inhibit 1'C-uridinie incorporation at this stage. Addition of abscisin to aleuroinelayers which are synthesizing a-amylase results in an inhibition o'f this synthesiswithin 2 to 3 hours. Cycloheximide oIn the other hand inhibits enzyme synthesisimmediately upon its addition. These data are consistent with the hyp.othesis thatthe expression of the GA effect requires the synthesis of enzyme-specific RNAmolecules. The similarity in the kinetics of inhilbition between abscisin on the onehaind and 8-azaguanine or 6-meth'yl;purineexert its action by inhibiting the synthesisor by preventiing their incorporation into

G(ibberellic acid gre,atly enhllanices the syiithesisof atmylolytic enzymes (and several other hydrolyticenzyinmes) in aleuiroine cells of barley (20, 23, 27),wildl oats (16), an(d rice (19). \Ve have stuidiedin detail the GA-enhanced synthesis and release ofa-amylase anid ribonlticlease (4) adI(I of protease(Jacobsen an(l Varner, in preparation) by isolate(daleuirone layers. The maniifestation of this actionof gibberellic acid is inhibited by inhibitors of pro-tein synthesis (p-fluorophenylalanine and( cyclo-heximide) and of ribonucleic acid synthesis (acti-nomyi)-cin D and 8-azagtuaninie). Eviden,ce obtainedby a nlew method in(licates that the appearanlce ofa-anivlase (6) and protease (jacobsen an(l \'arler,ill preparation) is (liie to the (de niovo svynthesis ofall the enzy,me which is produced. 'T'his de novosynthesis is depe,ndent oin the synthesis of onie ormore species of ribonuticleic aci.d.

Recently we reported that abscisinm ( abscisi 11or dormin) inhibits the GA-enhanced synthesis ofa-anmlase and the release of free amino acids byaletiromie layers (3). The inhibitiomi of enzyme

Ibis Uwork wsas supported by the United StatesAtomic Energy Commission under contract No. AT(1)1-1) 1338.

2 Present address: JohnIIMuir College. D)epartment ofBiology, UCSD, La Jolla, California 92307.

oni the other suggests that abscisin ma)of these enzyme-specific RNA molectulesan active enzyme-svnthesisiilg unit.

synthesis b))y abs'cisin cani be partially) o\vercomeyl)the addition of a larger amiiotunit of GA. Thekinetics of this interaction are neither competitive,nor non-competitive. The com,plexity of the systemmakes an interpretation of the kinetics data im-possible. The inhibitioni of enz me syinthesi s b)abscisin is different from, and( nmich miiore specificthan, that obtained by metabolic inhibitors. Indeed,abscisin does not affect respiration or phosphoryla-tion; neither does it inhibit the incorporationl ofradioactive precursors inlto proteini (other than thehydrolases) and RNA (3).

An infteractioni between GA and abscisiii hasbeen reporte(l ill several systems. A.bscisin inhibitsthe expansive growth of leaf sections of corni andttthis inhibitioni is reversed by G(A (2 I). It alsoiinhibits the G-A-enhanced elonigationi of niormial aniddxvarf peas (21). The elongationi ( normal aiilGA-enhan,ced) of lentil epicotyls is accomlpanied b)L)NA synthesis (16) boith elongationi and DNAs\nthesis are inhibited by abscisin andci this inhibi-tion ,can be partially reversedi by the addition of('A (Chrispeels, unpulblished).

WVe have studied in greater detail ,he effectso,f GA and abscisin on barley aleurone layers, aswell as the effect of RNA-synthesis-inhibitors onIa-amylase synthesis in order to learn more aboutthe mode of act,ion of the 2 hormones.

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CHRISPEELS AND VARNER-GA, ABSCISIN AND HYDROLASE SYNTHESIS IN ALEURONE CELLS 1009

Materials and Methods

Most of the materials and methods used in theseexperiments have been described in detail in ourprevious paper (4). Synthetic d,1-abscisin, a giftfrom Dr. J. van Overbeek (Shell), was used in allthe experiments reported here. A stock solution of0.2 mm abscisin in water was kept frozen or in therefrigerator.

