in the metabolism of cereal grains - plant physiology

Plant Physiol. (1969) 44, 1227-1232

A Survey of the Sequence of Some Effects of Gibberellic Acidin the Metabolism of Cereal Grains

Clifford J. PollardDepartment of Botany and Plant Pathology, Michigan State University,

East Lansing, Michigan 48823

Received February 4, 1969.

Abstract. The sequence in which a variety of enzymes and metabolites are affected bygibberellic acid after application of the hormone to aleurone layers of half seeds of barley(Hordeum vulgare var. Betzes) and half seeds of wheat (Triticum aestivum var. Gensee) wasinvestigated. With barley aleurone layers the first hormonal effect observed was the increasedsecretion of soluble carbohydrate, some of which appears to be a glucan containing some 1-1,3linkages. This was followed by increased oxygen consumption and increased secretion ofATPase, GTPase, phytase, phosphomonoesterase, phosphodiesterase, inorganic phosphate,carbohydrates other than amylase, peroxidase and amylase. Similar sequential effects wereseen in wheat half seeds. Increased activity of alcohol dehydrogenase in barley seeds waselicited by the hormone but there was no effect on glucose.6-phosphate isomerase.

It has been realized for some time that the activi-ties of several enzymes increase after treatment ofcereal grains with gibberellic acid (GA). It hasbeen impossible, however, to even approach the for-mulation of a unified concept relating to the mode ofaction of the hormone because of a lack of knowledgeof the identity of the enzymes affected, and perhapsequally important, of those not affected. Further-more, neither the sequence of metabolic events ac-companying the hormonal effects and their inter-relationships nor the universality of the hormonaleffects have been explored. Such knowledge isdesirable and probably necessary in attempts to relatethe phenomena to the operation of the entire cellularapparatus.

The studies reported here were initiated in orderto explore A) the identity of some of the enzymesaffected by GA as well as some that do not respond,B) the sequence in which they are affected, C) theuniformity of the responses as evidenced by a simi-larity of the responses in barley and wheat, D) theidentity of other metabolic effects preceding or ac-companying the effects of the secretion of enzymes.

In a recent note from this laboratory (8) it wasreported that the sequential increased secretion ofsoluble carbohydrate, ATPase, GTPase, and phvtaseare the earliest events we were able to detect in acomplex of responses noted after application of GAto barley aleurone layers. The relationship of theeffects to oxidative metabolism and RNA synthesiswas also given. The present paper documents morefully the sequence by giving the identification andposition of other enzymes and metabolites in thesequence noted witl barley. Also, the effects of GAon the respiration and secretion of enzymes fromwheat are given.

Materials and Methods

Barley seeds (Hordetuml ulydgare var. Betzes)were dehusked by treatment with 50% sulfuric acidas suggested by Coombe et al. (3). The acid treat-menit was followed by extensive treatmiienlt with dis-tilled water. Seeds were then allowed to imbibedistilled water at 30 for the periods indicated. \Vheatseeds (Triticiumt aestiz'umn var. Genesee) were ger-minated on moist paper towels at room temperature.Immediatelv prior to use all seeds were surfacesterilized with cold I % sodium hypochlorite solutionfor 15 min and then washed with copious quantitiesof cold distilled water. The embrvo ends from seedsselected for uniform size were remove(l wvith a razorblade and discarded. Aleurone layers were preparedas described previously (8).

Oxygen consumption was measured by conven-tional \Varburg manometry at 25°; the flasks con-tained 3.0 ml of solution. All experimental solutionsfor incubations contained 250 ,g streptomycin sul-fate/ml and in some instainces 20 lag l)enicillin G/mlwere used additionally.

