polyamine metabolism its relation response ...polyamine metabolism in barley aleurone *.b c de 0 2 4...

Plant Physiol. (1984) 74, 975-9830032-0889/84/74/0975/09/$01.00/0

Polyamine Metabolism and Its Relation to Response of theAleurone Layers of Barley Seeds to Gibberellic Acid'

Received for publication September 14, 1983 and in revised form December 12, 1983

PAUL P. C. LIN*Department ofAgronomy, University ofKentucky, Lexington, Kentucky 40546-0091

ABSTRACr

Polyamine metabolism and its relation to the induction of a-amylaseformation in the aleurone layers of barley seeds (Hordeum vulgare cvHimalaya) in response to gibberellic acid (GA3) has been investipted. Ahigh-performance liquid chromatographic system has been employed forqualitative and quantitative analyses of putrescine (Put), cadaverine(Cad), spermidine (Spd), spermine (Spm), and agmatine (Agm).

Active polyamine metabolism occurs in the aleurone cells of de-embryonate barley half seeds during imbibition. The aleurone layersisolated from fully imbibed half seeds contain about 880 nanomoles ofPut, 920 nanomoles of Spd, and 610 nanomoles of Spm as free form pergram tissue dry weight while the levels of Cad and Agm are relativelylow. The polyamine levels do not change significantly in the aleuronelayers in response to added GA3 (1.5 micromolar) during the 8-hour lagperiod of the growth substance-induced formation of a-amylase. Also,the polyamine levels are not altered by the presence of abscisic acid (3micromolar) which inhibits the enzyme induction by GA3. Kinetic studiesshow that both applied IU-"Cqornithine and lU-'4Carginine are primarilyincorporated into Put during 2 hours of incubation, but the incorporationis not significantly affected by added GA3. Additionally, added GA3 doesnot affect the uptake and turnover of 11,4-'4qCPut, nor does it affect theconversion of Put -. Spd or Spd -* Spm. Treatment of the aleuronelayers with GA3 for 2 hours results in no significant changes in the totalactivities or the specific activities of ornithine decarboxylase and argininedecarboxylase.

Experiments with polyamine synthesis inhibitors demonstrate that thelevel of Spd in the aleurone layers could be substantially reduced by thepresence of methylglyoxal-bis(guanylhydrazone) (MGBG) during imbi-bition. MGBG treatment does not affect in vivo incorporation of 18-'4(jadenosine into ATP. The lower the level of Spd the less a-amylaseformation is induced by added GA3. The reduction of GA3-induced a-amylase formation by MGBG treatment can be either completely orpartially overcome by added Spd,-depending upon the concentration ofMGBG used in the imbibition medium. The results indicate that the earlyaction of GA3, with respect to induction of a-amylase formation in barleyaleurone layers, appears to be not on polyamine metabolism. However,polyamines, particularly Spd, may be involved in regulation of the growthsubstance-dependent enzyme induction.

The mechanism ofaction ofGA3 on induction of the synthesisof several hydrolytic enzymes, particularly a-amylase, in aleu-rone layers of cereal grains has been the subject of extensive

Supported in part by a fund from the University of Kentucky FacultyResearch Committee. This paper (No. 83-3-198) is published with theapproval of the director of the Kentucky Agricultural Experiment Sta-tion.

research (18, 37, 38). Experiments with the aleurone layers ofbarley seeds have shown a number ofbiochemical events relatedto the synthesis of a-amylase in response to added GA3. Themass synthesis of a-amylase begins 8 to 10 h after exposure ofthe tissue to the growth substance (9). Syntheses and accumula-tion of a-amylase mRNA (17) and ER (38) start as early as 2 to4 h after treatment of the growth substance. For efficient expres-sion of a-amylase gene(s), it has recently been suggested that anewly synthesized protein is required (26). If GA3 has effects ontranslation, the synthesis ofthe unidentified protein factor couldoccur shortly after addition of the growth substance. Thus, itwould be interesting to investigate possible biochemical changeswithin 2 h after exposure of the aleurone layers to GA3, and todetermine whether any changes are related to the effects of thegrowth substance on the transcriptional and/or translationalevents described.The diamine Put2 and polyamines Spd and Spm are ubiquitous

in biological materials (1, 33, 35). These polyamines exist andfunction as organic cations in the cells at physiological pH.Although the physiological roles of the amines have not yet beenfully characterized, their syntheses and accumulation have beenshown to be closely associated with all growth responses studiedto date (1, 12, 32, 36). Several lines of evidence indicate thatpolyamines may be involved in regulation of structure, function,and/or biosynthesis ofmacromolecules (10, 16, 24, 28). In higherplants, polyamine metabolism has been related to the action ofplant growth substances on regulation of plant growth and de-velopment (7, 8, 13, 22). In the internodes of light-grown dwarfpeas the contents of Put and Spd per internode have been shownto increase during the elongation induced by exogenously appliedGA3 (13). Also, it has been reported that the addition of GA3results in 4-fold increase in the activity ofornithine decarboxylaseduring germination of barley seeds (22). It is not clear, however,whether the changes in polyamine metabolism and the action ofGA3 are interdependent or independent events.The objectives of this study were (a) to determine whether

added GA3 has any effect on polyamine metabolism in barleyaleurone layers during the lag period of its induction ofa-amylaseformation, and (b) to determine whether polyamines are involvedin the enzyme induction.

MATERIALS AND METHODSChemicals and Plant Materials. GA3, ABA, MGBG, PMSF,

and Cad were purchased from Sigma Chemical Co. Agm, Put,Spd, and Spm were purchased either from Fluka Chemical Co.or from Pfaltz and Bauer, Inc. Radiolabeled adenosine, ornithine,arginine, and Put were obtained from the New England Nuclear

2 Abbreviations: Put, putrescine; Cad, cadaverine; Spd, spermidine;Spm, spermine; Agm, agmatine; DFMO, a-difluoromethylornithine;DFMA, a-difluoromethylarginine; MGBG, methylglyoxal-bis(guan-ylhydrazone); PMSF, phenylmethylsulfonyl fluoride.

975 https://plantphysiol.orgDownloaded on May 7, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.

https://plantphysiol.org

Plant Physiol. Vol. 74, 1984

Co. Benzoylchloride (99+ % pure) was obtained from Aldrich.HPLC grade acetonitrile and water were purchased from FisherChemical Co. DFMO and DFMA were gifts from Dr. D. T-H.Ho (University of Illinois). All other chemicals were obtainedcommercially.Aleurone layers of barley seed (Hordeum vulgare L. cv Him-

alaya) were isolated from imbibed de-embryonate half seeds asdescribed (9) with modification. The barley seeds harvested in1979 and used in this study have -100% germination. Routinely,two hundred of the half seeds were imbibed under sterilizedconditions in a Petri dish containing two Whatman No. 1 filterpaper (9 cm) and 5 ml of distilled H20. For some experiments(e.g. treatment ofMGBG during imbibition), 20 barley half seedsper treatment were incubated in a solution in a 25-ml Erlenmeyerflask in a shaker water bath at room temperature.