The incorporation of 14C-letucine and "-C-uridinewas measured in the following way: Aleuronelayers were incubated as usual and 1 ,uc of 14C-leucine (200 mc/mmole) or of "4C-uridine (25mc/mmole) (both purchased from Schwarz Bio-research, Inc.) was added, and incorporation was

allowed to proceed for the appropriate period oftime. The aleurone layers were then rinsed 3 timeswith 1 mm cold, carrier leucine or uridine. When4C-leucine was used the aleurone layers were

homogenized in the cold in 5 ml of 0.2 N NaCl inthe ustual way, and the extracts were centrifugedfor 10 minutes at 2000 X g. An aliquot of 0.2 mlof the supernatant was plated on a glass planchetto determine the total uptake. Another aliquot of0.25 ml of the salt soluble proteins was mixed withan equal volume of 10 mm leucine and precipitatedwith 2 volumes of 15 % trichloroacetic acid. Theprecipitated proteins were collected by filtration on

Millipore filters and washed with 5 % trichloro-acetiic acid. The filters were dried and cotunted ina gas flow counter. When "C-uridine was usedthe aleuro-ne layers were homogenized in the cold in5 ml of 1 M NaCl with 5 mg of carrier RNA.Incorporation of the precursor into the salt-solublenucleoproteins was determined as described above,except that carrier uridine was used. This methodgives results similar to those obtained with more

elaborate extraction procedures for nucleic acids.

Results

Removal of Gibberellic Acid in Mfidcourse ofa-Amylase Production. The addition of GA toisolated aleurone layers of barley results in a linearsynthesis of a-amylase after a lag-period of 7 to 9hours. When aleurone layers are incubated in 0.05

Mt GA for 11 hours, frequent rinsing over a periodof 1 hour to remove as much GA as possible resultsin a substantial reduction of the a-aimylase syn-

thesized in the next 12 hour period (4). Gibberellicacid cain therefore not be considered as a triggerbecatuse its presence is required dturing the lag-periodas well as duiring the period of ca-amylase synthesis,if maximal enzyme synsthesis is to be obtained. Inthe present experiments the removal of GA was

improved by rinsing the aleurone layers 4 timesat half-hour intervals with a mediunm containing20 mm CaCI, and 1 mm sodium acetate buffer pH4.8. At the end of the rinsing period 1 set ofaleurone layers was further incubated with GA and1 without GA. The results of suich an experiment

are shown in figure 1. It is apparent that a-amy-lase syn,thesis is severely inhibited within a fe-whours, if GA is not added again after it has beenremoved.

\hen aleurone layers are incubated without GAfor 10 houirs, and 0.1 pm GA is added, no a-amylaseis produced in the first 5 to 7 hours indicating thatGA is required to overcome the lag-period. Thelag-period can be shortened, but not abolished byfirst incubating the aleurone layers in a low con-centration of GA (2.5 m/iM) and ad(iing a higheramount of GA (0.5 Mm) after 10 hours (fig 2).After such an increase in GA conceiitration thereis a lag-period of 3 to 4 hours before the aleuironelayers synthesize at-amylase at a rate equiivalent tothe one obtained with the higher GA concentration.

Once the lag-period has been overcome it cannotbe reintroduced by a prolonged incubation of thealeuirone layers in the absence of G.A. AleuroneIlayers were incubated for 8 hours in 0.05 JtM GA,and GA was removed as described above. Thealeurone layers were further incubated without GAfor 6 hoturs. The rate of enzyme synthesis hadslowed doown considerably at this time. Subsequentaddition of GA resulted in an immediate resuimptionof a-amylase synthesis at the control rate (fig 3).

Inhibition of GA Enhanced Synthesis of a-Amiiy-lase and Ribonuclease by Abscisin. The GA-en-hanced produiction of a-amylase by aleuirone layersis inhibited by abscisin (table I). Wkhen aleuironelayers are incubated with 0.1 Mm GA and 1 tom ab-scisin, very little enzyme is synthesized. Abscisin alsoinhibits the small amount of a-amylase synthesiswhich occuirs in the absence of GA. Addition of alarger amount of GA reverses this inhibition byabscisin, as shown earlier (3) and as (lemonstratedin a different way in table II. Aleurone layerswere inctubated with increasing amouint of GA(from 0.1 mPM to 0.1 mM) and either withoutabscisin or with 0.05 Mm or 5 umM abscisin. Theinhibitory effect of 0.05 FM albscisin is almost com-pletely reversed by 1 Mm GA. When 5 M/m abscisinare used reversal by GA never exceeds 30 % re-gardless of the amount oif GA used. This demon-strates that reversal is not merely a matter of themolar ratio of GA and abscisin.

