Plant Physiol. (1983) 71, 173-1760032-0889/83/7 1/0173/04/$00.50/0

In Vivo Synthesis and Turnover of a-Amylase in Attached andDetached Cotyledons of Vigna mungo Seeds'

Received for publication March 30, 1982 and in revised form September 27, 1982

TOMOKAZU KOSHIBA AND TAKAO MINAMIKAWADepartment of Biology, Tokyo Metropolitan University, Setagaya-ku, Tokyo 158, Japan

ABSTRACT

a-Amylase activity increased in attached cotyledons ofgerminated Vignamuago seeds until the 5th day after imbibition and decreased thereafter,whereas in detached and incubated cotyledons the activity continuouslyincreased and, at the 6th day, reached the value more than three times thatof the maximum activity of attached cotyledons. Zymograms of the activi-ties and Ouchterlony double inmunodiffusion test on the activities ofattached and detached cotyledons showed that the increase of activity indetached cotyledons was due to the identical enzyme as in attached tissues.a-Amylase contents, determined by single radial immunodiffusion method,changed in paraflel with enzyme activity in both attached and detachedcotyledons, which also suggested the de novo synthesis of a-amylase in V.mugo cotyledons.The rate of incorporation of the label from I3Hjleucine into a-amylase

and the ratios of dpm in a-amylase/dpm in trichloroacetic acid-insolublefraction did not show significant difference between attached and detachedcotyledons. The results indicated that in attached cotyledons fluctuation ofa-amylase activity was regulated by both synthesis and degradation of theenzyme, whereas in detached cotyledons a-amylase was synthesized andaccumulated, because of low degrading activity during incubation.

In cereal seeds, such as barley, it is well known that gibberellin(GA3) produced in the embryos moves to the aleurone layers andpromotes the de novo synthesis and secretion of a-amylase intoendosperm. Recent reports suggested that this enzyme inductiondepends on increase of translatable mRNA for a-amylase (7, 15,16), and that this increase is due to a direct enhancement oftranscription of a-amylase gene by GA3 (1).

In contrast, the mechanism of a-amylase formation in cotyle-dons of germinating leguminous seeds is not known. Many studieson effects of axis removal or plant hormones have been done, butthere are some discrepancies. For example, Yomo and collabora-tors (24, 26) reported that a-amylase activity increases in thecotyledons during seedling growth and during incubation of axis-free cotyledons of pea and bean. This was inconsistent with thefindings of others that this increase is partially inhibited by axisremoval (3, 11, 23). Furthermore, plant hormones such as GA3and kinetin showed little or no promotive effects on a-amylaseformation in bean and pea cotyledons (4, 21, 22), although inbarley aleurone layers a-amylase is very rapidly induced after 10to 12 h of GA3 treatment. There has also been no conclusiveevidence of de novo synthesis of the enzyme in leguminous coty-ledons. Because there is much difference in seed structure and in

'Supported partly by Grants-in-Aid (No. 554214 and 56740283) fromthe Ministry of Education, Science and Culture of Japan, and from ItoScience Foundation (1981).

processes of seed formation between cereal and leguminous seeds(2), we studied the mechanism of a-amylase formation in cotyle-dons of germinating bean seeds.We previously reported that amylolytic activity increases in

Vigna mungo cotyledons during germination and that the increaseis not delayed by axis removal (13). Histochemical observationrevealed that amylolytic and proteolytic activities appeared toincrease in tissue areas farthest from vascular bundles duringgermination (14). We also obtained pure a-amylase preparationfrom cotyledons of V. mungo seedlings using affinity chromatog-raphy (8). In the present study, using antibody toward the a-amylase obtained by immunization of a rabbit with purifiedenzyme, we investigated whether the increase of a-amylase is dueto de novo synthesis or the activation of preformed inactiveenzyme. We also compared rates of a-amylase synthesis andproteolytic degradation in attached and detached V. mungo coty-ledons.

