Plant Physiol. (1986) 82, 566-5740032-0889/86/82/0566/09/$0 1.00/0

Ca2 -Stimulated Secretion of a-Amylase During Development inBarley Aleurone Protoplasts1

Received for publication February 10, 1986

DOUGLAS Scorr BUSH*, MARIA-JESUS CORNEJO, CHUN-NONG HUANG, AND RUSSELL L. JONESDepartment ofBotany, University ofCalifornia, Berkeley, California 94720

ABSTRACT

The effects of gibberellic acid (GA3) and Ca2'on the synthesis andsecretion of a-amylase from protoplasts of barley (Hordeum vulgare L.cv Himalaya) aleurone were studied. Protoplasts undergo dramatic mor-phological changes whether or not the incubation medium contains GA3,CaCt2, or both. Incubation of protoplasts in medium containing both GA3and Ca2 , however, causes an increase in the a-amylase activity of bothincubation medium and tissue extract relative to controls incubated inGA3 or Ca2' alone. Isoelectric focusing shows that adding Ca2+ toincubation media containing GA3 increases the levels of a-amylase iso-zymes having high isoelectric points (pI). In the presence of GA3 alone,only isozymes with low pIs accumulate. The increase in a-amylase activityin the incubation medium begins after 36 hours of incubation, andsecretion is complete after about 72 hours. Protoplasts require continuousexposure to Ca2+ to maintain elevated levels of a-amylase release. Im-munoelectrophoresis shows that Ca2+ stimulates the release of low-pI a-amylase isozymes by 3-fold and high-pI isozymes by 30-fold over controlsincubated in GA3 alone. Immunochemical data also show that the half-maximum concentration for this response is between 5 and 10 millimolarCaC12. The response is not specific for Ca2+ since Sr2O can substitute,although less effectively than Ca2+. Pulse-labeling experiments show thata-amylase isozymes produced by aleurone protoplasts in response to GA3and Ca2+ are newly synthesized. The effects of Ca2+ on the process ofenzyme synthesis and secretion is not mediated via an effect of this ionon a-amylase stability or on protoplast viability. We conclude that Ca2+directly affects the process of enzyme synthesis and transport. Experi-ments with protoplasts also argue against the direct involvement of thecell wall in Ca2+-stimulated enzyme release.

Calcium stimulates the synthesis and secretion of a-amylasein isolated barley aleurone layers that have been pretreated withGA3. Although Ca>' does not by itself induce a-amylase produc-tion, it is required, for reasons that are not understood, formaximal enzyme synthesis and secretion (5). Aleurone layers ofHimalaya barley synthesize and secrete at least four isozymes ofa-amylase (13). These isozymes belong to two groups that arecoded on separate chromosomes (2) and are distinguished fromeach other by several properties including differences in theirpIs2 ( 14). Aleurone layers treated with GA3 alone synthesize andsecrete only one group of a-amylase isozymes, those having pIsbetween 4.5 and 5.2 (low-pI; 13, 14). When Ca>2 is added toGA3-treated layers, the secretion ofthe second group ofisozymes,those with pls between 5.9 and 6.3 (high-pI), is rapidly initiated,

'Supported by grants from the United States Department of Energy.2 Abbreviations: pl, isoelectric point; IEF, isoelectric focusing, CHA,

cycloheptaamylose.

and the secretion of the low-pI isozymes is unaffected (13, 19).Little is known about where in the process of a-amylase

synthesis and secretion Ca2" is required. It is clear, however, thatit is directly involved at some step since the Ca2" requirement isnot transient (4, 25). Withdrawal ofCa2" from the medium stopsthe secretion of high-pI isozymes within minutes (25), and theamount of high-pI a-amylase secreted is linearly proportional tothe external Ca" concentration in the millimolar range (20).Ca2+-stimulated secretion is not specific for Ca"; Sr2" at highconcentrations can substitute for Ca", but Ba , Mg", andmonovalent cations cannot (20).Although Ca2+-stimulated a-amylase secretion has been well

characterized, the mechanism by which Ca2> acts is not known.An early hypothesis that Ca>2 only appears to stimulate secretionby stabilizing an otherwise labile enzyme (5) has been disprovenby immunochemical data (19). Recently it has been shown that,unlike GA3, Ca2> does not influence levels oftranslatable mRNAfor high- or low-pI a-amylases (7, 8). Ca2> stimulation is thereforeat or between the levels of translation and release of the proteininto the medium. Varner and Mense (28) proposed that Ca>facilitated a-amylase diffusion through the cell wall, but Molland Jones (25) concluded that the mechanism for Ca" stimula-tion existed at the plasma membrane. Ca> may stimulate amy-lase release both at the cell wall and at the plasma membrane,but until recently it has not been possible to distinguish betweenthese two possibilities.The ability to isolate aleurone protoplasts that secrete a-

amylase in response to GA3 (15) provides an opportunity toseparate the effects of Ca2+ on enzyme release at the plasmamembrane and at the cell wall. We have characterized a-amylaserelease from protoplasts under various Ca> treatments andfound that the characteristics of Ca2+-stimulated secretion inprotoplasts are similar to those in isolated aleurone layers. Wehave also investigated the effect of Ca>2 on protoplast longevity,size, and morphology and have found that Ca>2 does not stim-ulate a-amylase release merely by increasing protoplast viability.We conclude that the cell wall is not obligatorily involved inCa2+-stimulated secretion.