Abscisin inhibits the GA enhanced production ofribonuclease in a similar fashion (table III). The

Table I. Inhibition of a-Amyl/ase Synthesis byA bscisin

Ten aleurone layers were incubated with buffer, 20mMi of CaCl, and with or without 0.1 ,uar GA and theconcentrations of abscisin indicated.

ca-AmvlaseTreatment per 10 aleurone layers

tgControl 44Control + 0.5 x\i abscisin 8GA 475GA + 1 Mu.i abscisin 67

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PLANT PHYSIOLOGY

300-

+ GA 500mMM GA

200-N.Shift

0 o

qa 100 0:3 ~~~~~~~GA *~%>

to | * o 2.5 mMM GA

| ~~~~~~~~~ ~ ~~~~~~~~~~I III * I

01300 -

+GA\* Control

200 O/I__TO

0- 0

0~~~~Abscisin

100 I Cycloheximide-GA

e GA -nh+ GA

8 16 24 8 16 24

HOURS OF INCUBATIONFIG. 1. Effect of removing GA at the end of the lag-period. Aleurone layers w~ere incubated fur 7 hours in 0.5 lAm

GA. GA was then removed by 4 consecutive one-half-hour rinses. The aleurone lay-ers were furthler incubatedeither with GA, or w ithout GA. anid a-amylase synthesis was measuired 6 and 14 hours la'ter.

FIG. 2. Shift from a low to a high concentration of GA. Aleurone layers were incubated in 2.5 mAuM GA or in500 mngM GA and ac-amylase synthesis was mneasured after 10, 16 and 22 hours. To 1 set of flasks the GA concen-tration ways increased to 500 mgm after the aleurone layers had been in 2.5 mAm GA for 10 hours (Shift).

FIG. 3. Effect of adding GA after it has been withheld for 6 hours. All conditions as in figure 1, except thatGA was added again at 15 hours to aleurone layers from which it had been withheld at 9 hours. Total a-amylasesynthesis was measured 15 and 23 hours after the start of the inctubation.

FIG. 4. Mid-course inhibition of ce-amvlase synthesis by abscisin and cycloheximide. Aleurone layers were incu-bated in 0.1 uM for 111 hours. At this time abscisin (5,UM) or cycloheximide (10 ,ug/ml) was added and a-amylasesynthesis was measured 2 and one-half, 5 and 10 hours later.

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CHRISPEELS AND VARNER-GA, ABSCISIN AND HYDROLASE SYNTHESIS IN ALEURONE CELLS 1011

Table II. Reversal by Gibberellic Acid of the AbscisinInhibition of a-Amylase Synthesis

Ten half aleurone layers were incubated in bufferwith 20 mm CaCl and the concentrations of GA andabscisin indicated. Activity of a-amylase was measuredin the medium and a tissue-extract after 24 hours.

a-Amylase per 10 aleurone layersGA concentration Abscisin concentration

0 0.05 ,u. 5 jM

lug P1 Pg0.1 muM 73 10 ...

1 muLM 248 7110mAM 522 280 220.1 pM 638 525 1011 ,AMt 662 584 189

10 AM 692 ... 1850.1 mM 580 ... 113

increase in ribontuclease which takes place in iso-lated aletirone layers in the absence of added GA,or in half seeds preincubating on moist sand (4)is also inhibited by abscisin.

In the experiments discussed abo-ve the 2 hor-mones, GA and abscisin, were added simultaneously.However, abscisin is also effective as an inhibitorof a-amylase synthesis if it is added in mid-course,while enzyme synthesis is in progress (fig 4).Aleurone layers were incubated for 11 hours in0.1 ,tm GA and 5 FM abscisin was added at thattime. Further synthesis of a-amylase was inhibitedcompletely after a delay of 2 to 3 hours. If bothhormones had been added at these concentratioins,at the same time, at the beginning of the incuba-tion, a-amylase synthesis would have been inhibitedcompletely. Addition of cycloheximide (10 jug/ml)at this time results in an imTmediate inhibition ofenzyme synthesis. It was also found that boththe production and the release of ribonuclease canbe inhibited by the addition of abscisin in mid-course (20-24 hrs after GA).