The respective para-nitrophenyl derivatives wereused as substrates for measuring the activities ofphosphomonoesterase, sulfatase and carbohvdrasesother than amylase. Bis para-nitrophenyl phosphateand the acetate derivatives were substrates for phos-phodiesterase and esterase activities, respectively.Aliquots of the incubation media were taken at inter-vals, centrifuged briefly and incubated with 0.11 mgof the para-nitrophenyl derivatives in 0.1 ml of waterand 1.0 ml of 0.05 Mi citrate buffer, pH 5.4 forone-half to 2 and one-half hr. The reaictions werestopped by the addition of either 0.5 ml of 0.1 Mutris buffer pH 8.5 (for esterase and sulfatase activi-

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PLANT PHYSIOLOGY

ties) or 0.5 ml of 1 % NaOH and read in 1.5 mlcuvettes in a Beckman D U. spectrophotometer at400 mi ol in a Klett colorimeter with a No. 42filter after samples were diluted to 6.0 ml with water.ATPase anld phytase were determined as previouslydescribed (8). Amylase activity was determined asgiven by Momotani and Kato (7), whereas perox-idase activity w,vas determined as outlined by Luck

Alcohol dehvldrogenase was measured as describedbv Racker (9) on aliquots of homogenates from halfseeds. Ten incubated half seeds were homogenizedwith 10 ml of cold 0.1 at potassium phosphate bufferpH 6.5 in an all-glass Tenbroeck homogenizer. Thehomogeniates were centrifuged at 3000g for 10 minatnd the snipernatant soltutions used as a source ofenizyme.

Glucose-6-p isomerase activity was assayed bythe appearance of fructose 6-p as measured with theSeliwanoff reagent (10). The homogenates were

prepare(l as \-was done for alcohol dehydrogenaseassays except that tris buiffer pH 9.0 was used as

the homogenizing medium.

Total soluble carbohydrate was determined by theanthrone method (12). Material reacting with theFolin Reagent was determined by the Lowry method(11). Inorganic phosphate was assayed as givenhy Ames (1). One ug G A\/ml -was used unless-,therwise noted.

Table I. The Effect of GA on the Secretion of EnzymesIntto the Incuibation Mediwiin by Barley Half Seeds

Experiment 1. Half seeds (150) were prepared fromseeds which had imbibed water for 72 hr at 30 wereplaced in 100 ml of solutions containing antibiotics givenin Materials and Methods; 1 ,ug GA3/ml was used intreated tissue. The half seeds were allowed to standfor 12 hr at 00 in these solutions. They were thenshaken at room temperature and aliquots of the incu-bation media were removed at the time intervals indi-cated and assayed for the activities of the enzymes given.Values in the table were obtained by dividing the ac-tivity found in the controls into the activity found inthe medium of the treated seeds.

Ratio of treated to controlactivity

HourEnzyme

PhosphomonoesterasePhosphodiesteraseEsteraseSulfatasea-Glucosidase/3-Glucosidasea-Galactosidase3-GalactosidaseAmylasePeroxidase

3 6 9 12

1.001.001.121.001.001.091.501.001.001.00

1.341.841.131.041.001.241.331001.001.00

1.001.001.041.001.001.001.001.001.001.00

4.703.861.131.131.572.321.751.671.671.44

Results

Thc SeqieqLce of GA:, Effects on Secretion ofEnz,W;nes anid Metabolites by Barley Seeds. TablesI and II slhow that the effects of the hormone on

enzymes and metabolites of half seeds may be dividedinto 3 categories; those that are elicited fairly early,those that are seen later and those not evident untilafter prolonged incubation. Among the enzymes

esterase and a-galactosidase appear to 'be affectedearliest; effects on phosphatases, sulfatase and,f-galactosidase are seen later whereas increasedexcretion of f8-glucosidase, peroxidase and amvlaseis noted still later. Both tables are included to indi-cate the variability in the time at which the effectswere noted in these studies. Cognizance should betaken, however, of the differences in condition ofimbibition of wvater given in the legends.

The pattern is more obvious in table I than intable II, although it is clear in both tables that theenzvmes are not affected in concert. Among themetabolites an effect on pi is seen earliest. Increasesin total soluble carbohydrate and Folin-reacting ma-

terial are observed at about the same time but beforeincreases in amylase, /8-glucosidase or peroxidase are

noted (table II).The sequence of events with aleurone layers

appeared to be slightlv different from that observed

withl half seeds, although increased amount of allenzymes appeared earlier than in half seeds. Themajor difference was the smaller amounts of esterase,

Table II. The Effect of GA3 on the SecretionEnzymes and Metabolites Into the Medium

by Barley Half Seeds

of

Experiment 2. Conditions the same as legend in tableI except that seeds were allowed to imbibe water atroom temperature for 20 hr and 300 half seeds in 250ml of incubation media were used.