MATERIALS AND METHODS

Protoplast Isolation. Protoplasts were isolated from barley(Hordeum vulgare L. cv Himalaya, 1979 harvest) caryopses. Theprocedures for protoplast isolation were similar to those usedand described by Jacobsen et al. (15), and all were carried outunder aseptic conditions in a laminar flow hood. Briefly, 20quarter grains (deembryonated grains cut along the suture) weresterilized with NaOCl (0.25%, w/v) for 30 min, rinsed in distilledH20, immersed in 10 mM HCI for 10 min, then rinsed again indistilled H20. Twenty quarter-grains were imbibed for 24 h in 2ml of 50 mM L-arginine and 20 mM CaCI2 in 25-ml flasks. Thestarchy endosperm was then removed and the quarter aleuronelayers returned to their flasks and incubated in 1.5 ml of the

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Ca2`-STIMULATED SECRETION DURING PROTOPLASTS DEVELOPMENT

protoplast isolation medium for 48 h; the isolation medium wasreplaced with fresh medium once after 24 h. The isolationmedium was that described by Jacobsen et al. (15) except thatCaCl2 was omitted. It consisted of Gamborg's B-5 medium ( 1)without 2,4-D or CaCl2 but with 0.35 M mannitol, 2% glucose(w/v), 10 mM L-arginine, 10 mm Mes ([2-(N-morpholino) eth-anesulfonic acid], obtained from Calbiochem), 4.5% (w/v) cel-lulase Onozuka R-10 (Yakult Pharmaceutical Industry Co. Ltd,Nishinomiya, Japan), and 1% (w/v) polyvinylpyrrolidone K25(Fluka A.G., Buchs S.G., Switzerland). During the imbibition ofthe quarter grains and the isolation of protoplasts (72 h), flaskswere kept in a desiccator flushed with N2 twice every 24 h; thefirst time for 45 min and the second time, 12 h later, for 15 min.

Protoplast Incubation. The protoplast isolation medium wasreplaced with incubation medium and the undigested seed coatswere removed. The incubation medium consisted of Gamborg'sB-5 without 2,4-D, but with 0.67 M mannitol, 2% (w/v) glucose,and 10 mM L-arginine to which were added 75 MlA 100 Mm GA3(Sigma Chemical Co), resulting in a final concentration of 5 gMGA-, and/or 0 to 150 ul 2 M CaCl2, resulting in 0 to 200 mMCaC12, or an equal volume of H20. Incubation was carried outin the dark at 23 ± 2°C for up to 96 h. Preliminary experimentsshowed that replicate samples of protoplasts sometimes differedin their ability to release amylase. To eliminate this variability,the flasks of freshly isolated protoplasts for each experiment werecombined and then dispensed into clean sterile flasks for incu-bation. To ensure that equal numbers of protoplasts were addedto each flask, three 500-gl aliquots of the combined protoplastsuspension were added to each flask while gently swirling thesuspension. Each flask finally contained 1.5 ml of the combinedprotoplast suspension.

After incubation, protoplasts were separated from incubationmedia by allowing a small volume of the suspension (about 300Mul) to stand in a 1.5-ml centrifuge tube for 30 min. The proto-plasts settled to the bottom of the tube, and the incubationmedium was removed and saved for analysis. The protoplastswere washed twice in fresh incubation medium (15) and lysed in10 mm CaCl2. The lysed protoplasts in 10 mM CaCl2 are referredto as 'protoplast extract.'

Radiolabeling, Radiocounting, and Fluorography. Protoplastswere incubated with 5 gM GA3 and/or 20mM CaCl2 until amylaserelease was detected (after about 40 h of incubation) and werethen pulse-labeled for 3 h with 1.25 x 106 Bq ml-' of 35[S]methionine (53.3 TBq mmolh', Amersham). Protoplast extractsand incubation media were separated as described above. Theamount of total incorporated label was measured by TCA pre-cipitation on filter paper strips (23). Incorporation of radioactiv-ity into a-amylase was measured after purification of a-amylaseby affinity chromatography on a CHA-sepharose 6B column(1.5-cm diameter, 2-cm long) according to the procedure de-scribed by Jacobsen and Higgins (14). Radioactivity was meas-ured in a liquid scintillation counter (LS7000, Beckman Instru-ments) as described by Melroy and Jones (24).The incorporation of radioactivity into specific proteins was

analyzed by SDS-PAGE. Equal volumes of protoplast extractsand incubation media were centrifuged and the supernatantadded to an equal volume of SDS-PAGE sample buffer (21).Proteins were separated on a discontinuous 12.5% polyacryl-amide gel as described by Laemmli (21). Gels were fixed, stainedwith Coomassie brilliant blue-R, soaked in 1 M sodium salicylatefor 1 h, rinsed briefly in distilled H2O, dried under a vacuum,and exposed to Kodak XAR-5 film with an intensifying screenat -70°C.Enzyme Assay. The total amylase activity ofprotoplast extracts

and incubation media was determined using the starch-iodinemethod (17).

essentially as described by Jacobsen et al. (13). Agar (0.8%, w/v)was prepared in 4 mm K-phosphate (pH 8.5). Bands of amylaseactivity were visualized with the starch-iodine procedure of Ja-cobsen et al. (13).

Isoelectric Focusing (IEF). a-Amylase isozymes were alsoexamined by IEF as described by Jacobsen and Higgins (14),using ampholine polyacrylamide gels, pH 4-6.5 (LKB ProdukterAb, Bromma, Sweden), and an Ephortec Isoelectric FocusingCell (Haake Buchler Instruments, Inc., Saddle Brook, NJ) cooledto 1C by a circulating water bath. After prefocusing the gel forapproximately 500 V-h, samples were applied directly to the gelon application papers (LKB) and focused for 3000 V-h. Bandsof amylase activity were visualized on IEF gels by soaking themin 2% (w/v) soluble potato starch (MCB Manufacturing Chem-ists, Cincinnati, OH) then flooding them with I2KI (14). Sincethe pattern of bands on IEF gels was identical to that reportedby Jacobsen and Higgins (14), we labeled the bands accordingly;for example, the two isozymes with isoelectric points above 5.5were labeled pI 5.9 and pI 6.3. Previously, a-amylase isozymeshave been labeled with the letters A and B and numerals 1 to 4based on their position on agar gels (13). We have determinedthat pI 4.5 and pI 4.7 correspond to group A isozyme 1. Similarly,pi 4.85 corresponds to group A isozyme 2, and pI 5.9 and pI 6.3correspond to group B isozymes 3 and 4, respectively.

Immunoelectrophoresis. The amounts of high- and low-pI a-amylase proteins were determined by Laurell rocket immunoe-lectrophoresis (29) using rabbit serum prepared against totalbarley a-amylase (7). Following electrophoresis at 60 V for 18 hat 4°C, proteins were stained with Coomassie brilliant blue. Low-and high-pI isozymes of a-amylase were identified on the rocketby adding known amounts of pure high-pI amylase to replicatesamples. The rocket heights that did not change after addinghigh-pI amylase were thereby identified as low-pI a-amylase. Theamount of a-amylase protein was quantified by comparing sam-ple rocket heights with those of pure high- and low-pI a-amylasewhose protein concentrations had been measured by the Brad-ford method (Bio-Rad Laboratories). A linear relation betweenrocket height and protein concentration was observed for purebarley a-amylase isozymes.