Induction ofa-Amylase Activity and Extraction of Polyamines.Unless otherwise stated, for each assay, 20 such aleurone layerswere incubated in a 25-ml Erlenmeyer flask containing 2 ml of20 mm Na-succinate (pH 5.0), 20 mm CaC12, and with or withoutthe addition of 1.5 uM GA3. After incubation in a shaker waterbath at room temperature, the medium was removed for deter-mination of the a-amylase activity with potato soluble starch assubstrate (9). The aleurone layers were rinsed with 5 ml distilledH20, transferred to a 15-ml Corex glass centrifuge tube, andhomogenized in 5 ml of chilled 5% HC104 with a Polytron tissuehomogenizer.

This homogenate was kept in an ice bath for at least 1 h priorto centrifugation at 20,000g for 20 min. The resulting superna-tant containing free polyamines was subjected to benzoylation.The pellet was extensively washed in the following order: 10 mlof 5% HC104 containing 0.2 M NaCl and 10 mm PPi; 10 ml ofacetone; and 10 ml of ether. The washed pellet then was incu-bated with 2 ml of 6 N HCI at 100°C for 16 h. After the acidhydrolysis, the solution was centrifuged at 20,000g for 20 min.The supernatant (1.0 ml) was mixed with 1.5 ml of HPLC waterand subjected to benzoylation. The acid hydrolysis should releasepolyamines from their conjugated forms.

Benzoylation and HPLC Analysis of Polyamines. Benzoylationof polyamine standards and plant tissue extracts were carried outas described (14, 31) with some modifications. The acid-extractedfree polyamine solution (2.5 ml) was mixed with 5 ml of 2 NNaOH in a 30-ml Corex glass centrifuge tube to which was added0.05 ml of benzoylchloride. The above solution was extensivelymixed by vortexing for at least 45 s to ensure a homogeneoussolution (or suspension) of benzoylchloride. The reaction ofbenzoylation was carried out at 28°C for 20 min and terminatedby pouring the solution into a 50-ml polypropylene centrifugetube containing 10 ml of saturated NaCl. This solution wasmixed extensively with 10 ml of diethyl ether to extract thebenzoylamines. After centrifugation at 5,000g for 10 min, 5 mlof the ether phase was collected in a polypropylene scintillationvial and evaporated to dryness with N2. Evaporation also can bedone in dry air.

Benzoylamines were dissolved in 0.1 ml of acetonitrile priorto fractionation by HPLC. HPLC analyses of benzoylamineswere carried out with a Varian 5000 liquid chromatograph on amicro-pak (MCH-10 N-cap), 10 ,um silica gel reverse phasecolumn (30 cm x 4 mm) with a UV detector at 254 nm andwith a mobile phase of H20 (pH 3.0, adjusted with a few dropsof concentrated phosphoric acid) and acetonitrile. The runninggradient was 15% to 100% acetonitrile over a period of 15 minat a flow of 4 ml/min. Sample of 0.02 ml was injected into theHPLC system for each analysis.

Incubation of Barley Aleurone Layers with '4C-Labeled Poly-amine Precursors. L-[U-'4CJornithine (316 mCi/mmol) and L-[U-'4Cjarginine (336 mCi/mmol) were used to study the effectsof added GA3 on in vivo incorporation of the amino acids into

Put and the conversion of Put -- Spd -. Spm. To the incubationmedium described for GA3-induced a-amylase activity wasadded 1.0 1Ci of ['4C]omithine/ml or 2.0 MCi of ['4C]arginine/ml. After a period of incubation, polyamines were extracted byHC104, benzoylated by benzoylchloride, and fractionated byHPLC as described. Fractions, 0.5 ml each, eluted from HPLCwere collected in scintillation vials. To each vial was added 15ml of Aquassure scintillation fluid (New England Nuclear) andthe radioactivity was determined by liquid scintillation spectro-photometry.

Incorporation of 18-'4CjAdenosine into ATP in MGBG-TreatedAleurone Layers. The aleurone layers were isolated from barleyhalfseeds imbibed for 3 d with or without the presence ofvariousconcentration of MGBG. Twenty such aleurone layers wereincubated in a 25-ml Erlenmeyer flask containing 2 ml of20 mmNa-succinate (pH 5.0), 20 mM CaCl2, and 2.0 uCi of (8-'4C]adenosine (52 mCi/mmol). After incubation in a shaker waterbath at 23°C, the aleurone layers were washed extensively withdistilled H20 and homogenized as described in 4 ml of chilled1% HC104 containing 2 mm each of ATP, ADP, 5-AMP, andadenosine. The homogenate was centrifuged at 20,000g for 20min. The supernatant (0.05 ml each) was subjected to celluloseTLC as described (23). The radioactivities associated with ATP,ADP, AMP, and adenosine were determined.Assay of the Activities of Ornithine Decarboxyase and Argi-

nine Decarboxylase. Sixty aleurone layers isolated from 3-dimbibed barley half seeds were incubated with or without thepresence of 1.5 uM GA3 for 2 h as described. The aleurone layerswere homogenized in a 6-ml buffer containing 50 mM Na-PO4(pH 7.4), 5 mm DTT, 0.01 mM pyridoxal-5-phosphate, 1 mMPMSF, and 5 mM EDTA. After centrifugation at 20,000g for 20min, 5 ml of the supematant was subjected to chromatographyon Sephadex G-25. The column (1.5 x 45 cm) was eluted in 4.3-ml fractions with the above buffer and without EDTA. Theeluted protein fractions were assayed for the activities of orni-thine decarboxylase and arginine decarboxylase as described (4)and with some modifications (Y. P. Yang, L. P. Bush, and P. P.C. Lin, manuscript in preparation). Briefly, the 0.2 ml ofenzymereaction mixture contained 10 mM Na-PO4 (pH 7.4), 2.5 mmDTT, 0.005 mm pyridoxal-5-P, 0.4 mM L-[1-'4C]omithine (1 x104 cpm/nmol), or 0.4 mM L-[U-'4C]arginine (1 x 104 cpm/nmol), and 0.1 ml of enzyme solution. The reaction was carriedout at 30C for 1 h and terminated by the addition of 0.2 ml of20% HC104. Protein concentration was determined by the pro-tein-dye binding method (6) with BSA as standard.