Requirenect for RNA Synthesis. ActinomycinD and various base analogues which inhibit RNA

Table III. Inhibition of Ribonuclease Production byAbscisin

Ten aleurone layers were incubated in buffer, 20mM of CaCl. and with or without 2 mjum GA and theconcentrations of abscisin indicated. Ribonuclease ac-tivity was measured after 48 hours in the medium andan extract of the tissue. Initial refers to the amountof ribonuclease present in the aleurone layers at thestart of incubation.

Units of riboniucleaseTreatment per 10 aleurone layersInitial 29Control 108Control + 0.5 pM abscisin 61GA 256GA + 5 Am abscisin 53

synthesis prevent a-amylase formation if they areadded at the same time as the hormone (4,23,24).The synthesis of a-amylase is inhibited to a muchlesser extent if actinomycin D is added 4 hoursafter GA, and is almost unaffected if the antibioticis added 8 hours after the hormone (table IV).A different effect is observed with 6-methyl-purine,a potent inhibitor of all RNA synthesis (11).When aleurone layers are inciubated with 1 UM

Table IV. Inhibition of a-Amylase SyntheSis and 14C-Uridine Incorporation by Actinomycin D Added 4 and

8 Hours After GATen aleurone layers were incubated in 0.1 jM GA

and after 4 or 8 hours actinomycin D (100 ,ug/ml)was added. Enzyme synthesis was measured at the endof the 24 hour incubation period. 1-C-Uridine (1 Aclflask) was added 4 hours after actinomycin D and in-corporation was allowed to proceed over a 4 hour period.

a-Amylase 14C-UridineTreatment per 10 aleurone layers incorporated

pg cpmGA 359 1380GA + Act. D

after 4 hrs 212GA + Act. D

after 8 hrs 325 466

Table V. Inhibition of a-Amylase Synthesis by6-Methylpurine

Ten aleurone layers were incubated in buffer, 20mM, CaCl2 and 0.5 pM GA. 6-Methyl,purine was addedat the same time as GA or 4 or 8 hours later and totala-amylase production was measured after 24 hours incu-bation.

Treatment a-Amylase per 10 aleurone layersTime of addition of 0.1 mm Of 1.0 mm Of

6-mnethylpurine 6-mnethylpurine 6-methylpurinepg Ag

0 hrs 38 94 hrs 115 558 hrs 208 140Control 384 426

GA and 0.1 mm 6-methylpurine a-amylase synthesisis inhibited by 90 %. However, if the analogue isadded 4 or 8 hours after the hormone enzyme syn-thesis is inhibited by only 70 % and 45 % respec-tively (table V). Larger inhibitions are obtainedwith 1 mm 6-methylipurine, and a-amylase synthesisis still inhibited by 66 % if this concentration ofthe analogue is added 8 hours after GA.

The failure of actinomycin D to inhibit a-amy-lase synthesis if the antibiotic is added 8 hoursafter the hormone could be due to its inability toinhibit RNA synthesis in general at this stage orto its inability to inhibit the RNA fraction which

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1012

is required for a-amylase synthesis. To test thefirst possibility aleurone layers were incubated in0.1 ,um GA, and 100 pg/ml of actinomycin D wereadded a(fter 8 hours. After 4 more hours 1 uc of'-C-uridine was added to each flask containing 10aleuro,ne layers an,d incorporation was allowed toproceed for 4 hours. During this period actino-mycin D inhibited the incorporation of '-C-uiridineby 66 % (ta,ble IV). \Ve can assume that theinhibition of RNA synthesis was even greater be-cause so-me of the incorporation represe-nts turrnoverof the terminal ends of the transfer RNA mole-cutles. (It was shoown by Chandra and Varner (2)that there is a rapid coniversion of ulridIine tocytidine in this tissuie). It appears then that actino-mycin D is a good inh bitor of '4C-uridine incor-poration whether it is added at the same time asGA (4) or 8 houirs after the hormone. This leadsto the conclusion that the inability of actinomycinD to inhibit a-amylase synthesis muist be duie toits inability to prevent the synthesis o,f the specificRNA fraction which is required for continuiieda-amylase synthesis.

