Plant Physiol. (1973) 52, 278-282

Cyclic Nucleotide Phosphodiesterase Activitv in Barlev Seeds1'2Received for publication August 1, 1972

JOZEF VANDEPEUTE,3 RAY C. HUFFAKER, AND ROBERT ALVAREZ4Department of Agronomy and Range Science and Department of Environmental Horticulture, Uniiversity ofCalifornia, Davis, California 95616

ABSTRACT

Barley seeds (Hordeum vulgare L. cv. Himalaya) contain anenzymatic activity which catalyzes the hydrolysis of adenosinecyclic 3':5'-monophosphate and adenosine cyclic 2' 3'-mono-phosphate. A large portion of the enzymatic activity is presentin the dry seed, existing in both soluble and particulate form.Secretion of the soluble phosphodiesterase from embryolessseeds is enhanced by gibberellic acid and inhibited by abscisicacid, dinitrophenol, and cycloheximide. Attempts to isolate ordetect a phosphodiesterase which specifically hydrolyzes adeno-sine cyclic 3': 5'-monophosphate were unsuccessful. Inhibitionexperiments indicate that probably one enzyme is involved inthe hydrolysis of both of these substrates.

During barley seed germination, GA is synthesized by tissuesof the embryo and translocated to the aleurone cells, thusacting as a hormonal signal (16, 21, 31). The aleurone cellssurround the storage tissue component of the endosperm. Theyare highly differentiated and respond to the GA stimulus bysynthesizing or secreting or both a number of hydrolytic en-zymes which degrade the starchy endosperm and provide thedeveloping embryo with essential metabolites. Much informa-tion is available concerning the biochemistry of the effect ofGA on aleurone cells (6, 7, 9, 28, 29); however, a precisemechanism of action has not yet been elucidated.

In contrast, studies on the mechanism of action of animalhormones indicate that in many cases adenosine cyclic 3': 5'-monophosphate (cyclic 3': 5'-AMP)' acts as an intracellularintermediate (10, 22, 23, 25). Hormones circulating in the

1 This research was supported by a National Defense EducationAct Title IV Fellowship.

2 Most of the research reported herein is derived from the doc-toral thesis of J. Vandepeute accepted by the Plant PhysiologyGroup of the University of California at Davis in partial fulfillmentof the requirements for the Ph.D. degree.

' Postdoctoral Fellow of the National Institutes of Health, Na-tional Institute of Arthritis and Metabolic Disease (AM 53253-01).Present Address: Department of Pharmacology, University of Colo-rado Medical Center, Denver, Colo. 80220.

' Postdoctoral Fellow of the National Institutes of Health, Na-tional Institute of General Medical Sciences (GM 50553-02). Pres-ent Address: Institute of Biological Sciences, Syntex Research, PaloAlto, Calif. 94304.

'Abbreviations: cyclic 3': 5'-NMP: cyclic nucleoside 3': 5'-mono-phosphates, e.g. cyclic 3': 5'-AMP, etc.; cyclic 2': 3'-NMP: cyclicnucleoside 2':3'-monophosphates, e.g. cyclic 2': 3'-AMP, etc.;cNPDE: cyclic nucleotide phosphodiesterase.

bloodstream activate the membrane-bound adenyl cyclase intheir target tissues which catalyzes the synthesis of cyclic3':5'-AMP from ATP. The increased cyclic 3':5'-AMP thenbrings about the physiological responses of the hormones. Thephysiological action of cyclic 3':5'-AMP is terminated by itshydrolysis to 5'-AMP via a specific cyclic 3': 5'-nucleotidephosphodiesterase (10, 22, 23).

Several investigators have examined the possible role ofcyclic 3':5'-AMP in barley seed metabolism (4, 5, 8, 18, 19,20). Galsky and his colleagues (5, 8, 18) have reported thatcyclic 3': 5'-AMP promotes the secretion of a-amylase andother hydrolytic enzymes from barley seeds in a manner similarto that evoked by GA under the same conditions. Pollard (19)has described a procedure which results in the production of a"C-labeled compound which cochromatographs with cyclic3': 5'-AMP in barley aleurone cells incubated with "C-adenine.Similar findings with other plant tissues have been reportedusing the same procedure (17, 24). Preliminary reports onthe degration of cyclic 3':5'-AMP in barley (12, 27) and otherplant tissues (11, 14, 30) have also been cited.