RESULTSBenzoylation and HPLC Analysis of Diamines and Polya-

mines. Figure 1, A and B, shows the typical separation of ben-zoylamines prepared from extracts of isolated barley aleuronelayers as compared to that from selected polyamine standards.The reproducible retention times in min are: Put, 5.45 ± 0.03;Cad, 5.91 ± 0.02; Spd, 6.71 ± 0.02; Spm, 7.52 ± 0.02; and Agm,9.8 ± 0.03. The retention times in min for benzoyl-norspermi-dine and diphenylamine are 6.40 ± 0.02 and 8.63 ± 0.03,respectively (data not shown).A linear relationship exists between the amounts of selected

polyamine standards and the peak area counts obtained fromHPLC analyses. This linear relationship is valid for amounts ofeach of the polyamines up to 100 nmol under the standard assayconditions. For reliable measurements, polyamine samples in-jected into HPLC should contain 20.1 nmol/0.02 ml. The usualrange of sensitivity for analysis in this study is from 0.5 to 5.0nmol. The resolution of an individual diamine or polyaminementioned is not affected by the presence of other polyaminesor the extracts of plant tissue.The highly reproducible separation (retention time) of the

976 LIN

https://plantphysiol.orgDownloaded on May 7, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.


POLYAMINE METABOLISM IN BARLEY ALEURONE

*.b c de

0 2 4 6 8 10 12 14 16RETENTION TIME (min)

FIG. 1. Separation of benzoylpolamines by HPLC. Operating condi-tions are as described in "Materials and Methods." A, HC104-extractedpolyamines from the aleurone layers of imbibed barley half seeds; B, amixture of 2 nmol each of the standard polyamines. Peaks: a, Put; b,Cad; c, Spd; d, Spm; and e, Agm.

benzoylamines by the reverse-phase HPLC coupled with thestandard procedures is reliable for qualitative and quantitativeanalysis of Put, Cad, Spd, and Spm in tissues of higher plants.Also, an accurate measurement ofAgm level in barley aleuronelayers is possible though, in many other plant tissues, the presenceof other amine-containing compounds (which benzoyl deriva-tives have retention times around 9-10 min) may make Agmmeasurement more difficult under the described HPLC opera-tion conditions (unpublished observation).Changes in Polyamine Contents in Barley Aleurone Layers

during Imbibition. For studies of GA3 effects, barley aleuronelayers usually are isolated from 3- to 4d imbibed half seeds.During imbibition, biochemical changes apparently occur in thealeurone cells, including polyamine metabolism. Increased im-bibition time results in increased polyamine contents in thealeurone layers. As summarized in Table I, the levels of Put,Spd, and Spm rapidly increase, especially during the 2nd d ofimbibition. After the 3rd d of imbibition, when the moisture

content ofthe tissue reaches near maximum, there are no furthersignificant increases of the polyamine levels in the aleuronelayers. The molar ratios of Put/Spd and Spd/Spm also changeduring imbibition. The ratio of Put/Spd, but not Spd/Spm,increases about 34% within the first 24 h ofimbibition, indicatingthat the majority of Put formed in this time period is notconverted into Spd. After l-d imbibition, however, more of thePut formed is converted into Spd and Spm, resulting in reductionof the ratios of Put/Spd and Spd/Spm (Table I).The aleurone layers isolated from imbibed barley half seeds

may be contaminated with starchy endosperm, particularly thoseisolated from l-d imbibed half seeds. However, contribution ofdiamines and polyamines from the starchy endosperm to theisolated aleurone layers can be neglected because the levels ofpolyamines in the starchy endosperm are relatively low. Thepolyamine levels in nmol per 20 starchy endosperms of 2-dimbibed barley half seeds are: Put, 15.5; Cad, 2.2; Spd, 2.6; Spm,10.6; and Agm, 1.2. Also, the seed coats contribute little to thepolyamine levels determined for the isolated aleurone layers.Additionally, no significant amounts of conjugated Cad, Spd,Sp, and Agm could be detected in the isolated aleurone layerswhile some Put are recovered as conjugated form (data notshown).

Effects of GA3 and ABA on the Synthesis of a-Amylase andPolyamine Levels. For studies ofthe effects ofGA3 on polyaminemetabolism, the aleurone layers prepared each time have to bechecked to insure their normal response to the added plantgrowth substance in terms of induction of a-amylase activity.With the 1979 harvested barley seeds used in this study, theinduction of a-amylase activity due to de novo protein synthesisby added GA3 and the inhibition of the GA3 action by ABA inisolated aleurone layers occur reproducibly (Fig. 2). As expected,the mass accumulation of a-amylase does not begin until 8 to10 h after the addition of GA3. Aleurone layers isolated from 2-to 4d imbibed barley half seeds show similar response to addedGA3 with respect to induction of a-amylase formation.Compared to the control, the addition of 1.5 iM GA3 has no

significant effect on the levels of Put, Spd, Spm, and Agm in thealeurone layers isolated from 4d imbibed barley halfseeds duringthe 8-h lag period of its induction of a-amylase activity (Fig. 3).The polyamine levels actually remain relatively constant throughthe lag period except for a slight increase in the level of Spmwithin 30 min after exposure of the aleurone layers to theincubation medium. Of the total amounts of HClO4-solublediamine and polyamine present in the aleurone layers about 4%Put, 10% Spd, and 15% Spm are released into the incubationmedium after 2 h incubation. The addition ofGA3 has no effecton the release of the polyamines into the medium.

Effects of added ABA on polyamine levels in the aleurone

Table I. Levels ofSelected HCIO4-Soluble Diamines and Polyamines in the Aleurone Layers of Water-Imbibed De-embryonate Barley HalfSeeds

Barley half seeds were imbibed in water as described in "Materials and Methods."

Time of Diamine Polyamine Put SpdImbibition Put Cad Spd Spm Agm Spd Spm

d nmol/20 aleurone layers? M ratio0 25.3 ± 6.2b 1.5 ± 0.6 15.6 ± 3.2 8.9 ± 2.1 9.2 + 1.2 1.62 1.751 37.0 ± 3.9 5.4 ± 1.2 17.0 ± 1.0 9.6 ± 2.8 7.3 ± 0.4 2.18 1.772 56.0 ± 3.1 2.9 ± 0.4 48.9 ± 2.7 29.8 ± 2.2 8.1 ±0.5 1.15 1.643 65.6 ± 3.5 2.5 ± 0.5 69.9 ± 7.1 44.4 ± 3.5 8.9 ± 0.5 0.94 1.574 63.8 ± 3.0 1.5 ± 2.9 64.6 ± 8.9 46.4 ± 3.7 10.2 ± 0.7 0.98 1.39

' The average dry weight of 20 aleurone layers is 73.7 mg.b For the aleurone layers of 0-d imbibed half seeds, the levels of diamines and polyamines were determined

by substraction of the polyamine levels in the half-seed from that in the starchy endosperm. The dry half seedswere soaked in 5% HC104 at OeC for 12 h before homogenization.