Elemental Analysis. Total Ca in the incubation medium wasdetermined after HCI04-HNO3 digestion ( 16) by atomic absorp-tion spectrophotometry (model AA6 Atomic Absorption Spec-trophotometer, Varian Associates, Palo Alto, CA). Free Ca2+ wasdetermined with murexide as described by Ohnishi (26). Phos-phorous was determined on digests using the Lachat Auto Ana-lyser (QuikChem method number 110518, Lachat Chemicals,Inc., Mequon, WI). pH was measured with a pH meter (modelPHM84, Radiometer, Copenhagen, Denmark) and a combina-tion glass electrode.

Microscopy. During incubation of protoplasts, samples weretaken each 24 h to monitor their numbers, viability, and stageof development. The number of intact protoplasts was deter-mined using a 200-Am deep Fuchs-Rosenthal hemocytometer;the initial yields were in the range of 3 to 5 x I05 protoplasts (mlofculture medium)-'. Protoplast viability was determined by theexclusion of Evans blue dye by intact membranes (6, 10) andalso with fluorescein diacetate (FDA). Viable cells fluoresced asfluorescein accumulated in the cytoplasm (22). Typically, 60 to70% of freshly prepared protoplasts were viable, and the coeffi-cient of variation in protoplast numbers was in the range of 5 to15%.To follow the morphological changes that protoplasts undergo

during incubation, four stages of development were classifiedbased on the number and size of vacuoles in the cytoplasm (Fig.1). Protoplasts were observed using a Zeiss standard photomicro-scope equipped with phase contrast and epifluorescence optics.

567

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Plant Physiol. Vol. 82, 1986

FIG. 1. Changes in the structure of barley aleurone protoplasts arbitrarily classified into four stages of development (x784).

Table I. Effect ofCa2+ and GA3 on Amylase Activity in ProtoplastExtracts and Incubation Media

Protoplasts were incubated in GA3, Ca2+, and GA3 plus Ca2" for 50 h,then incubation media and protoplast extracts were separated and amy-lase activity was measured.

Amylase ActivityTreatment

Medium Extract

units (JO5 protoplasts)-'Ca2+ (20 mM) 3 0.4GA3 (5 Mm) 6 3.0GA3 + Ca2+ 38 4.5

..,.~~~)

. _ ~~

!~~-NV ...

I iON to'... >;.|AMV; J

.4. _

~ ~ ~-A4, GA - -a G4 G-R'

EX TIR A.C T M EDfIUM

FIG. 2. IEF of protoplast extracts (lanes 1-3) and incubation media(lanes 4-6) of barley aleurone protoplasts incubated in 5 ,uM GA3, 20 mMCaC12, and 5 MM GA3 plus 20 mM CaCl for 50 h. The residual Onozukaamylase from the isolation medium and the pIs ofthe a-amylase isozymesare indicated.

RESULTS

Secretion and Synthesis of a-Amylase by Aleurone Proto-plasts. Incubation of barley aleurone protoplasts in a mediumcontaining 5 ,uM GA3 and 20 mm Ca2" results in an increase intotal amylase in incubation media and protoplast extracts relativeto that in media and extracts of protoplasts incubated in either

E

en

-J

2

14

7

20 36 52 68 84 100

TIME (h)

FIG. 3. Time course of the accumulation of amylase activity in theincubation media of washed (---) and unwashed ( ) protoplasts.Protoplasts were incubated in 5 gM GA3 with and without 20 mM CaC12for 40 h. At 40 h (l) some of the protoplasts were washed in Ca24-freemedium and incubated in GA3 with (0) and without (0) 20 mm CaC12while the controls were left in their original media with (A) and without(A) CaC12-

GA3 or Ca2" alone (Table I). IEF of incubation media andprotoplast extracts shows that, in the presence of extracellularCa24 and GA3, a-amylase isozymes with pIs of 5.9 and 6.3 areparticularly prominent (Fig. 2). Isozymes with high pIs are alsopresent, although at much lower levels, in media and extracts ofprotoplasts incubated in GA3 alone and in Ca2' alone (Fig. 2).a-Amylase isozymes with pIs of 4.5 and 4.85 accumulate in bothmedia and extracts of protoplasts incubated in GA3 alone,whereas only the isozyme pl 4.85 is found in media and extractsof protoplasts incubated in CaCl2 (Fig. 2). Protoplast extractscontain two isozymes with pis at 4.7 and 5.2 that are not foundin incubation media (Fig. 2). All incubation media contain an

amylase isozyme with a pI below 3.5 derived from the OnozukaR-l0 cellulose used to isolate the protoplasts (Fig. 2).The time course of a-amylase release from aleurone proto-

plasts incubated in GA3 in the presence and absence of 20 mmCa2" is shown in Figure 3. The effect ofCa2" on the accumulationof a-amylase in the incubation medium is evident after 36 h ofincubation, and the rate ofa-amylase secretion in this experimentwas greatest between 54 and 68 h of incubation (Fig. 3). Ca2`-promoted accumulation of a-amylase activity coincides with theaccumulation of high-pI isozymes in the incubation medium(data not shown). Agar-gel electrophoresis also shows that thedecrease in accumulation ofamylase activity in incubation mediawithout Ca2" is a result of a decrease in the level ofcontaminating

A<-CNTROL+Ca2+

WASHED +Ca2+

/0A~~~'o, CONTROL-Ca 2

4 ,2. R\O WASHED -Ca24If,{ ._

_

I

568 BUSH ET AL.

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Ca2`-STIMULATED SECRETION DURING PROTOPLASTS DEVELOPMENT

24

20

H-

z

U)

-Jin

HIGH-PI _LOW - PI -|

ONOZUKA _U Z.0 D Iu zu !:U IUU zUU

C0C12 CONCENTRATION

FIG. 4. The effect of Ca2" concentration on the accumulation ofamylase in incubation media of protoplasts incubated without GA3(-GA3) and in 5 yM GA3 with increasing concentrations of CaC12 for 72h. Total amylase activity was measured by the starch-iodine procedure(top), and a-amylase isozymes were separated by agar gel electrophoresis(bottom). The activities of Onozuka amylase and the low- and high-pIbarley a-amylase isozymes are indicated on the agar gel.