Ta,ble VI. Sensitivity of a-Aniyliase Synthesis toActinomzvcin D and 6-Methylpurine After

Removal of GATen aleurone layers were incubated in buffer, 20 mM

CaCl. and 0.5 JAm GA for 9 hours. GA was removedby 4 stubsequent one-half-hour rinsings in a mediumwithout GA. The aleurone la-ers wxere further incubatedfor 11 hours without GA or wxith 0.5 yM GA and actino-mycin D (100 ALg/ml) or 6-methylpurine (0.2 mM).Initial refers to the amount of a-amylaese present inthe aleuironie layers after the removal of GA (at theend of the washing out procedure).

Treatluieint

Initial-GA+GA+GA + actinomnvcin D+GA + 6-methylpurine

a-Amylase per 10aleurone layers

lg61128285221153

PHYSIOLOGY

\We have shown above that GA is not a t_igger,but is reqllired contintuously for maximal a-amylasesynthesis. The following experiment wias done totest whether the a-amylase synthesis which is de-pendent on the second addition of G al so requiirescontinuied RNA synthesis. Aleulrone iavers wereinctubatecl for 10 hoturs in 0.05 toi GA aulm the GAwas removed by 4 successive one-half-lhour rinses.The aleuirone layers were then further inculbatedwithout GA, or with GA and with actinomycin D(100 jLg/Aml) or 6-methylpturine (0.2 mat). Therestults show (table VI) thaSt the a-amylase syn-thesis which is depen,dent on the second additionof GA is not very sensitive to actinomycin D butis very strongly inhibited by 6-methylpuirine. Syn-thesis of a-amylase under these circuimstances pro-ceeds without delay uipon the addition of GA, evenif GA has been withheld for mainy houirs (fig 3).This indlicates that the aletirone layers are capableof a rapid synthesis of the RNA fraction necessaryfor a-amyl'ase synthesis. When aleuiroine layers areincuibated with GA there is normally a 7 to 9 hourlag before a-amylase synthesis begins.

The observations disctissed above stiggest thatthis lag is not necessitated by the requirement forthe sy'nthesis of the metabolically- unstable RNAfraction, the synithesis of which must atccompanya-amylase synthesis and can be inhiibited by 6-methylpurine. It seems likely that other biochemi-cal processes are associated with the lag-period andmlist occtur before a-amylase synthesis cani start.The possibility that a more stable RNA ineedls tobe synthesized dulring the lag-perio(d can, of couirse,noLt be rtiled ouit.

To test whether the requirement for R.NA syn-thesis can be satisfied by incuibating for a longtime with a small amouint of GA, half seeds werepreinciibated for 3 days on sterile sand( moistenedwith GA (0.01 or 0.1 /jm.) instea(l of water. Thealeurone layers were removeid from the starchyendosperm and fuirther incubated wxith GA (1 uM)oir with GA and actinomycin D (50 pg/ml). Thedata show (table VII) tha,t actinomyciin D inhibitsenzyme synthesis to approximately the same extenit,whether the half seeds were preincuibated in water

Table VII. Inhibition otf a- Ayl/se Synihiesis by Actinomycin D Aft Ic Preincubation of Ho/f S.(d.8in Gibberellic A cid

Half seeds xxere preincubated in water or in GA (0.01 1AI or 0.1 /cI) for 3 days. The aleuironie layers w-ereremoved land fuirtlher incubated inl buffer, 20 mm, CaCI2 and 1 ,AI GA, and with or without actinomncin D (50 ,g/rnl).0 houirs refers to the amount of a-amylase present in the aleurolne la-Yers a; the start of the incubatioll.

Treatmenlt and time

0 hrs GA8 hrs GA24 hrs GA24 hrs GA + actinomycin D

a-Amvlase per 10 aleuironie layersPreiincubation Preinciibation

in water in 0.01 Am GA

Lg

1135

408240

Mg

2264

445

270

Preincubat ionin 0.1 tIM GA

Lg(;0-)

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CTI ISPEELS AND VARNER-GA, ABSCISIN AND HYDROLASE SYNTHESIS IN ALEURONE CELLS 1013

or in GA. As a result of preincubation in 0.1 uMGA a-amylase synthesis is initiated at a slow rate(compare a-amylase svnthesis at 0 and 8 hrs afterpreincubation in 0.1 jM GA with preincubation inwater), but this does not obviate the necessity forthe initiation of more RNA synthesis if a higherconcentration of GA is added.