The findings on a possible role for cyclic 3': 5'-AMP inbarley seed metabolism have shown some negative results. Lackof success with attempts to detect adenyl cyclase in barley andpea tissues led Lin and Varner (13) to examine the enzymaticsystem for the degration of cyclic 3': 5'-AMP in peas. Theyhave characterized and partially purified a cNPDE from peaseedlings which hydrolyzed either cyclic 3': 5'-AMP or cyclic2':3'-AMP. The hydrolysis of both cyclic nucleotides is similarwith respect to pH, sulfhydryl reagents, temperature, urea, andmetal ions. These observations suggest that a single enzyme isinvolved in the hydrolysis of either substrate. It was concludedthat the cNPDE present in pea seedlings may function in thehydrolysis of cyclic 2': 3'-AMP during the degradation of RNA(13).A demonstration of a specific phosphodiesterase for cyclic

3': 5'-AMP would constitute supporting evidence for involve-ment of this nucleotide in plant biochemistry. Because barleyseeds have been reported to exhibit a response to exogenousapplications of cyclic 3': 5'-AMP, it was of interest to de-termine whether such an enzyme is present in these seeds.Furthermore, information on the development of cNPDEactivity and the effect of GA on this development would be ofgeneral interest in documenting the biochemical sequence ofevents in aleurone cells during germination.

MATERIALS AND METHODS

Enzyme Extraction Procedure. Embryoless barley seeds weresurface-sterilized in 1% sodium hypochlorite for 20 min,washed with sterile distilled water, and allowed to imbibe for12 to 18 hr in a sterile petri dish containing 8 ml of water. Tenhalf seeds were transferred to vials containing 0.1 ,M GA, 10mM calcium chloride, and 10 lug of chloramphenicol. All glass-

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CYCLIC NUCLEOTIDE PHOSPHODIESTERASE

ware and solutions were sterilized prior to addition of theembryoless seeds. The vials were placed in a metabolic shakeroperating at 40 rpm at 25 C for 48 hr. Aliquots of the incuba-tion medium were withdrawn and assayed directly or frozenat -20 C for later use. cNPDE activity was stable at thistemperature for several months. Crude enzyme from embryo-less seeds was obtained by homogenization in 4 ml of 10%(w/v) sucrose in a ground-glass homogenizer or with a mortarand pestle. Barley aleurone layers were isolated from embryo-less seeds in accordance with previously described procedures(3).

Assay for Cyclic 2':3'-AMP Phosphodiesterase Activity.This assay is based on the enzymatic hydrolysis of cyclic 2': 3'-AMP to 3'-AMP, which is then converted to adenosine byexcess snake venom nucleotidase. The incubation medium con-sisted of 50 mm MES buffer, pH 6.0, 1 mM cyclic 2': 3'-AMP,1 mm magnesium chloride, 4 mM mercaptoethanol, 50 jug/mlsnake venom (Crotalus ademanteus) nucleotidase, and crudebarley seed enzyme in a total volume of 0.3 ml. The snakevenom nucleotidase (Sigma Chemical Co.) contained no acti-vators or inhibitors for barley cNPDE activity. In all experi-ments, corrections were made for the small cNPDE activityassociated with this commercial preparation. The reactionmixtures were incubated for 120 min at 37 C, and the reactionswere terminated by adding 0.03 ml of 50% (w/v) trichloro-acetic acid. After centrifugation at 1800g for 10 min, thesupernatant fractions were assayed for Pi by a procedure de-scribed by Chen et al. (2).