Ratio of treated to controlHour

Enzyme or metabolite 9 12 15

Phosphomonoesterase 1.00 13.90 ...

Phosphodiesterase 1.00 12.00 ...

Esterase 1.53 3.13Sulfatase 1.00 1.15 ...a-Glucosidase 1.00 1.00 3.14/3-Glucosidase 1.00 4.58a-Galactosidase 1.00 2.383-Galactosidase 1.00 2.16Peroxidase 1.00 1.00 1.39Amylase 1.00 1.00 2.62Inorganic phosphate 1.12 1.26Soluble carbohydrate 1.00 1.42 3.24Folin reacting material 1.00 1.35 2.64

1 228

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POLLARD-SEQUENTIAL METABOLIC EFFECTS OF GIBBERELLIC ACID

Table IV. Timtle Course of Appearance of Enzymes in the Mediumiii of Barley Half Seed.sConditions identical to those listed in table I.

Activity'Phosphomonoesterase a-Galactosidase Peroxidase

Time Control +GA Control + GA Control +GAAOD 400 m,X103/hr'0.5 ml

168225470

2225

270285360520

,OD 485 niuX103/10 min50 uLl

250354480900

105

135100

100195200

a- and /8-galactosidase and a-glucosidase secretedby the layers. The first metabolic responses notedwere increased secretion of carbohydrates and thephosphatases as illustrated in part in table III anddocumented previously. That increased respirationoccurs after sugar secretion was also given (8).The times at which phosphomonoesterase and phos-phodiesterase were affected varied somewhat, ap-parently because the rates of secretion of these wereoften greater than other enzymes. Usually theywere affected after phytase. The carbohydrases otherthan amylase responded next as shown in table IIIfor 8-glucosidase. Peroxidase and amylase wereuniformly the last enzymes to respond. In half seedsincreased secretion of inorganic phosphate is one ofthe first effects of GA noted but this is somewhatdelayed in aleurone layers. On the other hand,effects on the secretion of soluble carbohydrates aredelayed in half seeds. These variations can probablybe explained on the basis of the relative ease or

Table III. The Effect of GA3 on the Secretion ofEntzymwes and Metabolites Into the Mediumn

by Barley Aleurone Layers

Thirty aleurone layers prepared from seeds that hadimbibed water for 8 days at 30 were shaken in 15 ml ofsolution containing 1600 Asmoles calcium chloride. Fourlayers and 2.0 ml medium were removed at intervals andassayed. Values for amylase are arbitrary since mediumfrom control showed no amylase activity.

Ratio of treated to controlHour

4 5 6 8 16

Soluble carbohydrate 1.13 1.98 2.23ATPase 1.15 1.97 3.20Phytase 1.00 1.64 4.20 ...Phosphomonoesterase 1.27 1.50 2.00 2.92 4.108-Glucosidase 1.00 1.04 1.08 1.10 1.22Peroxidase 1.00 1.00 0.85 8.90 9.00Amylase 0.00 0.00 0.40 2.10 2.70

difficultyt these metaholites have in traversing thestarchy endosperm in order to reach the incubationmedium. Other experiments demonstrated that all ofthe responses to GA application reside in the aleuronelayers; whereas dissected barley starchv endospermtissule may contain small amounts of the enzymes andmetabolites. it does not respond to the applicationof GA.

Table IV gives the time course of the appearanceof 3 of the enzymes in the medium. This table givesactual values obtained. The patterns of secretion ofphosphomonoesterase and a-galactosidase are similarbut the relative amounts secreted are different. Ingeneral. the magnitude and to some extent the patternof enzyme excretion varied.