Comitpairison of the Effect of Abscisin and In-hibitors of RNA Synthesis. We have shown thatthe addition of cycloheximide, a potent inhibitor ofprotein synthesis at the level of translation, toaletirone layers results in an immediate cessationof a-amylase synthesis and also of the incorpora-tion of 1-C-leticine into the celluilar proteins ofaleuirone layers (Varner, unpublished). AbDscisin,on the other hand, inhibits enzyme syntLhesis aftera lag of 2 and one-half to 3 hours. Experimentswith 6-methylpuirine show a continuous requiiremenitfor RN\A synthesis during the period of a-amylasesynthesis. It seemed, therefore, of interest to sttudythe mid-coturse inhibition of a-amylase synthesis byabscisin and base analogues and comipare theirkinetics.

Aleurone layers were incubated in 0.5 uM GAfor 11 hours to induce the GA enhanced synthesisoif a-amylase. At this stage the medlium was with-drawn, the aletirone layers were rinsed and furtherinculbated for 12 hours in a meditum containing GA(0.5 Nim) or GA and various inhibitors (10 FMabscisin, 0.5 or 5 mat 6-methylpurine, 5 mm 8-aza-gtuanine and 5 mm 5-azacytidine). The results intable AVIII indicate that abscisin and 5 mar 6-methyl-puirine give the same amount of inhibition (74 %)while 0.5 mmi 6-methylpurine and 5 mr 8-azagua-nine or 5-azacytidine are less inhibitory (58-39 %).The kinetics of inhibition of a-amylase synthesisby abscisin and the base analogues are very similar(fig 6). Abscisin inhibits a-amylase synthesiscompletely within 2 and one-halif to 3 hours, asshown earlier (fig 4). The base analogues also

Table VIII. Mid-course Inhibition of a-Anqlase byAbscisin and Base Analogue.i

Aleurone layers were incubated in buffer, 20 mMCa'Cl, and 0.5 xr GA for 11 hours. The medium wasdrawn off and the aleurone layers were rinsed. Thealeurone layers were further incubated for 12 hours in0.5 yIAu GA together with the inhibitors, as indicated.The figures represent the amount of al-amylase pro-duced in the 12 hour incubation.

a-AmtvlaseTreatment per 10 aleurone layers

Control 317Abscisin (10 ,uq) 826-Methvlpurine (0.5 mm) 1566-Methylpurine (5 mm) 845-Az<ac,'tidine (5 mar) 1948-Azaguanine (5 m-m) 132

c,,

-J

0lq

1o0F

50-

II 15HOURS OF INCUBA TION

19

FIG. 5. Mid-course inhibition of a-amylase synthesisby abscisin, 6-methylpurine and 8-azaguanine. Aleuronelayers were incubated in 0.05 SAM GA for 11 hours.At this time the medium was removed, the aleuronelayers were rinsed, and they were further incubatedwith 0.05 Am GA (*-*), or with GA and 10,Mm ab-scisiin (0-0), 5 msi 6-methylpurine (L-C), 0.5 ,UM6-methylpurine (U-U), or 5 mmi 8-azaguanine(A-A).

inhibit a-amylase synthesis completely within 2 andone-half to 4 hours depending on the concentrationused. The incorporation of "'C-uridine by thealeurone layers is inhibiited by 1 mm 6-methylpurinein the following way: 30 to 35 % within 2 andone-half hours of its addition and 65 to 70 % after4 hoturs of its addition. However, if the 6-methyl-purine concentration is incre'ased to 5 mM theinhibition of 14C-uridine incorporation is 65 to70 % within 2 and one-half hours. This may ac-count for the shorter lag time in the inhibition ofa-amylase synthesis observed with the higher con-centration of 6-methylpurine. The incorporation of1-C-leticine by the aleurone layers into the cellularproteins is inhibited by only 30 %, 4 hours after theaddition of 1 mw' 6-methy1purine. This is probablyan indirect effect mediated through its inhibition ofRNA synthesis. We therefore conclude that therapid inhibition of a-amylase synthesis caused by6-methylpuirine is not duie to a direct inhibition ofprotein synthesis.