Assay for Cyclic 3': 5'-AMP Phosphodiesterase Activity.This assay is based on enzymatic hydrolysis of 3H-cyclic3':5'-AMP to a mixture of 3'-AMP and 5'-AMP. The proced-ure is in accordance with a method described by Thompsonand Appleman (26). The nucleotides are subsequently con-verted to 3H-adenosine by excess snake venom nucleotidase.The incubation medium was identical to that used for cyclic2':3'-AMP phosphodiesterase activity (above), except that 1mM 3H-cyclic 3': 5'-AMP (3.3 X 105 cpm/4mole) was usedinstead of cyclic 2': 3'-AMP. After 120 min at 30 C, thereactions were terminated by adding 1.0 ml of a 1:3 (w/v)slurry of anion-exchange resin (Bio-Rad AG 1-8), and the tubecontents were immediately centrifuged at 1800g for 10 min.Charged nucleotides bind to the resin, whereas 3H-adenosineremains in the supernatant fluid. Radioactivity in the super-natant fluid was determined in a liquid scintillation spec-trometer. When "4C-cyclic 3': 5'-AMP was used as a substratethe reaction mixtures were chromatographed on paper. Afterdevelopment, the distribution of radioactivity was determinedwith a radio-chromatogram scanner or liquid scintillationspectrometer.

Protein Determinations. Protein content was estimated bythe method of Lowry et al. (15).Ammonium Sulfate Fractionation and Bio-Gel Chromatog-

raphy. Solid ammonium sulfate was added to a crude enzymepreparation (obtained from the incubation of embryoless seeds)until 30% saturation was achieved. After centrifugation, thesupernatant was brought to 70% saturation with ammoniumsulfate. Suspended material was precipitated by centrifugationat 10,OOOg for 10 min and redissolved in 0.5 ml of 0.01 Mtris-acetate buffer, pH 7.0, and applied to a Bio-Gel P-150column (0.7 X 30 cm) which had been equilibrated with 0.01M tris-acetate buffer, pH 7.0. The enzyme was eluted with thisbuffer and collected in 0.5-ml fractions at a flow rate of 6ml/hr.

Paper Chromatography. Whatman No. 1 filter strips wereused, and chromatography was performed at room tempera-ture. For descending chromatography, the solvent systems

were ethanol-1 M ammonium acetate, pH 7.4 (7:3) (solventsystem I); isopropanol-ammonium hydroxide-0.1 M boric acid(6:1:3) (solvent system II); and saturated ammonium sulfate-0.1 M phosphate buffer, pH 6.0-isopropanol (70:19:2) (solventsystem III). Authentic nucleotides and their derivatives wereused as markers and located under shortwave ultraviolet light.

Source of Barley Seeds, Hormones, and Nucleotides. Barleyseeds (Hordeum vulgare L. cv. Himalaya), 1969 harvest, wereobtained from the Department of Agronomy, WashingtonState University, Pullman. Synthetic (RS)-abscisic acid was agift of the R. J. Reynolds Tobacco Co., Research Department.Gibberellic acid and nucleotides were purchased from SigmaChemical Co. or Calbiochem. The 'H-cyclic 3':5'-AMP and4C-cylic 3': 5'-AMP were purchased from Schwarz/Mann.Paper chromatography of these labeled compounds in solventsystem I showed that there were impurities located in the areaof authentic adenosine, adenine, and 3'-AMP or 5'-AMP. Toremove these impurities the labeled cyclic 3':5'-AMP, stocksolutions were streaked on paper and chromatographed insolvent system I overnight. The cyclic 3':5'-AMP band waseluted free from bases, nucleosides, and other nucleotides withdeionized water (recovery rate of at least 80%) and was suffi-ciently dilute to use directly in the enzyme assays.

RESULTS AND DISCUSSIONProducts of Enzymatic Hydrolysis of Cyclic 2': 3'-AMP

and Cyclic 3':5'-AMP. Lin and Varner (13) have establishedthat cyclic 3':5'-AMP is hydrolyzed by a partially purifiedcNDPE from pea seedlings to a mixture of 5'-AMP and 3'-AMP. This is in contrast to the 3':5'-cNPDE isolated fromanimal tissues which hydrolyzes cyclic 3':5'-AMP exclusivelyto 5'-AMP (10). A cNPDE activity is present in embryolessbarley seed homogenates and in incubation medium, whichsimilarly catalyzes the hydrolysis of '4C-cyclic 3':5'-AMP toa mixture of 5'-AMP and 3'-AMP and converts cyclic 2':3'-AMP to 3'-AMP. The identity of these metabolites was ascer-tained by paper chromatography with solvent systems I, II,and III. To enhance the production of "C-3'-AMP and "4C-5'-AMP, their respective unlabeled nucleotides were added tothe reaction mixture. Since nucleotidase activity against 3'-AMP and 5'-AMP is present in these homogenates, adenosineis obtained as a major metabolite when either cyclic 2': 3'-AMPor cyclic 3': 5'-AMP is degraded by crude barley seed homoge-nates. The rate of 3'-AMP hydrolysis is considerably greaterthan 5'-AMP nucleotidase activity. Because of the presence ofthese nucleotidases, it was impossible to determine the exactratio of 5'-AMP to 3'-AMP in the hydrolysis of "4C-cyclic3':5'-AMP by cNPDE.The possibility that a transferase (1) may have catalyzed the