Onozuka R- 10 amylase activity (data not shown).To determine whether the Ca2+-stimulated secretion and syn-

thesis of a-amylase was transient or required continuous expo-sure to Ca2+, protoplasts were isolated, incubated in GA3 with orwithout Ca2+ for 40 h, then washed free of Ca2' and incubatedin GA3 with and without Ca2 . Figure 3 shows that when Ca2"was withdrawn from protoplasts, a-amylase production ceasedafter several hours. When Ca>2 is added back to protoplasts,however, they continue to release a-amylase at the rate of un-washed controls (Fig. 3). Agar gel electrophoresis of incubationmedia shows that Ca2 removal results in a reduction in accu-mulation of high pI a-amylase isozymes in the media, whilecontrol cells and cells that were washed but reexposed to Ca>2continue to secrete high-pI isozymes (data not shown).The effect of Ca>2 concentration on the accumulation of a-

amylase activity in media during 72 h of incubation of proto-plasts in the presence and absence of GA3 is shown in Figure 4.Calcium at 100 mm causes a 10-fold increase in a-amylaseaccumulation in the medium in the presence of GA3, and Ca>2at 20 mm produces a half-maximal response (Fig. 4). In theabsence of GA3, Ca>' does not promote a large increase in a-amylase activity. Agar-gel electrophoresis shows that Ca>2 stim-ulates the accumulation of a-amylase isozymes with high pIs(Fig. 4).

Because amylase activity from cell wall digesting enzymes ispresent in incubation media of all treatments (e.g. Figs. 2 and4), immunoelectrophoresis was used to examine changes inbarley a-amylase. Antibodies raised against a mixture of high-and low-pI barley a-amylase isozymes do not react with OnozukaR- 10 amylase in either Ouchterlony immunodiffusion (notshown) or in rocket immunoelectrophoresis (Fig. 5). Immunoe-lectrophoresis of incubation media from protoplasts incubatedin GA3 alone and in GA3 with increasing concentrations of Ca>2

I

E

0ca0-

-J

0

LIin

ARROW INDICATESHIGH - pl ISOZYMES

ONOZUKA 0 2.5 5 10 20 50 100 200

Ca Cl2 CONCENTRATION

FIG. 5. The effect of Ca2+ concentration on the accumulation of low-and high-pI barley a-amylases in incubation media of protoplasts incu-bated in 5 gM GA3 with increasing concentrations of CaC12 for 72 h asshown by Laurell rocket immunoelectrophoresis. The protein concentra-tions of the media and the identities of the low- and high-pI isozymeswere determined from rocket heights as described in "Materials andMethods." Immunoelectrophoresis of Onozuka cellulose is also shown.

Table II. Effect ofProtoplast Incubation on the Concentration ofCa2+in the Incubation MediumNominal Medium Ca2+ Concentration

Incubation 0 10Time

Observed Calculated Observed Calculated

h mm0 50.3 0.0 10.2 9.9

40 0.6 0.2 9.8 9.9

shows that high-pI isozymes are present in low amounts (about1 Atg protein ml-'), whereas low-pI isozymes are present at about10 qg protein ml-' (Fig. 5). Calcium increases the amount ofhigh-pI isozymes in the media by about 30-fold but that of low-pI isozymes by only 3-fold (Fig. 5). The half-maximal concentra-tion of Ca> required for the accumulation of low-pI a-amylaseisozymes is around 5 mm and for high-pI isozymes around 10mm. Saturation of the response for low-pI isozymes is at 10 mMCa>2 and for high-pI isozymes at 50 mM Ca>2 (Fig. 5). As is thecase for enzyme activity (Fig. 4), the accumulation of high-pIisozymes is inhibited above 100 mm Ca>2 (Fig. 5). There is agood linear relationship (r = 0.9, p = 0.01) between amylaseactivity and a-amylase protein (Fig. 5) confirming that Ca>2 doesnot affect the specific activity of released amylase.

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BUSH ET AL. Plant Physiol. Vol. 82, 1986

A\M. AS[E ACTIV Y 2 9 i6 8 6

HIGH-Ptl

LOW-RIP

ONOZUKA2+2+ 2+ 2+Z+ L',2± 2± 2+ 2± 2.+a +Sr Ca .r Cc "r C. Sr

C 0

FIG. 6. Agar gel electrophoresis of incubation media of protoplastsincubated in 5 gM GA3 with increasing concentrations of CaC12 or SrC12for 72 h. The total amylase activities were also determined and areindicated at the top of each lane.

Table III. Incorporation ofRadioactivity into Protoplast Extracts andIncubation Mediafrom Protoplasts Incubated in GA3, Ca2 , or GA3

plus Ca21Protoplasts were pulse-labeled with 35[S]methionine

Radioactivity

Protein a-AmylaseTreatment (TCA-precipitate) (CHA purified)

Extract Medium Total Extract Medium Total

cpm x 10-JCa2+ (20 mM) 104 587 691 1 9 10GA3(5 AM) 235 1157 1391 24 26 50GA3plus Ca2+ 117 1264 1381 55 86 141

Since incubation media from protoplasts incubated withoutGA3 or Ca" contain both low- and high-pI isozymes, the Ca>concentration of the media was monitored to determine howmuch Ca> our'minus-Ca"' media contained. Table II showsthat the nominal Ca> concentration of a medium preparedwithout GA3 or added Ca> was 300 ,M and it doubled after 40h of incubation with protoplasts. These results indicate that therewas little chelation of added Ca> in the protoplast medium.Accordingly, there was no difference between measured free Ca>and the amount of added Ca> in the 10 mM Ca2+ treatment(Table II). These observations were consistent with levels of freeCa> calculated with the computer program 'Geochem' (TableII; Ref. 27). The calculations were based on measurements oftotal Ca as determined by atomic absorption, total phosphorous,pH, and the composition of Gamborg's B-5 medium. Althoughthe Ca>2 concentration of Ca2+-free medium was 300 ,M, thislevel of Ca> could not account for the accumulation of a-

amylase isozymes in the medium, since titration of media withEGTA at pH 7.0 did not affect the pattern of isozyme accumu-lation (data not shown).The release of a-amylase activity from protoplasts is not spe-