Discussion

It is not yet possible to decide whether thesehormones work at the level of transcription or at

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PLANT PHYSIOLOGY

the level of translation. The data presented here,as well as the earlier results obtained by Varnerand coworkers (4, 22, 23, 24), are consistent withthe hypothesis that GA exerts its control at thelevel *of the gene, to bring about the synthesis o-fan RNA fraction specific for the proteins beingsynthesized. The similarity in kinetics for theinhibition of a-amylase synthesis by abscisin and6-methylpuirine or 8-azaguianine suiggests that ab-scisin exerts its control by inhibiting the synthesisof such a specific RNA fraction. However, mtuchcauition mtust be used in basing a model on the modeof action of the hormones on a study of the effectof inhibitors al,one. The data are equally consistentwith a control mechanism at the level of translationwith a requirement for continued RNA synthesis.

\Iany hormonal effects are inhibited by actino-mycin D (1, 9, 13, 17, 20,25), an(d this has oftenbeen interpreted as evidence that the hormonesaffect the synthesis of messenger RNA in a directway while all that can be concluided with certaintyis, that some kind of DNA directed RY.A svnthesismuist be allowed tLo go oIn before the hormonaleffect can express itself at the level of proteinsynthesis or of morphogenesis. Hybridlization ex-periments with bacterial ntucleic acids have shownregions of complementarity between DNA andmessenger RNA, ribosomal RNA, and transferRNA suggesting that the synthesis of all 3 majorclasses of cytoplasmic RNA is DNA dlirected. Thesynthesis of any or all of these 3 may be requiredfor the hormonal effect to become evident.

A second reason for cauition is that the hormone-enhance,d enzyme synthesis may resuilt in a morerapid tuirnover of one of the RNA's involved inthe synthesis of these enzymes (e.g. messengerRNA or a particular transfer RNA). This wouldresult in a requirement for R'NA synithesis, andsuich a process would be inhibited by actinomycin Dor by base analogues, even thoiigh the initial effectof the hormone is not at the lexel of transcription.

The principal, easilv observedl effect of GA onthe aleurone layers is the enhanceiment of the syn-thesis of several hydrolytic enzymes. RNA syn-thesis muist be allowed to occuir in ordler to obtainthis effect. A control mechanism at either tran-scription or transl'ation wo,uld probably involve thesynthesis of a new RNA. There dloes not appearto exist a great reserve of this critical RNA frac-tioIn. Indeed, base analoguies inhibit a-amylasesynthesis within 2 and one-half to 4 houirs andcontinued RNA synthesis must accompany enzymesynthesis. \Ve can also coniclu(le that this RNAfractioin is synthesized very rapAdly since additionof GA to aleurone layers from vxhich this hormonehas been withheld for several hours results in animmedliate resumption o,f a-amylase sylthesis whichis completely dependent oIn coIntinuedI RNA syn-thesis.

The principal, easily observed effect of abscis-in

is the inhibition of hydrolase synthesis (both normaland GA-enhan,ced). The kinetics of this inhibitionresemble those obtained with the base analogues6-methylpurine and 8-azaguanine. In the presenceolf 10 /uM ab,scisin or 5 mm 6-methyllpurine, at thisconcentration the 'base analogue inhibits '4C-uridineincorporation by 70 % within 2 and( one-half hours,a-amylase synthesis is completely inhibited within2 to 3 hours. \Vith 0.5 mm 6&methylpuirine or 5mm 8-azaguianine it takes somewhat longer beforeinhibition is complete. The incorporatioi of 14Cleucine is inhibited to a much smaller extent (30 %)than the incorporation of 14C-uridinle (70 % ) whenaleurone layers are incubated for 4 hours in 1 mm6-methylppurine. This suggests that tLhe inhibitionof protein synthesis is a result of the linhibitioon ofRNA synthesis, brought about by the (lecay ofshort-lived imessengers. This was confirmed byA. 'Morris (private communication) who showedthat 10 mm 6-methylpurine has nIo effect onl theincorporation of 14C-valine into the proteins ofrabbit retictulocytes, indicating that the anialoguehas no direct effect on protein synthesis in thissystem. (Reticulocytes have no nicleuis and pro-tein syinthesis is independent of contlinluedl RNAsynthesis). The 2 and one-half to 3 hour (lelayin the inhibition of a-amylase synthesis which isobserved with abscisin as well as with the baseanalogues could be due to the presenice of a suiffi-cient amount of the critical RNA fraction necessaryto suistain enzyme synthesis for this period of time.