formation of 3'-AMP from 5'-AMP (or vice versa) was ex-plored by separately incubating "4C-3'-AMP and "C-5'-AMPwith barley seed enzyme at 30 C for 2 hr and chromatograph-ing the reaction mixtures in solvent system II. No evidence oftransferase activity was observed.

Characteristics of Barley Seed cNPDE Activity. cNPDE ac-tivity against either cyclic 2':3'-AMP or cyclic 3':5'-AMP ispresent in both the supernatant and pellet fractions after cen-trifugation of barley seed homogenate at 100,000g for 60 min.The soluble and particulate enzymes exhibit similar pH optima,the same phosphodiesterase hydrolysis products and are simi-larly affected by EDTA and metal ions. A soluble cNPDE issecreted from embryoless seeds into their surrounding medium.This source of crude enzyme was used to examine some of thecharacteristics of cNPDE activity or alternatively it was frac-tionated with ammonium sulfate and passed over a Bio-GelP-150 column and then used for studies.

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VANDEPEUTE, HUFFAKER, AND ALVAREZ

iiu

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<-0A -

X-0

11) Cn

ar

12.0

10.0

8.0

6.0

4.0

2.0

u' 20 40 60 80 100TIME (min)

120 140

FIG. 1. Time course of the enzymatic hydrolysis of cyclic 2': 3'-AMP and cyclic 3': 5'-AMP by barley seed cNPDE. The reactionswere performed under standard conditions except that the volumewas increased so that aliquots equal to the standard assay volumecould be removed at the time intervals indicated. The enzyme wasobtained from the medium surrounding embryoless barley seeds.The crude enzyme preparation was fractionated with ammoniumsulfate, and cNPDE was isolated after Bio-Gel P-150 columnchromatography as described under "Materials and Methods."

U-

LI -0

cr ti

O

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aL

70.(

60.(

50.(

40.(

30.4

20.4

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vI 2 34 5 6 7 8

pH

FIG. 2. Effect of pH on cyclic nucleotide phosphodiesterase ac-

tivity. The reactions were performed under standard conditions,except that the pH was varied as indicated. Buffer used 50 mmMES (0); 50 mM tris (0); 50 mm sodium acetate (Q). Enzyme was

prepared as described in Fig. 1.

Under standard conditions the hydrolysis of either cyclic2': 3'-AMP or cyclic 3': 5'-AMP is proportional to enzyme con-centration and the rate is linear during the first 120 min (Fig.1). The optimal pH range for either reaction is between 5.0and 6.0 (Fig. 2).

Barley cNPDE activity was reduced in the presence ofEDTA. The EDTA had no effect on nucleotidase activity.Addition of magnesium chloride or zinc sulfate partially re-stores activity (Table I). A significant inhibition of cyclic 3':5'-AMP hydrolysis by sodium fluoride is obtained with little or

no effect on the hydrolysis of cyclic 2':3'-AMP. A similar ob-servation has been reported for cNPDE from pea seedlings(13).

Tests were also performed to examine the effect of themethylxanthines, caffeine and theophylline, on cNPDE. Thecrude enzyme preparations of both the soluble and particulatecNPDE activities showed inhibition with both compounds butthis property was lost with the purification of the medium en-

zyme indicating that their effect may be other than an interac-tion with cNPDE itself.