MATERIALS AND METHODS

Plant Material and Enzyme Preparation. Vigna mungo seedswere immersed in concentrated H2SO4 for 8 to 10 min at roomtemperature and rinsed thoroughly with tap water, then allowedto imbibe water for 8 h before sowing. Imbibed seeds weregerminated on layers of wet filter paper in the dark at 26 to 28°C.For experiments with detached cotyledons, dry seeds were cut intohalves and their cotyledons detached from the embryonic axes.These cotyledons were allowed to imbibe water for 6 h at 26 to28°C, then they were surface sterilized in 3% (w/v) solution ofcalcium hypochlorite fur 10 min. They were rinsed with sterilizedH20 and incubated on three layers of wet filter paper in Petridishes under aseptic conditions at 26 to 28°C in the dark. The 10to 20 pairs of cotyledons were harvested at various stages ofgermination and incubation and stored at -20°C until use. Thecotyledons were homogenized with 50 mm Tris-HCl buffer (pH7.4) in a cold mortar and pestle and centrifuged at 35,000g for 15min. The supernatant was used as enzyme solution.a-Amylase Assay. a-Amylase activity was determined according

to the method of Okamoto and Akazawa (17) with some modifi-cations, using fB-limit dextrin as substrate. The reaction mixturecontaining 0.5 ml of 0.3% (w/v) f-limit dextrin dissolved in 50mm Na-acetate buffer (pH 5.4) and 1.5 ml enzyme solutionappropriately diluted with the acetate buffer was incubated at30°C for 5 min. The reaction was stopped by adding 1.0 ml I2-KIsolution, followed by measuring the decrease in A at 620 nm. Oneunit of the activity was defined as the enzyme activity causing 10%Yoabsorbance decrease at 620 nm/min under the assay conditions.

Purification of a-Amylase and Preparation of Antibody. a-Am-ylase was purified using a simplified procedure as describedpreviously (8). Enzyme extraction from 4-d-old cotyledons wasfractionated by ammonium sulfate. a-Amylase was isolated fromthe protein by affinity chromatography on a fl-cyclodextrin Seph-arose 6B column (1.5 x 12 cm), preequilibrated with 50 mm Na-

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KOSHIBA AND MINAMIKAWA

acetate buffer (pH 5.4), containing 1 mm CaCl2 and 10 mm 2-mercaptoethanol. After washing the column with the acetatebuffer, the enzyme was eluted by using the same buffer containing10 mg/ml of fB-cyclodextrin. The active fractions were combinedand concentrated with an Amicon Diaflow membrane P-IO. a-Amylase was resolved from the /B-cyclodextrin by passing theenzyme through a Bio-gel P-4 column (1.5 x 17 cm), with subse-quent elution of the enzyme with 50 mm Tris-HCl buffer (pH 7.4).The active fractions were combined and used for the preparationof antibody.Antibody against a-amylase was prepared by subcutaneous

injection into a rabbit of the purified a-amylase emulsified withFreund's complete adjuvant at weekly intervals in four injections,each containing about 1 mg a-amylase. IgG2 was separated fromserum by ammonium sulfate precipitation at 40% and dialyzedagainst 20 mm Tris-HCl buffer (pH 7.5), containing 0.15 M NaCland 0.02% (w/v) NaN3.Ouchterlony Double Immunodiffusion and Single Radial Im-

munodiffusion. Double immunodiffusion was carried out accord-ing to the method of Ouchterlony (19) using 1% (w/v) agarosebuffered with 20 mm Tris-HCl (pH 7.5), containing 0.15 M NaCl,0.5% (v/v) Triton X-100, 0.5% (w/v) sodium desoxycholate, and0.02% (w/v) NaN3 (TD-TBS). Cross-reactions were tested byvarious antigen-antibody combinations, placing the antibody inthe center well and antigenic proteins in outer wells. The gel platewas incubated for 6 h at room temperature and then the gel platewas examined for precipitin lines.

Quantitative determination of antigen (a-amylase) in the crudeenzyme preparation was performed according to the procedure ofMancini et al. (12) using 1% agarose as above, except that 5 to 20IL IgG and 10 mm phenylmethyl sulfonyl fluoride were added.After 3 d incubation of the tested plate at 30°C, the area of theprecipitate was measured. In the range of about 4 to 200 jig a-amylase, a linear relation was observed between the area of theprecipitate and the concentration of the antigen.