Effect of Gibber ellic Acid Concentrationi on.Secretion of Barley Enzymes. The enzymes of bar-ley respond differently to varying concentrations ofgibberellic acid (table V). Responses by the phos-phatases are similar, saturation levels of the hormonebeing around 10-6 g/ml. The results from bothgalactosidases and peroxidase suggest 2 levels ofsaturation of GA::. The saturating concentration ofthe growth factor for esterase was around 10-5,whereas an increase in 8-glucosidase was still beingnoted at the highest concentration used.

Evidence for Heterogeneity of Soniic Enzynwcs.Few attempts have been made in these studies tocharacterize the enzy-mes secreted by the seeds.Because time course studies indicated increased ratesof secretion of some enzymes following a levellingoff of secretion and because certain enzymes showed2 apparent levels of saturation in response to varyingconcentrations of GA, it was suspected that severalspecies were being excreted. Upon chromatographyon DEAE cellulose, the incubation media fromtreated barley half seeds showed the presence of 2phosphomonoesterases. The media also showed 2apparent pH optima for hydrolyzing ATP. Thusthe activities measured for many of the enzymes areprobably due to the presence of several enzymes.Momotani and Kato (7), and recently Jacobsen andVarner (4), presented evidence for the secretion of

hr3691214

175285350480

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1230 PLANT PHYSIOLOGY

Table V. Effect of GA3 Concentration on the Secretion of Barle,y EnczymeiTwenty lhalf seeds prepared as in table I were incubated in 20 nil solutions for 36 hours. Aliquots of incubation

,lledia were assayed.

EnzyTme activity'Phosphio- Phospho- a A- -

('a., Conici. imionoesterase diesterase Esterase Galactosidase Galactosidase (Glucosidase Peroxidase

g/inl0 27 30 20 45 30 5 60

10-. 244 360 47 135 95 35 12010 292 410( 72 208 172 45 20810-' 300 420 80 200 161 75 15510- 294 405 85 218 158 93 17010-4 280 390 67 180 190 112 260

For peroxidase, activity AOD 485 m#i X 103/10 m-nin-50ul. For othier- eiz3 mes, activity -AOD 400 mwu X103/2 hrO0.5 ml.

a multiplicity of amylases 1b barley seeds and aleu-rone layers.

The Enhanceentw of ti/i Apparcnt Synthesis ofA41cohol Dehlvdrogcnase of Seeds by GA. Up tonow, all of the enzymes whose svnthesis have beenshown to be stimulated by gibberellic acid, with theexception of peroxidase. are hydrolases. Anothercommon featuire is that the enz\ymes are secreted intothe mediumi. Ani enzyme whose activity is increasedy- GA. that is niot a hvdrolase and, does niot appear

to be secreted into the medium is alcohol dehydro-grenase. It is evident fronm thle restilts shown intable VT that the hormone cauises upl) to a 2 foldincrease in the enznime in the seeds, the inlcreasebeing Iinear betw-een the eighteenith and twventy-fourthhr. It has not been p)ossible to deinonstrate thepresence of the enzymne in the meditim. yet losses in

Table VI. Effect of GA. on( Alcohol J)chydroyenaseActivity of Half Sceds

Half seeds treated as given in legenid in table 1.FHonmogeniates were prepared fromii 10 hlalf seeds after-iticubatioln. Aliquots (0.2 il) of 3000g supernatantsolutions w\ere used for- assays. (ivcloheximi(le contcenl-tration was 20 ,ug/nl.

activity occ-uLr in the seeds between the twenty-fourthaind thirtv-sixth hr to the extent that the activity- oftreated seeds is slightly less than control seeds.

The Failure of GA to Enhance the Activity ofGhlicose-6-P Isonmcrase of Barley Seeds. As part ofthis exploratory study- 1 have attempted to ascertainthe presence in barley seeds of certain enzymes thatare components. of major pathways or whose sub-cellular localizations are known with certaintv. TheIltimate goal being to determine whether suchenzymies reslpond to the hormone. Barley half seedsshowed measurable amlouints of glucose-t-P isomeraseactivitv. The data in table VII illustrate that theenizyme showed Ino appreciable response to GA overa 24 hr Incubation period. Equally interesting, theactivity of the control seeds did not increase overthat of unincubated seeds. The data is representa-tiv-e of 3 exl)eriments done specifically to note theeffect of the hormiiones on the isomerase. Otherenz\vnies that are usuall1 affected by the hormone-esponded as usual.