Effect of Imbibition and Plant Hormones on Secretion ofcNPDE. If cyclic 3':5'-AMP hydrolysis is due to the -ction ofan enzyme which is different from that involved in the degrada-tion of cyclic 2':3'-AMP, then there may be distinct patternsin their respective activities during imbibition or in response tohormones which influence enzyme synthesis and secretion inbarley seeds. To test this possibility, dry embryoless seeds were

incubated in the presence or absence of GA and cNPDE ac-

tivity was measured after various periods of incubation. Theresults indicate that cNPDE is present in the dry seed and thatits activity increases gradually throughout the 50-hr incubationperiod (Fig. 3). After 24 hr, cNPDE activity can be detectedin the incubation medium and GA increases the amount re-

leased (Fig. 3). In this system, the secretion of cNPDE is co-

incident with RNase release. GA-enhanced release is inhibitedby abscisic acid, cycloheximide, or dinitrophenol (Table II).The amount of enzyme secreted into the medium from isolatedaleurone layers increases as the concentration of GA is in-

creased to 0.1 4M (Fig. 4), at the same time no secretion ofcNPDE activity was detected from the accompanying endo-sperm tissue. Abscisic acid or GA caused neither direct inhibi-tion nor activation of cNPDE. In each case. patterns were

similar for the hydrolysis of either cyclic 3': 5'-AMP or cyclic2': 3'-AMP (Table II, and Figs. 3 and 4).

Table I. Effect of Iniorganiic Ionis onz cA\PDE Activity

Crude enzyme from the incubation medium surrounding em-

bryoless barley seeds was treated with EDTA and dialyzed prior tothe addition of MgCl2 or ZnSO4. cNDPE assays were performedas described under "Materials and Methods." The results are theaverages of duplicate determinations.

Phosphodiesterase Activ-ity

Cyclic 2': 3'-AMIP Cyclic 3': 5'-AMIP

Untreated' 100 1001.5 mM EDTA 34 603 mM MgC12 52 960.5 mm ZnSO4 79 930.001 mMNaF 100 1000.01 mM NaF 100 850.1 mM NaF 97 740.3 mM NaF 91 590.6 mM NaF 89 421 mM NaF 86 36

1 Enzymatic activities of the untreated enzyme were respectively27.0 and 3.7 nmoles of product/hr.0.01 ml of medium for cyclic2':3'-AMP and cyclic 3':5'-AMP.

cyclic 3'; 5'-AM P

cyclic 2': 3-AMP

° I

n

280 Plant-Pbysiol. Vol. 52, 1973

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Plant Physiol. Vol. 52, 1973 CYCLIC NUCLEOTIDE PHOSPHODIESTERASE

H20

E; a.u ._

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FIG. 3. Development and secretion of cNPDE in response toimbibition in water or GA. Dry embryoless seeds were transferreddirectly to the test solution. Assays were performed under standardconditions. Total phosphodiesterase activity within 10 embryolessseeds and 1.7 ml of the surrounding incubation medium is shownhere.

Table JI. Effect of GA, ABA, Dintitrophenol, and Cyclohex-imide oni cNPDE Activity in Embryoless Barley Seeds

antd Thleir Surrounding MediaSurface-sterilized dry embryoless seeds were transferred di-

rectly to the test solutions. cNPDE activities were determinedafter 48 hr of incubation. Total phosphodiesterase activity per 10half-seeds or per 1.7 ml of medium surrounding these seeds isshown below. The assays were performed under standard con-ditions. The following concentrations were used: GA, 0.1 jAM;ABA, 25,ui; dinitrophenol (DNP), 0.1 mm, and cycloheximide(CHI) 10lg/ml.

Phosphodiesterase Activity

Pi Adenosine

Half- Medium Total Half- Medium Totalseeds seeds

iLmoles produced/hr

H20 9.2 1.7 10.9 1.0 0.2 1.2GA 9.3 5.8 15.1 1.1 0.8 1.9GA + ABA 8.4 1.7 10.1 1.1 0.1 1.2GA + DNP 7.9 0.9 8.8 1.1 0.1 1.2GA + CHI 7.3 0.8 8.1 1.1 0.1 1.2

Attempts to Separate 3':5'-cNPDE Activity. Among the en-

zymes which might be responsible for the observed cNPDEactivity, prominent ones in barley are RNase, 3'-nucleotidase,and ATPase. Bio-Gel fractionation separates cNPDE activityfrom RNase. The separation of cNPDE from 3'-nucleotidasehas been attempted with no success using the following tech-niques: gel filtration, isoelectric focusing, sucrose density gra-dient centrifugation, and gel electrophoresis (12). In additionto some of these techniques, heat inactivation, acetone frac-tionation, cellulose powder (Whatman CF 1 1) chromatography,and Dowex column (Bio-Rad AG 50-W-4) chromatographywere also not capable of effecting a separation from nucleo-

1.0

0.5

-9 8 -7

GA. log (g /I)

FIG. 4. Effect of GA concentration on the amount of cNPDEsecreted from isolated aleurone layers. Fifteen aleurone layers eachwere incubated in 2 ml of test solution. The assays were conductedunder standard conditions.