cific for Ca2+, since Sr` is an effective substitute (Fig. 6). Withincreasing concentrations of Sr2> both the total released amylaseactivity and the level of high-pI a-amylase isozymes increase(Fig. 6). Strontium is less effective than Ca> in stimulating thesecretion of a-amylase isozymes, however (Fig. 6).To determine whether proteins released into the incubation

medium resulted from new synthesis, aleurone protoplasts prein-cubated in Ca-", GA3, or GA3 plus Ca> for 40 h were pulse-labeled with 35[S]methionine. Incorporation of radioactivity intototal TCA-insoluble protein was determined in aliquots of theincubation media and protoplast extracts (Table III). Radioactiv-ity was highest in protein isolated from media and extracts ofprotoplasts incubated in GA3 and GA3 plus Ca>. When incor-poration into affinity-purified a-amylase was monitored, how-

ever, the highest level of incorporation of radioactivity was intomedia and extracts of protoplasts incubated in GA3 plus Ca2"(Table III). Label incorporated into CHA-purified media andextracts of protoplasts incubated in GA3 alone was 35% of theGA3-plus-Ca2" treatment, whereas incorporation by Ca2"-treatedlayers was only 7% of the GA3-plus-Ca2" treatment (Table III).Labeled proteins were also separated by SDS-PAGE, and afluorograph oflabeled protein is shown in Figure 7. In incubationmedia and protoplast extracts incorporation of radioactivity intoa band corresponding to the position of a-amylase is highest inGA3-plus-Ca2+-treated protoplasts (Fig. 7). The amount of labelincorporated into media and extracts of Ca2"-treated protoplastscontrasts markedly with GA3 and GA3-plus-Ca2+ treatments (Fig.7). Thus, in the absence of GA3, incorporation of label into aband corresponding to a-amylase is negligible, although otherproteins that appear in the media label equally in the presenceand absence of GA3 (Fig. 7).

Effect of Ca2" on a-Amylase Stability. To determine if insta-bility of the enzyme could account for the absence of high-pI a-

amylase in the minus-Ca" medium, we measured the rate ofdegradation of a-amylase added to protoplast incubation media.Protoplasts were incubated in GA3 with and without Ca" for 48h, by which time there was a large amount of high-pI a-amylasein the medium of GA3- plus-Ca2+-treated protoplasts and littlein the medium of GA3-minus-Ca2+-treated protoplasts (data notshown). The protoplasts were removed from the media, and themedia were each divided into two aliquots. a-Amylase wasremoved from one of the aliquots by CHA-affinity chromatog-raphy ( 14), both aliquots were cold-sterilized, and pure high- andlow-pI a-amylase was added to a final concentration of about 10

,gg ml-', together with chloramphenicol (final concentration 10mM) and NaN3 (final concentration was 0.05%). High and low-pI a-amylase were slightly more stable in the plus-Ca" comparedto the minus-Ca" medium; however, the rates of degradation inboth media were quite low. The activity of amylase detectableby enzyme assays as well as the amount of a-amylase proteindetectable by Laurell rocket immunoelectrophoresis declined

4-

:A 3 GA . A

X T' 4"4§ M -: t.

FIG. 7. The effect of the composition of the incubation medium onthe incorporation of 35[Sjmethionine into proteins in protoplast extractsand incubation media of protoplasts incubated in 5 gM GA3, 20 mMCaC12, and 5 Mm GA3 plus 20 mm CaC12 for 4 h as indicated by SDS-PAGE and fluorography. The position of a-amylase on the gel wasdetermined by Coomassie blue staining (not shown). The arrows markthe positions of proteins that show enhanced labeling in the presence ofGA3 plus Ca>.

570

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Ca2+-STIMULATED SECRETION DURING PROTOPLASTS DEVELOPMENT

lLLu

I-0w

C/)-J

z

wToru)CO

I

TIME(h)0

500

50

4

4-'

Ca2+(10 mM) high pi low pi OZ

+o

3

21

16!

0

A

? 12 t

E AM...

80

00 10 20 30 40 50

TIME (h)

FIG. 8. The stability of amylase activity and a-amylase protein inprotoplast incubation media. Protoplasts were incubated in 5 MM GA3 orGA3 plus 20 mm Ca24 for 48 h. The incubation media were separatedfrom the protoplasts, cold-sterilized, and pure high- and low-pi a-amylaseisozymes were added to them. Aliquots of the media were taken over a50-h period for measurements of a-amylase isozyme activities (using IEFelectrophoresis), total amylase activity (using the method of Jones andVarner [ 17]), and the amount of high- and low-pI a-amylase isozymes(using Laurell rocket electrophoresis). The IEF gel shows the activity ofhigh-pI and low-pI a-amylase isozymes and Onozuka amylase (OZ) atthe beginning (0 h) and end (50 h) of the experiment for the twoincubation media. Total amylase activity (top graph) is shown for plus-Ca24 (-) and minus-Ca24 (---) media. The amount of protein for a-

amylase isozyme (bottom graph) is shown for plus-Ca2" (- ) andminus-Ca24 (---) media; high-pI isozymes in the two media are indi-cated with open symbols (0, A) and low-pI isozymes by the solid symbols(0, A).

slowly in minus-Ca24 media (Fig. 8). The degradation of high-pIa-amylase was slightly faster than that of low-pI a-amylase, butthis difference was not statistically significant (Fig. 8; Table IV).The average rate of degradation for both high- and low-pI amy-lases was 67 ng protein h-' in the minus-Ca24 medium and 4 ngh-' in the plus-Ca24 medium. Removing secreted a-amylase fromthe media by CHA-affinity chromatography had no significanteffect on the rates of degradation of the added enzyme (data notshown).

Table IV. Degradation Rates ofa-Amylase in Media in WhichProtoplasts Had Been Incubated

Protoplasts were incubated in 5 AM GA3 or 5 uM GA3 plus 20 mmCaC12 then removed from the incubation media after 48 h. The mediawere cold sterilized, and pure high- and low-pI a-amylase were added,and the degradation rates monitored. Rates are the absolute values ofthe slopes of the linear regressions shown in Figure 8.