977




w

z

\wLII

-J

400[

300F~

200w

io00

o 5 10 15 20 25TIME (h)

FIG. 2. Effects of added GA3 and/or ABA on the formation of a-amylase. Twenty aleurone layers isolated from 3.5-d imbibed barley halfseeds were incubated with the presence of the growth substances asdescribed in "Materials and Methods." The total amount of a-amylasein both the medium and tissue extracts was determined (9). (0-0),+ 1.5 Mm GA3; (@ @), + 1.5 uM GA3 and 3.0 uM ABA; (@-*),control.

(A) Putrescine

0 0*0 0

0

I I I I I I I

2 4 6 8

(C) Spermine

P0 o 0

'F

(B) Spermidine

0

I I I l lI

2 4 6 8

_ (D) Agmatine

I I .-L IL *1 I I I I_ I I I I I I I2 4 6 8 0 2 4 6 8

TIME OF INCUBATION (h)

FIG. 3. Effects of added GA3 on the level of Put, Spd, Spm, and Agm.Twenty-five aleurone layers isolated from 4-d imbibed barley half seedswere incubated with (O-O) or without (@ ) the addition of 1.5AM GA3. The polyamine levels were determined as described in 'Mate-rials and Methods."

layers isolated from 2-d imbibed half seeds are summarized inTable II. As compared to the control and GA3 alone, the additionof 3.0 Mm ABA together with 1.5 uM GA3 at 0 time does notresult in any significant changes in the levels of Put, Cad, Spd,Spm, and Agm in the early phase (cl h) of the lag period ofGA3-induced formation of a-amylase.

Table II. Effects ofABA and GA3 on Selected Diamine and PolvamineLevels (HCIO4-Soluble) in Barley Aleurone Layers

The aleurone layers were isolated from 2-d imbibed barley half seeds.

Time Diamines PolyaminesTreatment'Incubation Put Cad Spd Spm Agm

h nmol/20 aleurone layers0 Control 57.0 2.6 38.2 36.8 9.8

0.5 Control 63.1 2.4 37.3 35.6 7.3+GA 66.8 2.5 31.5 40.4 9.3+GA/ABA 59.4 2.5 32.6 42.4 7.9

1.0 Control 63.1 2.3 35.8 35.8 8.6+GA 65.2 2.5 28.9 35.8 9.5+GA/ABA 62.7 2.1 32.6 34.2 7.4

13.5 Control 62.6 2.5 30.2 40.2 9.5+GA 64.7 1.9 23.8 28.5 9.0+GA/ABA 63.7 2.0 27.6 22.7 6.4

18 Control 62.6 2.5 28.9 41.6 10.3+GA 44.8 2.1 32.7 19.4 5.2+GA/ABA 53.6 2.1 30.5 19.9 4.7

a GA3 (1.5 Mm) and ABA (3.0 AM) were added at 0 time.

During the course of accumulation of a-amylase, 10 h afterthe addition of GA3, the level of Spm is substantially reduced inthe growth substance-treated aleurone layers. However, the pres-ence of ABA, which inhibits GA3-induced formation of a-amy-lase, does not prevent the reduction of Spm level, nor does itaffect the levels of other polyamines described.

Effects of Added GA3 on Incorporation of "4CiOrnithine and1'4CjArginine into Polyamines. To determine any effect of GA3on polyamine metabolism in barley aleurone layers, radiolabeledpolyamine precursors, such as ornithine and arginine, were addedto the incubation medium. With the aleurone layers isolatedfrom 2-d imbibed barley half seeds, a substantial amount of L-[U-'4C]ornithine or L-[U-'4C]arginine is taken up and subse-quently incorporated into polyamines within 1 h of incubation.However, neither the uptake of the polyamine precursors northe total amount of radioactivity incorporated into polyamine issignificantly affected by the presence of GA3.

Analysis of benzoyl derivatives of polyamines shows that mostof the radioactivity incorporated into polyamines from either['4C]ornithine or ['4Clarginine is recovered as Put and with about5% as Spd (Fig. 4, A and B). Spm, Cad, and Agm are not labeled.Kinetic studies indicate that added GA3 has no significant effecton the rate of incorportion of ['4CJornithine into Put and theconversion of Put -* Spd during 1.5 h incubation (Fig. 5). Similarresults were obtained with ['4CJarginine as polyamine precursor(data not shown).The in vivo incorporation of ['4C]ornithine and [1'4C]arginine

into Put could be catalyzed by ornithine decarboxylase (EC4.1.1.17) and/or arginine decarboxylase (EC 4.1.1.19) because ofpossible interconversion of the two amino acids via the ureacycle. It is noteworthy, however, that added DFMO and DFMA,potential inhibitor of omithine decarboxylase and arginine de-carboxylase, respectively, have no inhibitory effects on the in-corporation of ['4CJornithine and ['4Clarginine into Put in thealeurone layers (Fig. 6). Additionally, Table III shows that treat-ment of the aleurone layers with GA3 for 2 h results in nosignificant changes in the total activities or the specific activitiesof omithine decarboxylase and arginine decarboxylase assayedin vitro. The aleurone layers apparently contain about 8 timesmore activity of arginine decarboxylase than that of ornithinedecarboxylase. Whether the in vitro activities of ornithine decar-

+ GA3

/ GA3 + ABA

2/'Control

it100

8000

oy 60w

40

LLJ 20z0

ow-J< 100IC)

K 80wz 60

< 40U)0 20E

C

_0

978 LIN




RETENTION TIME (min)

z

0

U

VU

12 16 20 24FRACTION

FIG. 4. Effects of added GA3 on the incorporation of ['Clornithineinto polyamines. Twenty aleurone layers isolated from 2-d imbibedbarley half seeds were incubated with the presence of L-[U-"C]ornithine(1.0 MCi/ml) or L-[U-'4C]arginine (2.0 MCi/ml). After incubation, polya-mines were extracted, benzoylated, and fractionated by HPLC as de-scribed in "Materials and Methods." The amounts of '4C cpm incorpo-rated represent 0.02 ml aliquot of a total volume of 0.1 ml, which wasinjected into HPLC. Incubation conditions: control (-@); + 1.5 AMGA3 (O-O). Incubation time: A, 60 min with the ['4C]omithine; B,30 min with the ["C]arginine.