I/E[S]IxoFIG. 5. Inhibition of cyclic 2': 3'-AMP hydrolysis by cyclic

3': 5'-AMP. A Lineweaver-Burk plot of cNPDE activity with cyclic2': 3'-AMP as substrate and V as nrmoles Pi/hr. The assay for cyclic2': 3'-AMP hydrolysis was conducted under standard conditions, ex-

cept that substrate (S = cyclic 2':3'-AMP) and inhibitor (I = cyclic3': 5'-AMP) concentrations were varied as indicated. Enzyme was

prepared as described in Fig. 1.

tidase activity nor a separation between 3': 5'-cNPDE and2': 3'-cNPDE activity.

In view of the lack of success with these procedures, inhibi-tion experiments were devised to test whether 3'-nucleotidaseor ATPase might be responsible for the cNPDE activity.Neither 3'-nucleotidase nor ATPase activities are inhibited bycyclic 3': 5'-AMP 1 mm, using substrate concentrations of 0.1mm for 3'-AMP and ATP. Tests with cyclic 2':3'-AMP couldnot be made because its rate of hydrolysis at these concentra-tions is sufficient to produce Pi itself, hence preventing an ac-

curate determination of the 3'-AMP and ATP hydrolysis rates

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cyclic 3':5'-AMP1.5

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282 VANDEPEUTE, HUFFAKER, AND ALVAREZ

via Pi determination. In addition, the results from the experi-ments with metal ion treatments (Table I) were also obtainedconcomitantly for the substrates, 3'-AMP and ATP. The rateof hydrolysis of neither of these two substrates was affected by1.5 mm EDTA. While zinc sulfate restores cNPDE activityfrom EDTA inhibition, it brings about a drastic reduction ofnucleotidase and ATPase activity. These experiments supportthe possible existence of a true cNPDE with no phosphatase ornucleotidase activity, as was demonstrated for pea cNPDE byLin and Varner (13).

Inhibition of Hydrolysis of Cyclic 2':3'-AMP and Cyclic3': 5'-AMP. The Michaelis constant for cyclic 2': 3'-AMP hy-drolysis (0.7 mM) differs from that of cyclic 3': 5'-AMP hy-drolysis (0.4 mM). If one enzyme is involved in the degradationof both nucleotides, then the hydrolysis of one substrate wouldbe expected to be inhibited by the addition of the other sub-strate. Cyclic 3': 5'-AMP competitively inhibits the hydrolysisof cyclic 2': 3'-AMP (Fig. 5), and cyclic 2': 3'-AMP inhibitsthe hydrolysis of cyclic 3': 5'-AMP.

These results suggest that the soluble cNPDE in barley seedsis due to the action of a single enzyme and that it is nonspecific,catalyzing the hydrolysis of either cyclic nucleotide. If ribo-nuclease action in plants results in an accumulation of cyclic2': 3'-NMP, this phosphodiesterase may function to completethe degradation of RNA to 3'-AMP as previously suggested(13). Should unequivocal evidence be obtained for cyclic 3':5'-AMP in barley seeds, then it is possible that the soluble barleyseed enzyme is involved in its hydrolysis. In a series of pre-liminary experiments, however, we have been unable to dem-onstrate the presence of cyclic 3': 5'-AMP, adenyl cyclase, or acyclic 3':5'-nucleotide-dependent protein kinase in barley seeds.The presence of a nonspecific cNPDE, therefore, does notnecessarily implicate the involvement of cyclic 3': 5'-AMP inbarley seed metabolism.

Acknowledgsnents-We wN-ish to express our appreciation to Drs. R. W.Breidenbach, R. M. Sachs, and J. R. Whitacker for their helpful suggestions. Wealso thank Drs. P. P. C. Lin and J. E. Vamer for sending us a copy of theirmanuscript while it was in press.

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