Degradation of a-AmylaseIncubation Activity ProteinaMedium

Total' High-pI Low-pIunits h-' fg h-'

GA3 0.005 0.081 0.053GA3 + Ca2+ 0.025 0.007 0.001

a Determined by Laurell rocket electrophoresis. b Includes amylaseactivity from high-pl, low-pI, and Onozuka amylases as determined bythe assay of Jones and Varner ( 17).

w >-- I'n~-

(>>-HH742 z

(n(I)

cl:

a.(9z

24 48 72 96TIME (h)

FIG. 9. The effect of medium composition on released amylase activ-ity and the number of living protoplasts as a function of incubation time.Protoplasts were incubated in 5 uM GA3 (0), 20 mM CaCI2 (0), GA3 plus20 mm CaC12 (A), or in the absence of both GA3 and CaCl2 (A). Errorbars represent 95% confidence intervals of the plotted mean.

Effect of Ca2( on Protoplast Viability. To determine if Ca24stimulated a-amylase release indirectly through an effect onprotoplast viability we monitored protoplast viability, size, num-ber, and structure. During incubation there is a steady decline inthe number of living protoplasts in all treatments (Fig. 9). Thisdecline was most rapid for GA3-treated protoplasts whether ornot Ca24 was also present. Calcium slowed the rate of decline inprotoplast number in both GA3-treated and untreated proto-plasts, but the difference between the number of living proto-plasts in GA3 and GA3-plus-Ca24 treatments was not significantuntil 72 h, by which time a-amylase synthesis and release wasnearly complete (Fig. 9).

After isolation, aleurone protoplasts undergo dramatic mor-phological changes (15). Initially, protoplasts are densely packedwith aleurone grains which gradually enlarge to form vesicleswhich fuse and ultimately produce one or a few large vacuoles(Fig. 1). We examined the effect of Ca24 on this process byarbitrarily dividing it into four stages, based on the degree ofprotoplast vacuolation (Fig. 1), and determining the percentageof protoplasts in each of these stages (Fig. 10). Protoplasts vacu-olate whether or not they are treated with GA3 and/or Ca24, butGA3 accelerates and synchronizes the process. Thus by 72 h,

571

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Plant Physiol. Vol. 82, 1986

r~~--GA3 + GA3

+ Ca2+ - Ca2+r- 'I

1=I '

11 III IV 11 III IV

I o

I1Eh__

+ Ca2+

I1r-

11 III IV 11 III IV

DEVELOPMENTAL STAGE

FIG. 10. The effect of medium composition on the tempo of proto-plast development. Protoplasts were incubated with or without 5 ,Mm GA3and 20 mm CaC12 and the percentage of protoplasts in developmentalstages I-IV (see Fig. 1) assessed after 24. 48, and 72 h.

uJ

rl

0

(0

z

U)

n

(n

-J

0o0

0~

150

125

100 _

75

50

G

25

0 _0

TIME (h)

FIG. 11. The effect of medium composition on the number of pro-

toplasts in developmental stages II or III (Fig. 1) relative to the period ofamylase release. Protoplasts were incubated in 5 AM GA3 (0), 20 mmCaC12 (E). 5 AM GA3 plus 20 mm CaC12 (A), or in the absence of both

GA3 and CaC12 (A). The data are derived from those presented in Figures9 and 10.

protoplasts treated with GA3 were predominantly in develop-mental stages III and IV, irrespective of Ca>2 treatment, whilethose not treated with GA3 were evenly distributed across devel-opmental stages I to IV (Fig. 10). This effect of GA3 on thetempo of vacuolation was not modified by Car2 (Fig. 10).

ca-Amylase release from a protoplast population is not detect-able until vacuolation begins, that is after stage I, and secretionis essentially over by the time most protoplasts have becomefully vacuolate, that is by stage IV (Figs. 9 and 10). The rate ofa-amylase secretion is highest when protoplasts are in develop-mental stages II and III, but Ca>2 does not significantly affect the

FIG. 12. The effect of medium composition on the percentage oflumpy protoplasts. Protoplasts were incubated in the presence of 5 ,uMGA3 (0), 20 mM CaC12 (E), 5 pM GA3 plus 20 mM CaC12 (A), or in theabsence of both GA3 and CaC12 (A). Inset, a typical lumpy protoplast; x

600.

number ofGA3-treated protoplasts in these stages (Fig. 1 1). Thus,the difference in the amounts of amylase released by GA3 andGA3-plus-Ca2 treated protoplasts is not due to a difference inthe number of cells that have reached a particular stage ofdevelopment. Figure 10 also shows that when protoplasts are nottreated with GA3, large numbers of them remain in stages II andIII after 48 h, further evidence that GA3 accelerates protoplastdevelopment.

Although there was no evidence that Ca2' altered the timecourse of protoplast development, by 72 h of incubation a highpercentage of the protoplasts in the GA3-minus-Ca2+ treatmentdeveloped an unusual lumpy morphology (Fig. 12). These lumpyprotoplasts were present in all treatments at all times, but by 72h they were the dominant form in the GA3-minus-Ca2+ treatment(Fig. 12). Neither Car2 nor GA3 affected protoplast size. Theaverage diameter of protoplasts in each treatment was: H20, 46.6,um; GA3, 45.5 Mm; Ca2, 44.7 Mm; and GA3 plus Ca2', 48.9 ,um.

DISCUSSION

In this paper we have presented evidence that a-amylasesynthesis and release by GA3-treated barley aleurone protoplastsdepend on the concentration of Ca2+ in the external medium.We interpret this to mean that protoplasts, like intact aleuronelayers, have a mechanism for a-amylase secretion that is stimu-lated by Ca . We justify this interpretation by showing similar-ities between our data and that previously reported for intactaleurone layers and by eliminating the possibility that Ca2stimulates a-amylase secretion indirectly by preserving enzymeactivity or by influencing protoplast viability. We conclude fromour data that the cell wall is not obligatorily involved in Ca2-stimulated a-amylase secretion.The Ca2+-stimulated release of a-amylase that we observed in

barley aleurone protoplasts is similar to that reported for intactaleurone layers. In both layers and protoplasts Ca2+-stimulation

LUi(5

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>0-0

11LUI

(-3LU(I

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Uf)

-J

0

0

10

24 h

z0

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z

. a U U --Z,:

572 BUSH ET AL.

72 h

I a - - -j

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Ca2'-STIMULATED SECRETION DURING PROTOPLASTS DEVELOPMENT

is dependent on GA3 (Table I; Fig. 4; Refs. 5, 20) and is largelyconfined to the high-pI a-amylase isozymes (Figs. 2, 5; Refs. 13,19, 20). In the presence of Ca>2 alone, both layers and protoplastsrelease little a-amylase, most of it the pl-4.85 isozyme (Fig. 2;Ref. 20). With GA3 alone protoplasts and layers release low-pIisozymes pI 4.5 and pI 4.85 (Fig. 2; Refs. 13, 19, 20), but whenboth GA3 and Ca> are present in the medium, high-pI isozymespI 5.9 and pI 6.3 are released in addition to the low-pI isozymes(Figs. 2, 4, 5; Refs. 13, 19, 20).