Incorporation of I3H]Leucine into Protein and a-Amylase. Tenpairs of the cotyledons were harvested at various stages of seedlinggrowth or incubation, and were sectioned transversely into fourparts. Each section was fed with 3 ,ul [3H]leucine (3 ,tCi, 52 Ci/mmol) and incubated for 2.5 h at 26 to 28°C. After washing withdistilled H20, the sections were quickly frozen by liquid N2 andstored at -20°C until use. The sections were homogenized with50 mm Tris-HC1 buffer (pH 7.4), and the homogenate was centri-fuged at 100,000g for 40 min. The supernatant solution was usedfor the measurements of the radioactivities in total soluble, TCAinsoluble, and a-amylase fractions, and also provided the samplefor SDS-polyacrylamide gel electrophoresis and fluorography.The radioactivity of total soluble fraction was measured directly

using 10 ,ul of the supernatant solution with tT21 liquid scintillatormade of 667 ml toluene, 333 ml Triton X-100, and 3 g PPO. Theradioactivity of TCA-insoluble protein fraction was measured as

follows: an aliquot (100 1tl) of the supernatant solution was mixedwith 100 ,P of 20% (w/v) TCA, and resulting precipitates werecollected by filtration on a Toyo glass fiber filter GC-50. Thepaper was rinsed with 5% TCA solution and 99% (v/v) ethanol,and then dried. The radioactivity of the filter was measured withtoluene liquid scintillator.

Immunoprecipitation and measurement of the incorporation of[3Hjleucine into a-amylase were performed as follows: 1.0 ml ofthe supernatant solution was mixed with 100 ,ul IgG and 1.0 mlTD-TBS. After 30 min of incubation at 37°C, some amount ofthe pure cx-amylase was added as carrier to make antigen contentin the mixture constant (approximately 50,ug) and incubation was

continued for 30 min at 37°C, then overnight at 4°C. The resultingimmunoprecipitates were collected by centrifugation and washed

2Abbreviation: IgG, immunoglobulin G.

four times with TD-TBS. This immunoprecipitation procedurewas repeated two times, and the obtained immunoprecipitateswere combined and used for SDS gel electrophoresis. Electropho-resis was performed as described below. After electrophoresis, thegel was stained for protein with Coomassie blue. The stained bandcorresponding to a-amylase was cut out with a razor, dissolved in33% (v/v) H202 solution, and the radioactivity was measured withtT21 liquid scintillator.

Polyacrylamide Slab Gel Electrophoresis and Fluorography.Polyacrylamide gel electrophoresis and detection of a-amylaseactivity on the gel were performed as described previously (8),using 7.5% polyacrylamide gel with 2 mm thickness. Proteinsamples were loaded with 20% (w/v) sucrose and electrophoresedat4C for 6 h.

SDS-polyacrylamide slab gel electophoresis was carried outaccording to the method ofLaemmli (9) using 10%7o polyacrylamidegel with 1 mm thickness. Protein samples and immunoprecipitateswere treated with 25 mm Tris-HCl buffer (pH 6.8), containing 4%(w/v) SDS, 1o (v/v) 2-mercaptoethanol, 0.002% (w/v) bromo-phenol blue, and 20% (v/v) glycerol for 2 to 5 min in boilingwater. Electrophoresis was performed at 25 mamp constant cur-rent. The gels were stained for protein with Coomassie blue andwere prepared for fluorography using Enhance (10) and wereexposed to x-ray film (Fuji Rx) for 2 weeks.

Chemicals. Epoxy-activated Sepharose 6B was purchased fromPharmacia; fi-cyclodextrin from Sigma; [3H]leucine and Enhancefrom New England Nuclear. fl-Limit dextrin was prepared byhydrolyzing a potato starch solution (17).

RESULTS

Change in a-Amylase Activity and Content during Germinationand Incubation. During germination, a-amylase activity of Vmungo cotyledons increased from the 2nd to 5th d, and decreasedthereafter. In contrast, during incubation of detached cotyledonsthe activity increased almost the same as that of attached cotyle-dons until the 3rd d, but still rapidly increased until the 6th d and

-A

0 24 6

da vsFIG. 1. Polyacrylamide slab gel electrophoresis pattern of a-amylase in

attached and detached cotyledons of V. mungo. Ten pairs of the cotyledonswere harvested at various stages of germination (A) and incubation (B).Extraction of enzyme, gel electrophoresis, and detection ofamylase activityon the gel are described in the text.