7The Natuire of the Soluble Carbohydrate Secreted.lHecau-se of the possibility that the increased carbo-hvdrate secreted (tables II, III) may be unique inorigin and structure and because this apparent pri-miary respoinse may have been elicited from the site

Series 1 Coiltrol

hr242424

Series 21518222436

Activity+ GA.A

+ GA,, Cycloheximide

OD 340 in.p >X 108 per 10 min

200280220

405355355

205240275

165 245 14516; 260 175200 355235 395 255165 135 110

Table VII. Absence of Respontse of Glucose 6-PIsoincrase of Barley Half Seeds to GA

Seeds treated as given in legend in table I. Homo-genates made from 10 half seeds were incubated for24 hr. Unincubated seeds were kept at 30.

Enzyme activityug fructose 6-P/hr*02 ml

Uninicubated half seedsControl+ GA+ GA + cycloheximiiide (3 /Ag/iml)

142143151153



POLLARD-SEQUENTIAL METABOLIC EFFECTS OF GIBBERELLIC ACID

of action of GA, preliminary experiments were con-ducted to determine the nature of the sugar.

A sizeable portion of the soluble carbohydrateconisists of a polysaccharide. The material is pre-cipitated in cold 80 % (v/v) ethanol; it was purifiedby repeated suspension in cold 50 % (v/v) ethanol.It was not hydrolyzed by dilute acid, however, uponhydrolysis at 1000 in 2.5 N HCl 85 % of the poly-saccharide was liberated as glucose. as determinedwith glucose oxidase. Shorter periods of hydrolvsisresulted in lesser amounts of glucose being liberatedand longer periods resulted in the destruction ofglucose. So the actual glucose content is probablygreater than 85 %. The product of reaction of thematerial with carbazole gave an absorption spectrumidentical to that given bv glucose and was distinctlvdifferent from the ones given by mannose and galac-tose.

The polymer appears to possess an average of 1

glucose reducing equivalent per 25 glucose residtues.It was not attacked by several preparations of cellu-lase which were all active toward carboxymethvlcellulose, nor by emulsin, a-glucosidase or amylasefrom plant and bacterial sources. On the other hand.treatment of Lhe material with f8-1,3 glutcanase fromSclerotinia libertiana (obtained from Dr. A. Kivilaan,who originally obtained it from Professor Y. Sato-mura) resulted in the liberation of 25 % of theglucose content as free glucose. It is tentativelyconcluded, thnerefore, that the material is a glucancontaining some /-1,3 linkages.

On the Genesis of the Enlzymes Secreted. In theprevious communication, evidence was given thatsome of the enzymes that are secreted are preformned,whereas others are activated or synthesized (8').Hence the effects of GA appear to be solely on

secretion in some instances and on secretion atnd

synthesis of enzymes in others. More extensiveevidence of this is given in table VIII. The datain the table demonstrate that in GA treated aleuronelayers supplied with labeled leucine there was in-creased secretion of ATPase activity as early as thethird hr and bv the sixth hr the activity 'lad increasedmore than 11 fold over the control. Secretion oflabeled proteins began at about the fourth hr but theradioactivity in these was not significantly differentin treated tissues until the sixth lhr. Tlhus theATPase secreted earlier than the sixtlh lhr was prob-ably preformed and unlabeled. Note also that thesecretion of ATPase reached a plateau around theeighth hr, which suggests that the source of theenzyme became depleted. Since the appearance ofradioactive protein in the medium precedes that ofamylase, it is possible that this enzyme is labeled,hence is synthesized, de oiiooV. a fact that appears tobe well established (12).

In an experiment designed to ascertain xwhetherthere was a net increase in ATPase and phytase intreated aleiirone layers, the activity of homogenates

prepared from layers as well as aliquots from themedium was determined. The values (arbitraryunits) for ATPase at the second, fourth and sixthhrs were 5.3, 5.5, and 5.2, respectively. Correspond-ing values for phytase were 3.0, 4.3 and 8.0, re-spectively. Increased amounts of ATPase over un-treated controls were found in the medium at thesecond hr. Phytase responded at the fourth hr.Thus, although GA effected increased secretion ofboth enzymes, there was a net increase in phytaseonly.