Both protoplasts and layers require mm levels of Ca> formaximal a-amylase release (Figs. 4, 5; Refs. 13, 20) and in bothSr2> can substitute for Ca> (Fig. 6; Ref. 20). The concentrationof extracellular Ca>2 required for maximal a-amylase secretionis 50 mM in protoplasts (Figs. 4, 5) compared to 5 mm in layers(20). This higher Ca>2 requirement in protoplasts is not due toincreased chelation of Ca>2 in the protoplast medium (Table II).It may reflect real differences in the ionic environment of theplasma membrane in the absence of the cell wall. The aleuronecell wall contains fixed acidic groups (1, 3), which might increasethe concentration of free Ca>2 at the plasma membrane (9). Thehigher Ca>2 requirement of protoplasts may also be due to thesolute potential of the medium. Experiments with intact layersare usually done in media of low solute concentration, whereasprotoplast experiments are done in 600 mm mannitol. Thesecretion of a-amylase in layers is known to be influenced bysolute potential (18), but the Ca>2 requirement at high solutepotential is not known.

In intact aleurone layers the effect of Ca>2 is reversible. WhenCa>2 is withdrawn from the medium, Ca2+-stimulated secretionof the high-pI a-amylase stops with a half-time of 10 min, andwhen Ca>2 is added back, Ca2+-stimulated secretion rapidlyresumes (25). In protoplasts also the effect of Ca>2 is reversible:when Ca>2 is withdrawn, a-amylase release ultimately stops (Fig.3). Protoplasts differ from layers in the speed of the response toCa>2 withdrawal since release does not stop or even slow forseveral hours (Fig. 3). This difference may point to a role of thecell wall or turgor in modulating Ca2"-stimulated amylase release.The data of Varner and Mense (28) suggest that electrolytesfacilitate a-amylase release from intact layers, and these authorsconcluded that in the absence of Ca>2 the wall acts as a barrierto amylase release. Although our data show that the wall is notobligatorily involved in Ca2+-stimulated secretion, extracellularCa>2 may be necessary for amylase movement through the wall.This would explain why Ca>2 withdrawal causes a rapid inhibi-tion of amylase release in intact layers but not in protoplasts.

Ca'2-stimulated a-amylase release from aleurone protoplastsand intact aleurone layers is strikingly similar. This stronglysuggests that a-amylase release is a Ca2+-regulated secretaryprocess in protoplasts as it has been shown to be in layers.However, an important question to address is whether Ca>2stimulates a-amylase release because of a specific requirement inthe secretary process or because it increases either enzyme sta-bility or cell viability.There is evidence that Ca-2 preserves the activity of the high-

pI a-amylase isozymes (Fig. 8; Refs. 13, 28). However, we rejecton several grounds the hypothesis that Ca>2 only appears tostimulate a-amylase release from aleurone protoplasts becausereleased protein is degraded in its absence. First, there is not asignificant difference in the effect of Ca>2 on the stability of thehigh- and low-pI amylases. Since low-pI a-amylase is present inthe minus-Ca>' medium, the high-pI a-amylase would have todegrade faster than low-pI a-amylase if degradation of high-pI a-amylase were to account for its absence. Second, the degradationrates in the minus-Ca> media are too low to account for theobserved difference in a-amylase synthesis and secretion in plus-Ca> and minus-Ca>2 media. Ca2+-treated protoplasts secrete

(data not shown, but see Fig. 5) which is much higher than therates of degradation observed in the medium of minus-Ca2+-treated protoplasts (Fig. 8). Thus, degradation could only accountfor the absence of high-pI a-amylase in minus-Ca2" media if therate of secretion were considerably lower than in Ca2"-treatedprotoplasts. Somewhat higher rates of amylase degradation wereobserved after washing protoplasts (Fig. 3). This is expected sincewashing damages protoplasts (15) and may result in the releaseof proteases that are normally sequestered inside the cell. Third,Ca2" also greatly stimulates the incorporation of 35[S]methionineinto affinity-purified a-amylase and into proteins which comi-grate with a-amylase during SDS-PAGE (Fig. 7; Ref. 19). Thisis true for both the medium and extract (Fig. 7). Since it isunlikely that free Ca2" ever exceeds 10 AM inside the aleuronecell under any conditions of incubation (D Bush, RL Jones,unpublished observation), differential changes in cell Ca2" per seare not likely to influence enzyme stability inside the cell. Finally,additional evidence that the effect of Ca2" on enzyme stabilityalone is insufficient to explain Ca2"-stimulated secretion of a-amylase is that Ca2" also stimulates the secretion of severalproteins that are not known to require Ca2" for their activity(Fig. 7; Ref. 20).

Ca2" is usually included in protoplast isolation and incubationmedia at mm levels to increase protoplast viability (12). Wefound no significant effect of Ca" on protoplast viability in theabsence of GA3 (Fig. 9). However, in GA3 without added Ca2living protoplasts were fewer in number and, after 72 h ofincubation, were lumpy in appearance when compared to thosein GA3 plus Ca2 (Figs. 9, 12). These observations point to a roleof Ca>2 in maintaining normal protoplast morphology especiallyin the later stages of development, but they do not suggest thatCa>2 stimulates secretion by maintaining protoplast health.The evidence for a specific effect of Ca>2 on secretion that is