8000* CONTROL

0 t

0 G30 0 9

w6WO-

~~~~put

0U

z~~~TM4000)

U 00

Spd

0 30 60 90

TIME (min)

FIG. 5. Kinetic studies of the incorporation of ['4C]ornithine intopolyamines. The experimental conditions are as described in Figure 4.Following the time course, the amounts of 14C radioactivity incorporatedinto Put, Spd, and Spm fractionated by HPLC were determined. Nodetectable radioactivity is associated with Agm. Incubation conditions:control (-.*); + 1.5 jAM GA3 (0-C O).

w

cr

I

a.

41

12000

_ (A)

r Put

8000 _

40001_

0 25 50 10

DFMA (mM)

600o

4000

(B)

Put

20001-

zspd O

0 25 50 10DFMO (mM)

FIG. 6. Effects of Put synthesis inhibitors on incorporation of ["C]arginine and ["C]ornithine into polyamines. As described in Figure 4,the incubation media contained varied concentrations of DFMA orDFMO. After preincubation of the aleurone layers with the inhibitorsfor 2 h, (A), L-[U-14C]arginine (2.0 M&Ci/m1) or (B) L-[U-'4C]ornithine(1.0 uCi/ml) was added and incubated for 30 min prior to extractionand analysis of polyamines.

Table III. Effects ofAdded GA3 on the Activities ofOrnithineDecarboxylase and Arginine Decarboxylase in the Aleurone LayersSixty aleurone layers were incubated with or without the presence of

1.5 Mm GA3 for 2 h. After homogenization and gel filtration on SephadexG-25, the activities of the decarboxylases were determined as describedin "Materials and Methods."

Omithine Decarbox- Arginine Decarbox-ylase ylase

TreatmentTotal Specific Total Specific

activity activity activity activity

unitsa units/mg units' units/mgprotein protein

Control 2.4 1.2 ± 0.2 19.7 7.5 ± 0.5+1.5 Mm GA3 2.5 1.1 ± 0.2 19.1 7.2 ± 0.5a One unit of enzyme activity is defined as that amount of enzyme

which catalyzes 1.0 nmol ofC02 release per h at 30°C. The total activityrepresents the activity in the extracts (after filtration through SephadexG-25) of 60 aleurone layers.

boxylase and arginine decarboxylase from barley aleurone layersare DFMO and DFMA sensitive, respectively, have not beendetermined.

Effects of Added GA3 on the Conversion of L-l,4-'4CPut intoPolyamines. Although the uptake of exogenous polyamines byliving cells is generally believed to be limited (15, 33, 35), asignificant amount of radioactivity is taken up and/or adsorbedby isolated barley aleurone layers after incubation with L-[ 1,4-'4C]Put for 30 min at room temperature. Analysis by HPLCreveals that most ofthe radioactivity associated with the aleuronelayers is recovered as the parent compound Put. and with littleas Spd or Spm (Fig. 7).

After the incubation with the ['4C]Put, the aleurone layerswere rinsed and subsequently incubated with or without thepresence ofGA3. As shown in Figure 7A, about 15% ofthe ["4C]Put associated with the aleurone layers, presumably the adsorbedfraction, is released into the incubation medium within 30 min.The remaining ['4C]Put could be partially converted into Spd ata slow rate; the estimated half-life of Put is 8 h. No significantamount of radiolabeled Spm could be detected under the exper-

979

16000* 80CMOO




300C

2500[

Ia-u

(A) CONTROL

Put

2000_

1500_

1000_

500 Sp1

0 60 120

(B) + GA3

Put

Spd

0

0 60 120

TIME (min)

FIG. 7. Effects ofadded GA3 on the conversion of L-[1,4-14C]PUt intopolyamines. As described in Figure 4, the aleurone layers were incubatedwith L-[ 1,4-'4C]Put (1.0 gCi/ml) for 30 min, rinsed, and subsequentlyincubated (A) with or (B) without the presence of 1.5 Mm GA3 for varioustime periods.

(A)(B

Lii ~ Put

Wi 100_400~~~~~~~~~qGA3+Spd

(N

L \ SPd 2

< R ColGA3

E CDic :z Control

0 1 2 3 4 5MGBG (mM)

0 1 2 3 4 5MGBG (mM)

FIG. 8. Effects of MGBG on polyamine levels of GA3-induced for-mation of a-amylase. A, Barley half seeds were imbibed with variedconcentrations of MGBG for 2.5 d. After imbibition, aleurone layerswere isolated and analyzed for polyamine contents as described in Figure3. B, The above isolated aleurone layers were continuously incubatedwith varied concentrations of MGBG for 24 h and with the followingadditions: 1.5 AM GA3 (-); 1.5 AM GA3 plus 2.5 mM Spd(@ @); none (control, *-*). The activity of a-amylase was deter-mined at 24 h after the addition of GA3 as described in 'Materials andMethods."

imental conditions. The presence ofGA3, however, has no effecton the half-life of the absorbed ['4C]Put in the aleurone layersduring the incubation (Fig. 7B).

Effects of Polyamine Synthesis Inhibitors. As described earlierin Table I, imbibition results in increase of polyamine levels inthe aleurone layers. Attempts were made to reduce the levels ofpolyamines by using potential polyamine synthesis inhibitors,such as DFMO, DFMA, and MGBG. Figure 8A shows that theamine levels, particularly Spd, in the aleurone layers could besubstantially reduced by the presence of MGBG during imbibi-tion. MGBG at 2 and 5 mM reduce the level of Spd by about38% and 55%, respectively, after a 2.5-d imbibition, while the

presence of MGBG results in marked increase in accumulationof Put. Spm level decreases slightly in response to added MGBG.Other polyamine synthesis inhibitors, DFMO and DFMA, arenot as effective as MGBG in reduction ofthe amine levels duringimbibition.

Figure 8B shows that GA3-induced formation of a-amylase isreduced in the aleurone layers isolated from MGBG-imbibedbarley half seeds. The degree of reduction of the enzyme induc-tion, like that of the level of Spd, is a function of the concentra-tion ofMGBG present in the imbibition medium. It is notewor-thy that a continuous presence of MGBG is required for maxi-mum reduction ofGA3-induced a-amylase formation. Addition-ally, 2.5 mm MGBG does not affect the activity of a-amylasedetermined from the medium which contains proteolytic activ-ity. To determine whether the decreased formation of a-amylaseis due to low level of Spd, exogenous Spd was applied togetherwith GA3 to the incubation medium. Also, the addition of 2.5mM Spd could either completely or partially restore the growthsubstance-induced formation of a-amylase in the MGBG-treatedaleurone layers (Fig. 8B), depending upon the concentration ofMGBG used during imbibition of the half seeds. As expected,uptake of exogenous Spd by the aleurone layers is limited.Incubation of 20 aleurone layers with 2.5 mM [1,4-'4C]Spd (420cpm/nmol) in the presence of 2.5 mm MGBG and 1.5 uM GA3for 1 to 3 h results in uptake of about 30 to 40 nmol of ['4C]Spd(data not shown). The ['4C]Spd taken up is not extracted bywater and is not exchanged by an excess amount of unlabeledSpd. The ['4C]Spd taken up by the aleurone layers remains asthe parent compound as judged by HPLC analysis.