Effects of GA on Enzymes of 'heat Seeds. Thedata in table IX illustrate that the GA3 effects seenin wheat seeds germinated at room temperature aredistinctly different from those seen in barley. Itappears that the response of such seeds to GA3 isalso different in wheat half seeds which are allowedto imbibe water at 3°. With the seeds germinatedat room temperature there was absolutely no effecton a-galactosidase activity over a 17-hr period. Thislack of effect was noted also for /8-gala ctosidase andboth glucosidases (data not shown). It should bepointed out that the absence of GA3 effect did notderive from the inability of the seed to secrete theenzymes. There were large increases in activitiesof the enzymes in the medium of control and treatedtissue. A small but definite effect was seen onphosphomonoesterase beginning at the fourth hr, butthere was no suggestion of an effect on phospho-diesterase until the ninth hr. Unlike barley whereincreases in amylase and peroxidase were noted atabout the same time, the latter enzyme was affectedby the seventh hr, whereas increased amylase activityappeared between the ninth and seventeenth hr.

In contrast, when dry, embryoless wheat halfseeds were allowed to imbibe water at 30 for 24 hrand then treated with the hormone definite increasedsequential secretion of the phosphatases and carbohy-(Irases was noted. For example, in the case of phos-phomonoesterase the ratio of control to tested activitywas 1.25, 1.33, 1.61, and 2.12 at 14, 17, 21, and 24 hr.respectively. (Parenthetically, in this experimentthe secretion of peroxidase was completely dependentoIn the presence of the hormone and the enzyme couldnot be detected in the treated medium until after20 hr of incubation). One interpretation of thedifferenices in responses due to different treatmentof wheat seeds is that the seeds allowed to germinateat room temperature produced enough endogenoushormlone to affect certain enzymes, such as thephosphatases, but not enough to affect others, suchas amylase and peroxidase. Presumably, when halfseed, are allowed to imbibe water at the cold tem-perature no hormone is produced.

Wheat half seeds pretreated at 30 as above alsoslhowed increased oxygen consumption after treatmentwith GA beginning around the thirteenth hr. In anexperiment the cumulative oxygen consumption of10 half seeds from the thirteenth through the six-teenth hr was 115 ml for control of 150 for treated.

1231



PLANT PHYSIOLOGY

These values were averages from 4 flasks. So, theability of the hormone to enhance respiration and thesequential secretion of metabolites in these cerealgrains appears to be a general property of GA.

Discussion and Conclusions

Several points slhould be emphasized. Firstly,this work has been concerned primarily with secre-

tion of the enzymes, since it was found that differ-ences in treated and control tissues are first seen inthe medium. Secondly, the metabolic events we

report may be preceded by others, since there are

probably no criteria that can be applied to ascertainwhether the earliest event we have noted here lhasabsolute primacy.

In studies related to the present ones the follow-ing were noted: MacLeod et al. (6) observed thesequential formation of endo /3-glucanase, acid plhos-phatase, amylase and protease in barley endospermslices treated with GA. Chrispeels and Varner (2)demonstrated that the dose response curve of ribo-nuclease to GA as well as the time which the enzyme

was secreted into the medium differed greatly, fromamylase. Jacobsen and Varner (4), on the otlherhand, noted a parallelism in the time and rates ofrelease, dose response to GA and effect of inlhibitorson the production in protease and amylase. Thissuggested to them that the enzymes are produced as

a unit. The apparent discrepancies in all of thesestudies can probably be explained on the basis ofvarietal differences and the possibility that some ofthe studies involved synthesis and others involvedsecretion or a combination of these.