separable from its effect on protoplast viability is as follows: (a)The pI-4.7 isozyme (Fig. 2) is not secreted by intact layers but isassociated with protoplast extracts (15). It is possible that thisisozyme is a precursor to a secreted isozyme. Since the protoplastextract contains the pI-4.7 isozyme and the medium containsonly a small amount of it (Fig. 2), a-amylase release in bothplus- and minus-Ca2+ treatments was not due to cell lysis. (b)The minus-Ca2+ protoplasts secrete large amounts of low-pIamylase and this secretion is dependent on GA3 (Figs. 2 and 4).The secretion of low-pI a-amylases indicates that protoplasts inthe minus-Ca2+-media are capable of controlled amylase synthe-sis and secretion. (c) The differences in protoplast number andmorphology that occur in GA3-treated protoplasts in the presenceand absence of Ca>2 appear only 3 d after isolation (Fig. 9). Bythis time a high percentage of the protoplasts in both treatmentshave undergone complete vacuolation and have ceased to secretea-amylase (Figs. 9, 10).The similarity of Ca2+-stimulated secretion in aleurone proto-

plasts and intact aleurone layers lead us to conclude that thealeurone cell wall is not obligatorily involved in Ca2+-stimulatedsecretion. This conclusion is consistent with that of Moll andJones (25) based on a kinetic analysis of a-amylase release fromintact layers that Ca2+ regulation is at the plasma membrane.Our data do not eliminate the possibility that Ca>2 regulatesamylase release from the aleurone by facilitating its movementthrough the cell wall as suggested by Varner and Mense (28).This, however, would be an additional and secondary point ofCa2+ regulation rather than the principal point.

Acknowledgments-The authors want to thank Eleanor Crump for her help inpreparing the manuscript, and Teri Takehiro for her help in the laboratory.

LITERATURE CITED

1. BACic A, BA STONE 1981 Chemistry and organization of aleurone cell wallcomponents from wheat and barley. Aust J Plant Physiol 8: 474-495high-pI amylase at a rate of 0.5 to 1 Mg h-' (106 protoplasts)-'

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574 BUSH ET AL.

2. BROWN AHD, JV JACOBSEN 1982 Genetic basis and natural variation of a-amylase isozymes in barley. Genet Res Camb 40: 315-324

3. BUSH DS, RL JONES 1985 Calcium uptake and exchange in barley aleuronelayers and protoplasts during Ca-stimulated amylase secretion (abstract). InMolecular and Cellular Aspects of Calcium in Plant Development. NATOAdvanced Workshop, Edinburgh, p A 1

4. CARBONELL J, RL JONES 1984 A comparison of the effects of Ca2" andgibberellic acid on enzyme synthesis and secretion in barley aleurone. PhysiolPlant 63: 345-350

5. 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

6. CRIPPEN RW, JL PERRIER 1974 The use of neutral red and Evans Blue forlive-dead determinations of marine plankton. Stain Technol 49: 97-104

7. DEIKMAN J, RL JONES 1985 Control of a-amylase mRNA accumulation bygibberellic acid and calcium in barley aleurone layers. Plant Physiol 78: 192-198

8. DEIKMAN J, RL JONES 1986 Regulation of the accumulation of mRNA foramylase isoenzymes in barley aleurone. Plant Physiol 80: 672-675

9. DEMARTY MC, C RIPOLL, M THELLIER 1980 Ion exchange in plant cell walls.In J Dainty, ed, Plant Membrane Transport: Current Conceptual Issues.Elsevier/North-Holland Biomedical Press, Amsterdam, pp 33-47

10. GAFF DF, 0 OKONG'O-OGOLA 1971 The use of non-permeating pigments fortesting the survival of cells. J Expt Bot 22: 756-758

11. GAMBORG OL, RA MILLER, K OJIMA 1968 Nutrient requirements of suspen-sion cultures of soybean root cells. Exp Cell Res 50: 151-158

12. HOOLEY R 1982 Protoplasts isolated from aleurone layers of wild oat (Avenafatua L.) exhibit the classic response to gibberellic acid. Planta 154: 29-40

13. JACOBSEN JV. JG SCANDALIOS, JE VARNER 1970 Multiple forms of amylaseinduced by gibberellic acid in isolated barley aleurone layers. Plant Physiol45: 367-371

14. JACOBSEN JV, TJV HIGGINS 1982 Characterization of the a-amylase synthe-sized by aleurone layers of Himalaya barley in response to GA3. Plant Physiol70: 1647-1653

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15. JACOBSEN JV, JA ZWAR, PM CHANDLER 1985 Gibberellic-acid-responsiveprotoplasts from mature aleurone of Himalaya barley. Planta 163: 430-438

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17. JONES RL, JE VARNER 1967 The bioassay of gibberellins. Planta 72: 155-16118. JONES RL, JE ARMSTRONG 1971 Evidence for osmotic regulation of hydrolytic

enzyme production in germinating barley seeds. Plant Physiol 48: 137-14219. JONES RL, JV JACOBSEN 1983 Calcium regulation ofthe secretion ofa-amylase

isoenzymes and other proteins from barley aleurone layers. Planta 158: 1-920. JONES RL, J CARBONELL 1984 Regulation of the synthesis of barley aleurone

a-amylase by gibberellic acid and calcium ions. Plant Physiol 76: 213-21821. LAEMMLI UK 1970 Cleavage of structural proteins during the assembly of the

head of bacteriophage T4. Nature 277: 680-68522. LARKIN PJ 1976 Purification and viability of plant protoplasts. Planta 128:

213-21623. MANS RJ, GD NoVELLI 1961 Measurement ofthe incorporation of radioactive

amino acids into protein by a filter-paper disc method. Arch BiochemBiophys 94: 48-53

24. MELROY D, RL JONES 1986 The effect of monensin on intracellular transportand secretion of a-amylase isoenzymes in barley aleurone. Planta 167:252-259

25. MOLL BA, RL JONES 1982 a-Amylase secretion by single barley aleuronelayers. Plant Physiol 70: 1148-1155

26. OHNISHI ST 1978 Characterization of the murexide method: dual-wavelengthspectrophotometry of cations under physiological conditions. Anal Biochem85: 165-179

27. SPOSITO G, SV MATTIGOD 1979 Geochem: a computer program for thecalculation of chemical equilibria in soil solutions and other natural watersystems. The Kerney Foundation of Soil Science, University of California,Berkeley

28. VARNER JE, RM MENSE 1972 Characteristics of the process ofenzyme releasefrom secretory plant cells. Plant Physiol 49: 187-189

29. WEEKE B 1973 Rocket immunoelectrophoresis. In B Weeke, ed, A Manual ofQuantitative Immunoelectrophoresis. Universitetsforlaget, Oslo, pp 37-46

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