Effects ofMGBG Treatment on ATP Synthesis. Experimentswere carried out to determine whether MGBG treatment duringimbibition of barley half seeds has any effect on the liability ofthe aleurone cells with respect to ATP synthesis. Any inhibitoryeffect on ATP synthesis may subsequently result in reducing a-

amylase formation induced by GA3.As demonstrated previously (21), exogenously applied adeno-

sine is readily taken up and subsequently incorporated into ATPin isolated barley aleurone layers. Figure 9A shows that, ascompared to that in the control tissue, the rates of uptake of [8-'4C]adenosine in the aleurone layers isolated from MGBG (2.5

i03-UZ

a'o1-

z

C]

0

11

0~llJ

IL

4 6 8 10

TIME (h)

1-%

'o

-J

U)

I~

H7

0 2 4 6 8TIME (h)

FIG. 9. Effects of MGBG treatment on uptake of [8-'4C]adenosineand its conversion into ATP in barley aleurone layers. The aleuronelayers were isolated from barley halfseeds imbibed for 3 d in the presenceof waiter (0), 2.5 mm MGBG (0), or 5.0 mm MGBG (0). For 20 aleuronelayers, the amounts of [8-"C]adenosine taken up and ['4C]ATP formedas a function of incubation time are shown in A and B, respectively. Thedashed line shown in B represents the percentage of incorporation of['4C]adenosine taken up into ATP.

980 LIN




and 5.0 mM)-treated barley half seeds after a 3-d imbibition aresignificantly reduced by 30 to 50%. The total amounts of ['4C]adenosine taken up are also reduced in the MGBG-treatedaleurone layers during 7 h incubation. Since only a trace amountof adenosine is added to the incubation medium, the endoge-neous pool of adenosine is not saturated by added adenosine.This can be judged by the observation that the majority (about75%) of ['4C]adenosine taken up is converted into ATP (Fig.9B). Although the rates of ['4CJATP synthesis or accumulationare reduced in the MGBG-treated aleurone layers, as expected,due to reduced rates of uptake of ['4C]adenosine, the MGBGtreatment apparently does not affect the endogenous synthesisof ATP from adenosine (Fig. 9B). Thus, the aleurone layersisolated from barley half seeds inhibited for 3 d with or withoutthe presence of 2.5 to 5.0 mM MGBG are metabolically nodifferent with respect to ATP biosynthesis.

DISCUSSIONIn this study a well-known system, GA3-induced formation of

a-amylase in the aleurone layers of barley seeds, has been chosenfor the investigation of a possible relation between polyaminemetabolism and the action of a plant growth substance. One ofthe unique features of this system is that each of the aleuronelayers comprises three layers of differentiated, uniform cellswhich undergo no cell division and/or growth in response toGA3 treatment (33). Thus, unlike the other plant systems studiedto date (7, 12, 18), polyamine metabolism in relation to theaction ofGA3 in the aleurone layers could be investigated withoutconcerning cell proliferation and growth. This study has yieldedseveral lines of evidence that in barley aleurone layers the earlyaction of GA3 on induction of the synthesis of a-amylase or itsmRNA appears to be not on polyamine metabolism, but thelevels ofpolyamines, especially Spd, may be crucial for the actionof the growth substance.

Polyamine Metabolism in the Aleurone Layers. With thereliable HPLC system employed in this study, the cellular con-tents of diamines (Put and Cad) and polyamines (Spd, Spm, andAgm) can be accurately determined. Substantial amounts of Put,Spd, and Spm are present in the aleurone layers isolated fromimbibed barley half seeds, which are routinely used for study ofGA3 effects. The diamine Put and polyamines Spd and Spmapparently are synthesized and accumulated primarily as freeform during imbibition. The aleurone layers contain smallamounts of Cad and Agm. With the possible exception of Put,most of the polyamines are not covalently conjugated withmacromolecules. After 3 to 4 d imbibition, the contents of acid-soluble polyamines remain relatively unchanged and with littleturnover in the aleurone layers as judged by the fact that theconversion of Put -- Spd -- Spm is very slow.

In most of the plant tissues studied to date, the diamine Put isprimarily synthesized from arginine by arginine decarboxylase(EC 4.1.1.19) via the intermediate Agm (15, 33, 34). In thealeurone layers of barley seeds, however, Put apparently can besynthesized from ornithine by ornithine decarboxylase (EC4.1.1.17) as judged by the following observations. First, added[U-'4C]ornithine is readily converted into Put without lag period(Fig. 5). Second, the lack of detectable ['4C]Agm indicates that['4C]ornithine is probably not converted into arginine via theurea cycle prior to formation of Put. Third, the isolated aleuronelayers contain the activity of ornithine decarboxylase. The bio-chemical nature of incorporation of [U-'4C]arginine into Put inthe aleurone layers is not clear. The observed activity of argininedecarboxylase, which is greater than that of ornithine decarbox-ylase in the tissue extracts, appears to indicate that arginine couldbe decarboxylated and subsequently converted into Put in barleyaleurone layers (Table III). However, Agm, the intermediate ofthe conversion of arginine -. Put via arginine decarboxylase, is

not radiolabeled when the aleurone layers are incubated with [U-'4C]arginine. This suggests either that the conversion of Agminto Put via putrescine synthetase complex (34) is a fast reaction(not rate limiting) or that arginine could be directly convertedinto ornithine by arginase and then converted into Put byornithine decarboxylase. Since the substrate specificities of thedecarboxylases are not determined, the nature of enzymic for-mation of Put from arginine in the aleurone layers remains tobe clarified. The attempts to use DFMO and DFMA, the poten-tial enzyme-activated competitive inhibitors of ornithine decar-boxylase (19, 25) and arginine decarboxylase (20), respectively,for determination of whether Put could be formed from decar-boxylation of arginine in the aleurone layers have been unsuc-cessful (Fig. 6). The lack of effects of the inhibitors on Putformation could be due to poor uptake of the chemicals or lackof sensitivity of the decarboxylases of barley aleurone layers tothe chemicals. It has been reported that not all of the ornithinedecarboxylases isolated from bacteria (19) and plant tissues (15)are DFMO sensitive.The other potential polyamine synthesis inhibitor, MGBG,