The large number of enzymes and metabolitesaffected by GA in aleurone layers has been emplha-sized by these studies; it is likely that they representa small fraction of the total numbers under actualcontrol. This suggests that the effects of GA are

probably exceedingly complex. It seems that thecritical findings reported here are A) the effects are

not evoked in concert, B) the first effects that have

been noted are on the secretion of soluble carbohy-drates and phosphatases and C) the sequential effectsappear in barley and wheat. These may be signifi-cant, since it may be possible to determine whetherthey are independent on corollary responses, hence,

whether the hormone is acting catalytically to set intomotion a chain of events. Further, it may be pos-

sible to determine the origin of the metabolites and

enzymes, hence the probable site of action of thehormone. The existence of the responses in barleyand wheat suggests that the effects are generalproperties of the hormone and therefore legitimatefor studies on the mode of action of the lhormone.

Literature Cited

1. ANIES, B. 1966. Assay of Inorganic Phosphate,Total Phosphate and Phosphatase. In: Methodsin Enzymology. Vol. VIII. S. P. Colowick andN. 0. Kaplan, eds. Academic Press, New York.115-118.

2. CHRISPEELS, M. J. AND J. E. VARNER. 1967. Gib-berellic acid-enhanced synthesis and release ofa-amylase and ribonuclease by isolated barleyaleurone layers. Plant Physiol. 42: 398406.

3. COOMBE, B. G., D. COHEN, AND L. G. PALEG. 1967.Barley endosperm bioassay for gibberellins. I Para-meters of the response system. Plant Physiol. 42:105-12.

4. JACOBSEN, J. V. AND J. E. VARNER. 1967. Gib-berellic acid-induced synthesis of protease by iso-lated aleurone layers of barley. Plant Physiol. 42:1596-1600.

5. LeCK, H. 1963. Peroxidase. In: Methods of Enzy-matic Analysis. H. U. Bergmeyer, ed. AcademicPress, New York. 895-97.

6. MAcLEOD, A. M., J. H. DUFFUS, AND C. S. JOHNS-TON. 1964. Development of hydrolytic enzymes ingerminating grain. J. Inst. Brewing LXX: 521-28.

7. MOMOTANI, Y. AND J. KATO. 1966. Isozymes ofa-amylase induced by gibberellic acid in embryo-less grains of barley. Plant Physiol. 41: 1395-98.

8. POLLARD, C. J. AND B. N. SINGH. 1968. Earlyeffects of gibberellic acid on barley aleurone layers.Biochem. Biophys. Res. Commun. 33: 321-26.

9. RACKER, E. 1955. Alcohol delhydrogenase frombakers yeast. In: Methods in Enzymology Vol. I.S. P. Colowick and N. 0. Kaplan, eds. AcademicPress, New York. 500-03.

10. SLEIN, M. W. 1955. Phosphohexoisomerase frommuscle. In: Methcds in Enzymology Vol. I.S. P. Colowick and N. 0. Kaplan, eds. Aca-demic, New York. 299-306.

11 SPIES, J. R. 1967. Colorimetric procedures foramino acids. In: Methods in Enzymology, Vol.III. S. P. Colowick and N. 0. Kaplan, eds. Aca-demic Press, New York. 467-77.

12. UMBREIT, W. W., R. H. BURRIS. AND J. F. STAUF-FER. 1964. Manometric Teclhniques. Burgess Pub-lishing Company, Minneapolis, Minnesota.

1232



Plant Physiol. (1970) 45, 365

Corrections for Volume 44 (1969)

Page 264. Fig. 4. legend should read "Effect of glycerol on super-natant NADP-IDH."

Page 274. Column 1, 4th paragraph. "Significant differences (5%level) were found for OD260 fraction numbers 5 and 6 between11 and 14 days), OD260....." should read: "Significant differences(5% level) were found for OD260 fraction number 3 (increasedbetween 11 and 14 days)...."

Page 343. Column 1, line 15. .... was developed with l-butanol:pyremidine. . ." should read: .... was developed with 1 butanol:pyridine. . ."

Page 343. Column 2, last paragraph. "The bathochromic shiftwith AlCI3 to a 291 nm peak. . ." should read: "The batho-chromic shift with AlCl3 to a 305 nm peak. . ."