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SYNTHESIS AND TURNOVER OF BEAN a-AMYLASE

A B

FIG. 2. Ouchterlony double diffusion test. The center wells contained25 Id of anti-a-amylase IgG, and surrounding wells contained 25 t1 ofcrude extracts or purified a-amylase. Wells contained as follows: 1, 7,crude extracts from dry seed cotyledons; 2, 3, 4, 5, crude extracts fromcotyledons of 2-, 3-, 4-, 6-d germinated seedlings, respectively; 6, 1,

purified a-amylase; 8, 9, 10, crude extracts from detached cotyledons of 2-, 4-, 6-d incubation, respectively; 12, crude extract from cotyledons of 5-dgerminated seedlings.

S0 2 4 6 0 2 4 6

c0

*0

v0*-

In

a

E4

DaysFIG. 3. Quantitative analysis of changes in a-amylase content in at-

tached and detached cotyledons of V. mungo. Assay of a-amylase activityand determination of a-amylase content by single radial immunodiffusionmethod were carried out as described in "Materials and Methods."(O-O), a-amylase activity; ( -- 0), a-amylase content.

reached a value of 60, more than three times that of the maximumactivity of attached tissues (Fig. 3). Zymograms ofamylase activityshowed that the a-amylase in detached cotyledons has the same

region of migration as that of intact cotyledons (Fig. 1). Immu-nochemical identity of these enzymes from attached and detachedcotyledons was investigated by the Ouchterlony double diffusiontest as shown in Figure 2. Anti-a-amylase IgG gave a continuoussingle precipitin line against purified a-amylase and crude extractsfrom various periods after germination and incubation. No pre-

cipitin line was formed with a crude extract from dry seed coty-ledons, but it appeared after the 2nd to 3rd d of germination andincubation. Furthermore, quantitative determination ofa-amylaseby single radial immunodiffusion method revealed that the en-

zyme contents changed in parallel with the activity in both at-tached and detached cotyledons (Fig. 3).

Incorporation of I3HILeucine into Immunoprecipitate and a-

Amylase. To investigate the incorporation of label into a-amylase,SDS-polyacrylamide gel electrophoresis of the immunoprecipitatewas performed. Only one stained protein band corresponding tothe pure a-amylase was observed, in addition to two stained bandsof light (L) and heavy (H) chains of IgG, and the label of [3H]

H

1-

a b c d C dFIG. 4. SDS-polyacrylamide slab gel electrophoresis of radioactive im-

munoprecipitate. [3H]Leucine was administered to the sections of cotyle-dons from the seedlings of 3-d germination, which were then incubatedfor 5.5 h. Enzyme extraction and immunoprecipitation are described inthe text. Gel was stained for protein with Coomassie blue (A) and itsfluorogram (B). a, Purified a-amylase; b, immunoprecipitate from crudeextract (4-d germination); c, d, radioactive immunoprecipitate, 25 tul and50 Id, respectively; H, heavy chain of IgG; L, light chain of IgG. The arrowindicates position of a-amylase.

Table I. Rates of Incorporation of [3HlLeucine Label into Protein anda-Amylase

Three to five mm thickness sections of 10 pairs of cotyledons were fedwith 240 tCi I3Hlleucine for 2.5 h at 270C. The sections were homogenizedand fractionated, and the radioactivities were measured.

Incorporation

Day Total a/bSoluble TCA Insoluble (a) a-Amylase (b)

dPm x 10-6 dpm x 10-3 %Attached 2 205 12.48 63.2 0.51

4 231 8.34 40.7 0.496 238 5.23 24.7 0.47

Detached 3 213 8.97 30.2 0.345 211 6.96 39.3 0.56

leucine incorporated into only the band of a-amylase (Fig. 4). Theresults show that the IgG preparation reacts specifically with a-amylase and that no cross-reactive material other than a-amylaseis detected in crude extracts.