appears to affect polyamine metabolism in the aleurone layers.Although the mechanism by which MGBG brings about amarked decrease in Spd content (Fig. 8A) has not yet beendetermined, the compound may inhibit the activity of S-adeno-sylmethionine decarboxylase in the aleurone layers as it does inanimal tissues (27, 29, 30). Inhibition of the decarboxylaseactivity wouid reduce the amount of decarboxylated form of S-adenosylmethionine available for the conversion of Put -- Spd-- Spm and thus result in decreased levels of Spd and Spm withincreased level of Put in the aleurone layers (Fig. 8A).

Polyamine Metabolism in Relation to the Action of GA3.Despite the high levels of Put, Spd, and Spm, the aleurone layersisolated from imbibed barley half seeds still require the presenceof GA3 for induction of a-amylase formation. This result leadsto the question of whether the action of GA3 is mediated bypolyamines as suggested by numerous investigators (13, 15). Toinvestigate this problem, experiments have been conducted todetermine whether added GA3 affects polyamine metabolismduring its induction of a-amylase formation, and whetherchanges in polyamine levels may affect the action of the growthsubstance on the enzyme induction.

Evidently, during the 8-h lag period ofGA3-induced formationof a-amylase, the endogenous levels of Put, Spd, and Spm in thealeurone layers do not change significantly in response to theaddition of GA3 (or ABA). This is in contrast to the recentobservation from Bernal-Lugo (5) that GA3 treatment results in66% more total polyamine level than control during the first 30-min incubation and added ABA prevents this increase. Since theauthor used ion exchange chromatography to separate acid-soluble polyamine and ninhydrin reaction to quantitate polya-mines, contamination of other compounds in the polyaminedetermination is likely. Lack of effect of added GA3 on polya-mine metabolism can be further judged by the following lines ofsupporting evidence: (a) added GA3 has no significant effect onthe incorporation of '4C-labeled ornithine and arginine into Putand the limited conversion of Put -. Spd -* Spm during the 2-h incubation; (b) the presence of GA3 does not affect the ratherslow turnover of absorbed ['4C]Put in the aleurone layers; and(c) treatment of the aleurone layers with GA3 for 2 h does notaffect the total activities and specific activities of ornithine de-carboxylase and arginine decarboxylase. The above results alsoindicate that the induction of a-amylase mRNAs, which beginsat 2 h after exposure of the aleurone layers to GA3 (17, 26), isnot a result of changes in polyamine metabolism or polyaminelevels. Thus, the diamine Put and polyamines Spd and Spmapparently do not act as 'second messenger' for the growthsubstance as implied recently by other investigators (15). How-

981




ever, the present results do not rule out the possibility thatpolyamines may be required for or involved in the action of thegrowth substance on induction of a-amylase formation. Sincethe endogenous levels of polyamines are not affected by thepresence of GA3 or Put biosynthesis inhibitors (DFMO andDFMA), the above suggested possibility could be examined byusingMGBG to lower the levels of polyamines, particularly Spd,or applying exogenous polyamines to increase the cellular levelsof polyamines.

It appears that the levels of polyamines, especially Spd, maybe crucial for the action of GA3 on induction of a-amylaseformation in the aleurone layers. This view is supported by theobservation that, in MGBG-treated barley aleurone layers, thelower the level of Spd the less a-amylase formation is inducedby added GA3. Additionally, application of exogenous Spd to-gether with MGBG could either totally or partially restore theinducible effect of the growth substances in the aleurone layersisolated from barley half seeds imbibed with various concentra-tions of MGBG. Comparison of the data obtained with thealeurone layers isolated from MGBG-imbibed and the control(water-imbibed) half seeds indicates that the approximately 2.5nmol of Spd per aleurone layer are required for the optimuminduction of a-amylase activity by added GA3. Recently, it hasbeen reported that GA-induced synthesis of a-amylase is in-hibited by MGBG (5). The author, however, did not mentionwhat percentage of inhibition caused by MGBG. Routinely, Ihave observed that the addition of 2.5 mM MGBG together with1.5 gM GA3 results in about l10% reduction of a-amylaseactivity after a 24-h incubation of 3-d imbibed barley aleuronelayers (unpublished observation). Thus, once the level of Spd inthe aleurone layers, such as those isolated from 3- to 4-d water-imbibed half seeds, reaches 2.5 nmol per aleurone layer, theaddition of MGBG has little effect on the enzyme induction.The above view is further supported by the observation (Fig. 8B)that exogeneously applied Spd has no significant effect on theGA3-induced formation of a-amylase in the aleurone layerswhich already contain substantial amounts of the polyamineafter imbibition (Table I).The mechanism by which the low level of Spd brings about

decreased GA3-dependent induction of a-amylase activity is notunderstood. Spd has been suggested to be important in regulationof protein synthesis (3, 31) and the structure and/or function ofDNA (24, 28). Thus, in the aleurone layers of barley seeds, thelevel of Spd may affect the synthesis of a-amylase at transcrip-tional and/or translational levels. My preliminary data indicatethat Spd level may be crucial for GA3-induced synthesis of a-

amylase mRNAs. This is judged by the observation that in thealeurone layers isolated from 2.5 mm MGBG-imbibed barleyhalf seeds (34 imbibition), more a-amylase is produced at 24 hafter the addition of GA3 when 2.5 mm Spd is added at t = 0instead of t = 6 after the addition of the growth substance(unpublished observation). Thus, it would be important to de-termine how the MGBG treatment and added Spd might affectGA3-induced synthesis of a-amylase mRNAs in barley aleuronelayers. For efficient expression of a-amylase gene(s), it mayrequire GA3-dependent synthesis of protein factor(s) as suggestedrecently (26). If Spd added to MGBG-treated aleurone layerscould restore GA3-induced synthesis of a-amylase mRNAs, theadded polyamine might be involved in the regulation ofsynthesisof the unidentified protein factor(s).

In this study the GA3-induced formation ofa-amylase is basedon quantitation of the enzyme activity. The enzyme activity hasbeen shown to be due to de novo synthesis ofa-amylase in barleyaleurone layers (38). Though the MGBG treatments show nodirect effect on the activity of a-amylase assayed with the mediaor the tissue extracts which may contain proteolytic activity (38),experiments are needed to determine whether the endogenous