Page 349. Column 1, lines 7 and 8. "Apparent KA values for K+,Rb+, NH4+, and Nat were respectively: 2.4 X 10-2, 9.3 x 10-3,3.7 X 10-3 and 1.6 x 10-M. should read: "Apparent KA valuesfor K+, Rb+, NH4+, and Na+ were respectively: 2.4 X 10-2,9.3 x 10-3, 1.6 X 10-2 and 3.7 X 10-3 M."

Page 487. Table I, Expt. 4, column headed "Mean potential."Delete -191 + 6.

Page 624. Table I, next to last line. "24 hr R 1.61 ± 1.27"should read: "24 hr R 16.1 :i 1.27."

78. Column 2, line 34. "not likely" should read: "mostlikely."

Page 875. Table I. Tracheids X 104 in the no kinetin columntreatment was "7.70 + 0.66" should read: "1.70 i 0.66."

Page 1141. Running head. "APP- Aminoacyl-tRNA. . ." shouldread "Craker and Abeles-Aminoacyl-tRNA. . ."

Pages 1143, 1144, 1145, 1147, and 1149. "Cracker" should read"Craker."

Page 1147. Table 3, last line. "Abscisic acid + C2H2 60 44a"should read: "Abscisic acid + C2H2 20 44a."

Page 1161. Column 1, 2nd paragraph, last line. "NAA" insteadof "IAA."

Page 1209. Reference 9 should read: "Edstrom, J-E. 1964. Micro-electrophoretic determination of deoxyribonucleic acid contentand base composition in microscopic tissue samples. Biochim.Biophys. Acta 80: 399-410."

Page 1328. Column 1, lines 5 and 7. "photorespiration was intro-duced by Krotkov (3). . ." should read: "photorespiration wasintroduced by John P. Decker (J. Agric., Univ. Puerto Rico, 43:50-55, 1959) and defined by Krotkov (3). . . ."

Page 1414. Table I: "A, B, 16:0, 16:1, C, 16:2, 18:0, 16:3, 18:1,

16:4, 18:2, D, 18:3" should read: "A, B, 16:0, 16:1, C, 16:2,16:3, 16:4, 18:0, 18:1, 18:2, D, 18:3 E."

Pages 1227 and 1232. Tables VIII and IX were omitted.

Table VIII. Sequence of Appearance ATPase, Amylase, andLabeled Protein in the Medium during the Incubation of

Barley Aleurone Layers with Leucine-l-14CFifty aleurone layers from seeds that had imbibed water at 3°

placed in 30-ml solutions containing 15 dcl of DL-leucine-l-14Cand 0.5 g of CaCl2. Enzymes assayed as given in "Material andMethods." Radioactivity in protein measured after coprecipita-tion of protein in 1-ml aliquots with 20 mg of egg albumen bytrichloroacetic acid, washing of precipitate 6 times by homogeni-zation, and centrifugation followed by rinsing with acetone anddrying.

Amount or Activity'

ATPase Amyla&e Radioactivity in ProteinHour

-GA +GA -GA +GA -GA +GA

cpm

3 0.02 0.07 0.10 0.10 0 04 0.03 0.15 0.07 0.15 39 405 0.08 0.29 0.12 0.12 60 636 0.04 0.59 0.11 0.11 70 1207 0.08 0.91 0.11 0.25 218 2988 0.07 1.17 0.11 0.25 341 48710 0.08 1.18 0.08 0.33 422 729

1 ATPase activity = AOD 820 m,u/90 min/0.3 ml; amylaseactivity = AOD 620 m,u/10 min/0.5 ml.

Table IX. Effect of GA on the Secretion of Enzymes intothe Incubation Medium by Wheat Half-seeds

Wheat seeds were allowed to germinate for 48 hr at room tem-perature; 160 half-seeds prepared therefrom were incubated in100 ml of solutions and incubation media assayed at times indi-cated.

Ratio of Treated to Control ActivityHour

Enzyme

4 7 9 17

Phosphomonoesterase 1.05 1.09 1.14 1.13Phosphodiesterase 1.00 1.00 1.05 1.14a-Galactosidase 1.00 1.00 1.00 1.00Amylase 1.00 1.00 1.00 152.00Peroxidase 1.00 1.20 1.40 5.90

365

in the metabolism of cereal grains - plant physiology

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