Howevrer, similar kinetics might be cxpectedl ifabscisin exerted its action at the level of translationby inhibiting the incorporation of this RN\ iintoan active enzyme-synthesizing uilit. JIn this casethe delay w%ouild reflect the half-life of this activecomplex.

The inhibition of a-amylase sy-nthesis h! abscisillcan be overcome, at least partially, b1 the addclitionof a larger amotint of GA. How\ever, if too higha concelntration of abscisin is uised, llo rev-ersal isobtaine(l. Similar observations \ ere mla(le hy J.van Oveerbeek (personal communication) who stuld-ied the inhibition of growth of Lc, nim minor byabscisin aind the reversal of this ilhibition bybenzyladenine. Reversal by beuzy-ladenie onilytakes place if growth has not been comlpletelv in-hihitedI by abscisin.

A large nuimlber of sttudlies indicate thatl planthormones increase the rate of prote in anid RNAsynthesis in the tisstues to which they are appliedl.II some systems this can be measlire(l Vy ani ill-crease in the total amotunt of prote n anid RNA\,while in others there is only an increarse in the rateof incorporationi of radioactive precursors inltothese macromolectules. Hormone applicationi alwaysrestults in a general derepressioni of RN-X syinthesisand in Ino instanIce has a system been fo(und in whichthe hormone preferentially increases t1he sx uthesi sof 1 particuilar RNA fraction.

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CHRISPEELS AND VARNER-GA, ABSCISIN AND HYDROLASE SYNTHESIS IN ALEURONE CELLS 1015

Gibberellic acid does not enhance the incorpora-tion of 14C-uridine into the RNA3 or of '-C-leucineinto the cellular proteins of isolated aleurone layers,dissected from the half seeds after the customary3-day preincubation period (Chandra, Chrispeelsand Varner, unpublished); neither does abscisininhibit these processes (3). Although the hor-mones appear to have no effect on protein synthesisas measured by precursor incorporation, they doaffect the synthesis of specific enzymes. Thisobservation ihas not been matched at the RNAlevel, and we have been unable to find a specificRNA fraction (after extraction of the RNA andchromatography on methylated albumen kieselgtihrcolumns), the synthesis of which is evoked by GAand inhibited by abscisin. Recent improvements inthe methods used to extract nucleic acids fromanimal tissues (12) and to separate she variousRNA's (5, 14) will be applied in our search forstuch an RNA fraction. The possibility that GAand abscisin exert their control at the level oftranslation can be checked by studying the effectwhich these hormones have on the in vitro svn-

thesis of a-amylase by a subcellutlar fraction iso-

lated from aleurone layers.

Acknowledgments

We acknowledge with pleasure the skillful technicalassistance of Miss Nancy Joseph. We thank R. A. Nilanfor supplying the Himalaya variety of barley, and A.Morris for determining the effect of 6-methylpurine onamino acid incorporation in raibbit reticulocytes.

The actinomycin D was a gift from Merck, Inc.

Literature Cited

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2. CHANDRA, G. R. AND J. E. VARNER. 1965. Gib-berellic acid controlled metabolism of RNA inaleurone cells of barley. Biochim. Biophys. Acta180: 583-92.

3. CHRISPEELS, M. J. AND J. E. VARNER. 1966. In-hibition of gibberellic acid induced formation ofa-amylase by abscision II. Nature 212: 1066-67.

3 Chandra and Varner (2) and Varner et al. (24) re-ported a GA-enhanced incorporation of radioactive pre-cursors into the RNA of aleurone layers 8 to 24 hoursafter the start of imbibition of half seeds. Navlor (15)observed a GA-enhanced incorporation of 3H-cytidineinto the nuclei of aleurone cells from wild oats, 24 hoursafter the stirt of imbibition. It seems likely that in thesecases GA is accelerating processes which are associatedwith inbibition or with biochemical changes which followit. It is exactly for this reason, to avoid all biochemicalchanges associated with imbibition, that half seeds arepreincubated for 3 days before we study the effects ofGA and zibscisin on aleurone layers.

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18. OGAWVA, Y. AND S. IMAMURA. 1965. Effect ofplant extracts and gibberellin A3 on a-aTnylaseproduction in embryoless rice endosperm in rela-tion to growth promoting activity. Proc. JapanAcad. 41: 842-45.

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