level of Spd affects incorporation of '4C-labeled amino acid(s)into a-amylase protein in GA3-treated barley aleurone layers.Additionally, added GA3 also induces the aleurone cells to syn-thesize several hydrolases, including protease and a-glucosidase(38). It would be important to determine whether the synthesesof the hydrolases induced by GA3 are all related to the endoge-nous Spd level in the aleurone layers.

Acknowledgments-I thank Y. P. YangandG. M. Li for their help in preparationof barley half seeds and aleurone layers. I am also grateful to Dr. R. A. Nilan(Washington State University) for providing the barley seeds used in this study.

LITERATURE CITED

1. BACHRACH U 1979 Metabolism and function of spermine and related polya-mines. Annu Rev Microbiol 24: 109-134

2. BAGNI N, E CORSINI, DS FRACASSINI 1971 Growth factors and nucleic acidsynthesis in Helianthus tuberosus. I. Reversal of actinomycin D inhibitionby spermidine. Physiol Plant 24: 112-117

3. BAGNI N, DS FRACASSINI 1973 The role of polyamines as growth factors inhigher plants and their mechanism of action. In Plant Growth Substances.Hirokawa Publishing Co., Tokyo, pp 1205-1217

4. BEAVEN MA, G WiLcox, GK TERPsTRA 1978 A microprocedure for themeasurement of '4CC2 release from ['4C]carboxyl-labeled amino acids. AnalBiochem 84: 638-641

5. BERNAL-LuGo I 1983 Relationship of polyamines endogenous content andGA3 effect in barley aleurone layers. Plant Physiol 72: 5-473

6. BRADFORD M 1976 A rapid and sensitive method for the quantitation ofmicrogram quantities of protein utilizing the principle of protein-dye bind-ing. Anal Biochem 72: 248-254

7. CHo SC 1983 Effects of cytokinin and several inorganic cations on thepolyamine content of lettuce cotyledons. Plant Cell Physiol 24: 27-32

8. CHO SC 1983 Enhancement by putrescine ofgibberellic-induced elongation inhypocotyls of lettuce seedlings. Plant Cell Physiol 24: 305-308

9. CHRISPEELS MJ, JE VARNER 1967 Gibberellic acid-enhanced synthesis andrelease of a-amylase and ribonuclease by isolated barley aleurone layers.Plant Physiol 42: 398-406

10. Cocucci S, N BAGNI 1968 Polyamine-induced activation of protein synthesisin ribosomal preparations from Helianthus tuberosus tissue. Life Science 7:115-120

1 1. COHEN E, SM ARAD, YM HEIMER, Y MIZRAHI 1982 Participation of ornithinedecarboxylase in early stages oftomato fruit development. Plant Physiol 70:540-543

12. COHEN SS 1982 The polyamines as a growth industry. Fed Proc 41: 3061-3064

13. DAi YR, R KAUR-SAWHNEY, AW GALSTON 1982 Promotion by gibberellicacid ofpolyamine biosynthesis in internodes oflight-grown dwarfpeas. PlantPhysiol 69: 103-105

14. FLOREs H, AW GALSTON 1982 Analysis of polyamines in higher plants by highperformance liquid chromatography. Plant Physiol 69: 701-706

15. GALSTON AW 1983 Polyamines as modulators of plant development. BiobScience 36: 382-388

16. HUNG DT, LU MARTON, DF DEEN, RH SHAFER 1983 Depletion of intracellularpolyamines may alterDNA conformation of9L rat brain tumor cells. Science221: 368-370

17. JACOBSEN JV, JA ZWAR 1974 Gibberellic acid causes increased synthesis ofRNA which contains poly (A) in barley aleurone tissues. Proc Natl Acad SciUSA 71: 3290-3293

18. JONEs RL 1973 Gibberellins: their physiological role. Annu Rev Plant Physiol24: 571-598

19. KALLIo A, PP MCCANN 1981 Difluoromethylornithine irreversibly inactivatesornithine decarboxylase of Pseudomonase aeriginosa, but does not inhibitthe enzyme of Escherichia coli. Biochem J 200: 69-75

20. KALLIO A, PP MCCANN, P BEY 1981 DL-a-(difluoromethyl)arginine: a potentenzyme activated inhibitor ofbacterial arginine decarboxylase. Biochemistry20: 3163-3166

21. KEATES RAB 1973 Evidence that cyclic AMP does not mediate the action ofgibberellic acid. Nature 244: 355-357

22. KYRIAKIDIS DA 1983 Effect of plant growth hormones and polyamines onomithine decarboxylase activity during the germination of barley seeds.Physiol Plant 57: 499-504

23. LIN PPC, JE VARNER 1972 Cyclic nucleotide phosphodiesterase in pea seed-lings. Biochim Biophys Acta 276: 454-474

24. LiQuoRI AM, L CONSTANTINO, V GRESCENZI, A ELIA, E GIGIo, R PUTLITI,M DE SANTI SAVINO, V VITAGLIANO 1967 Complexes between DNA andpolyamines; a molecular model. J Mol Bio 24: 113-122

25. METCALF BW, P BEY, C DANZIN, MJ JUNG, P CASARA, JP VEVERT 1978Catalytic irreversible inhibitionofmammalina ornithine decarboxylase (E.C.4.l.1.17) by substrate and product analogs. J Am Chem Soc 100:2551-2553

26. MUTHUKRISHNAN 5, GR CHANDRA, ES MAXWELL 1983 Hormonal control ofa-amylase gene expression in barley: studies using a cloned cDNA probe. J

982 LIN




Biol Chem 258: 2370-237527. PEGG AE, JE SEELY, H Poso, FD RAGINE, IS ZAGON 1982 Polyamine biosyn-

thesis and interconversion in rodent tissues. Fed Proc 41: 3065-307228. PORTER CW, RJ BERGERON 1983 Spermidine requirement for cell proliferation

in eukaryotic cells: structural specificity and quantitation. Science 219: 1083-1085

29. Poso H, AE PEGG 1981 Differences between tissues in response of S-adenosyl-methionine decarboxylase to administration of polyamines. Biochem J 200:629-637

30. Poso H, AE PEGG 1982 Comparison of S-adenosylmethionine decarboxylasefrom rat liver and muscle. Biochemistry 12: 3116-3122

31. REDMON JW, A TSENG 1979 High-pressure liquid chromatographic determi-nation of putrescine, cadaverine, spermidine and spermine. J Chromatogr170: 479-481

32. RUSSELL DH, BGM DURIE 1978 Polyamines as Markers of Normal andMalignant Growth. Raven Press, New York

33. SMITH TA 1977 Recent advances in the biochemistry of plant amines. In LReinhold, JB Harborne, T Swain, eds, Progress in Phytochemistry, Vol 4.Pergamon Press, Oxford, pp 27-81

34. SRIVENUGOPAL KS, PR ADIGA 1981 Enzymatic conversion of agmatine toputrescine in Lathyrus sativus seedlings: purification and properties of mul-tifunctional enzyme (putrescine synthetase). J Biol Chem 256: 9532-9541

35. TABOR CW, H TABOR 1976 1,4-Diaminobutane (putrescine, spermidine, andspermine). Annu Rev Biochem 45: 285-306

36. TABOR CW, H TABOR, AK TYAGI, MS COHN 1983 The biochemistry, genetics,and regulation of polyamine biosynthesis in Saccharomyces cerevisiae. FedProc 41: 3084-3088

37. VARNERJE, T-HD Ho 1976 The role ofhormonesin the integration ofseedlinggrowth. In J Papaconstantinou, ed, Molecular Biology of Hormone Action.Academic Press, New York, pp 173-194

38. YoMo H, JE VARNER 1971 Hormonal control of a secretory tissue. In AAMoscona, A Monroy, eds, Current Topics in Developmental Biology, Vol 6.Academic Press, New York, pp 11-144

983



polyamine metabolism its relation response ...polyamine metabolism in barley aleurone *.b c de 0 2 4...

Documents