Incorporation of [3H~leucine into protein and a-amylase wasinvestigated at various stages of germination and incubation(Table I). Incorporations into total soluble fraction of attachedand detached cotyledons were relatively constant, of which valuesare about 40%o of the fed radioisotope. The incorporation intoTCA-insoluble protein fractions was the highest at the 2nd d inattached cotyledons and then decreased, which was similar to thatof detached cotyledons. Incorporation of [3H]leucine into a-amy-lase was determined by measuring the radioactivity of the stainedband corresponding to a-amylase on SDS-polyacrylamide gelzymogram. Changes of the values showed the same pattern asthose in incorporation into proteins in attached cotyledons, but indetached cotyledons the values slightly increased from the 3rd to

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KOSHIBA AND MINAMIKAWA

5th d. Because the contents of amino acid were changing in thecotyledons (13), the values of incorporation do not directly indi-cate its rate of in vivo synthesis. However, the ratios of dpm in a-amylase/dpm in TCA-insoluble fraction also showed no signifi-cant difference between attached and detached cotyledons (TableI). Thus, it is likely that in attached cotyledons the increase of a-amylase activity is due to the accumulation of the enzyme and thedecrease occurring after the 5th d of germination is due to theproteolytic degradation of the enzyme, which may already startbefore the 5th d of germination. On the other hand, in detachedcotyledons, a-amylase is continuously synthesized and accumu-lated throughout incubation, because of the low level of theproteolytic degradation in this tissue.

DISCUSSIONThe present results show that, even when the embryonic axes

were removed from cotyledons before imbibition, a-amylase ac-tivity of V. mungo cotyledons increases during incubation andreaches the value more than three times that of the maximumactivity of attached cotyledons. Yomo and Varner (26) reportedsimilar results in pea cotyledons. They demonstrated that duringgermination a- and ,B-amylases in the cotyledons increased rapidlyfrom the 4th to 10th d and then leveled off, while during incuba-tion of excised cotyledons the activities steadily increased up tothe 12th d. They suggested the possibility that ABA is involved inthe regulation of a- and ,8-amylase formations in pea cotyledons.In this work, we have shown that the increase in detached V.mungo cotyledons is due to the identical enzyme increase as inintact tissue and that the high activity in detached cotyledons isthe result of the accumulation of a-amylase in the tissue, in whichthe level of proteolytic activity is low throughout incubationperiod. The low level of protease activity during incubation of.detached cotyledons was also described previously in V. mungo(13) and pea (26). These findings support the concept that proteo-lytic activity is mainly involved in the regulation of cotyledonarya-amylase fluctuation, and the proteolytic activity may be con-trolled by the existence of axis organs or hormonal action. Inmany other seeds, existence of a-amylase isozymes and ,B-amylasewas reported, but in V. mungo cotyledons there was only one typeof a-amylase on zymograms. We also reported previously that /-amylase was not detected in this tissues (8). In V. mungo cotyle-dons, it is likely that only one type of a-amylase mainly acts instarch mobilization.

Histochemical observations in some leguminous seeds (6, 14,25) have shown that amylolytic and proteolytic activities appearedto increase in tissue areas farthest from vascular bundles and thatmobilization of starch and protein reserves also proceeded in theseareas. Recently, in rice seeds, it has been reported that an impor-tant site of the formation of amylase and other hydrolases is thescutellar epithelium (17, 18). In barley seeds, in contrast, Palmer(20) suggested a minimal role for the scutellum in secretion ofhydrolytic enzymes. Thus, there are a number of problems to besolved in the mechanism of a-amylase formation of germinatingleguminous seeds, in contrast to cereal seeds. For example: (a)Why is there a different control mechanism between the synthesisof a-amylase and protease in bean cotyledons? (b) Although inbarley aleurone layers occurrence of transcriptional control of a-amylase synthesis by GA3 was suggested (1), is there the samecontrol mechanism in bean? (c) Whether a-amylase is synthesized

in individual cells, or the site of a-amylase synthesis is restrictedto some regions from where it is secreted to whole cells incotyledon during germination? We are presently pursuing studiesalong this line.

Acknowledgment--The authors wish to thank Prof. S. Yoshida for his valuablediscussions and advice.

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