Plant Physiol. (1979) 63, 336-3400032-0889/79/63/0336/05/$00.50/0

Enzymic Mechanisms of Starch Breakdown in GerminatingRice Seeds7. AMYLASE FORMATION IN THE EPITHELIUM

Received for publication July 25, 1978 and in revised form September 21, 1978

KAZUO OKAMOTO AND TAKASHI AKAZAWAResearch Institutefor Biochemical Regulation, School ofAgriculture, Nagoya University, Chikusa, Nagoya 464Japan

ABSTRACT

The time sequence analysis of the starch digestion pattern of the thinsectioned germinating rice (Oryza satva L.) seed specimens using thestarch film method showed that at the initial stage amylase activity wasalmost exclusively localized in the epithelium septum between the scuteliumand endosperm. Starch breakdown in the endosperm tissues began after-ward; amylase activity in the aleurone layers was detectable only after 2days. Polyacrylamide gel electrofocusing (pH 4 to 6) revealed nearly thesame zymogram patterns between endosperm and scutellum extracts,although additional amylase bands appeared in the endosperm extracts atlater gennination stages (4 to 6 days). These are presumably attributableto the newly synthesized enzyme molecules in the aleurone cells.

Germination of starchy cereal seeds is of classical interest inplant biological research. Despite extensive investigations in avariety of disciplines (e.g. anatomical, physiological, biochemical,and enzymic), the precise nature of starch breakdown is notentirely clear. Although there is a good possibility that eachspecific cereal seed has its characteristic machinery for hydrolyzingreserve materials, so far no unified mechanism has been presentedin different kinds of cereal seeds relating to the enzymic hydrolysisof reserve starch.

Based on histological observations of germinating barley seeds,Brown and Morris (3) reported in 1890 that the site of synthesisof diastatic enzyme is in the epithelium of the scutellum; sincethen this unique feature of the epithelium has received muchattention by several investigators (17, 23). In 1960, Dure (6)reported that in germinating maize seeds, the site of a-amylase(EC 3.2.1.1) biosynthesis was restricted to the scutellum, whereas/3-amylase (EC 3.2.1.2) was localized in the endosperm. Thecrucial role of embryonic tissues (including the scutellum) in thedigestion of starch of germinating cereal seeds has been proposedby several investigators. Briggs (2) reported that in malt, the a-amylase activity located in the embryo occupied about 7% of thetotal activity of the seed, and that 6.5% of the endospermic a-amylase was of embryonic origin. The scutellum is part of theembryo, and is clearly distinguishable from the embryonic axis,but analytical study of enzyme reactions occurring in epitheliumis technically quite difficult because of the small size of the tissue.

Since the discovery of gibberellin, enhanced biosynthesis of a-amylase and other hydrolases in aleurone cells of barley seeds (4,7), many investigations have focused at the molecular level on theintrinsic role of the hormone in the de novo synthesis of theenzyme (4, 8, 10, 12). There is ample experimental evidence

showing the site of GA' synthesis to be in the embryo; it ishypothesized that the hormone is transported to aleurone cellswhere hydrolytic enzymes including amylase are synthesized insitu (9, 14, 19, 20). The important role of aleurone layers in theenzymic starch digestion revealed by such studies appears to beconsistent with the classical report of Linderstr0m-Lang and Engel(13), dealing with the ultramicroscopic examination of amylaselocalization in malt. The over-all picture is not necessarily con-sistent with the above described pattern of starch digestion initi-ating at the embryonic tissues, studied by previous workers. Inwork reported in this communication, we attempted to reexamine,employing a starch film technique, the initial site of amylaseformation in germinating rice seeds.

MATERIALS AND METHODSGrowth of Plants and Preparation of Crude Enzyme. Rice seeds

(Oryza sativa L. cv. Kimmaze) germinated in a dark chamber at30 C were harvested at the appropriate stage (15), scutellum andendosperm tissues dissected from the seeds were used for analyticalstudies (16). It is relatively easy to separate the two tissues afterthe 2nd day of inhibition. To avoid contamination with endo-sperm, scutella were thoroughly washed with 50 mm Tris-HClbuffer (pH 7.0) containing 3 mm CaCl2 and 4 mm NaCl. Onehundred each of isolated scutellar and endospermic tissues werethen homogenized using 2 and 5 ml, respectively, of the buffersolution; the homogenates were centrifuged at 15,000g for 15 min.An aliquot of the supernatant fraction was passed through acolumn of Sephadex G-25 (1.0 x 9.0 cm) equilibrated with theabove buffer solution. The eluate served as the crude enzymepreparation.Enzyme Assays. a-Amylase activity was determined according

to the method of Okamoto and Akazawa (18), using fl-limitdextrin as substrate. ,8-limit dextrin was prepared by hydrolyzingpotato starch solution (2% [w/vJ in 50 mm acetate buffer [pH5.3]), using crystalline potato fi-amylase free from a-amylase (24hr, 30 C). 0.3% fl-limit dextrin dissolved in 50 mm acetate buffer(pH 5.3) containing 1 mm KH2PO4-2 mm CaCl2.2H20 served asthe substrate. The reaction mixture containing 0.2 ml each ofsubstrate and enzyme solution was incubated at 37 C for 5 min;the reaction was stopped by adding 0.5 ml of 12-KI solution,followed by measurement of the decrease in A at 620 nm uponaddition of 2 ml H20. One enzyme unit was defined as the enzymeactivity causing 10%Yo absorbance decrease at 620 nm under thestated assay condition.

,8-Amylase activity was assayed by a modified method of Bern-

'Abbreviation: GA: gibberellin (gibberellic acid).336

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AMYLASE FORMATION IN RICE SEED EPITHELIUM

feld (1), using 1% potato starch solution dissolved in 50 mm Na-citrate buffer (pH 3.6) containing 1 mm EDTA as substrate. Thesame assay conditions as for a-amylase were used. At the end ofthe reaction 0.5 ml of 3,5-dinitrosalicylic acid reagent was addedto the assay mixture, and the A increase at 540 nm was measuredafter heating the whole mixture. One enzyme unit was defined as

the enzyme activity forming reducing sugars equivalent to I mgmaltose during 5-min incubation.

In order to determine the reducing power of each amylase band(A, B, and C) (cf. Table II), amylase preparations were elutedfrom gel segments according to our previous method (18). Eachrespective enzyme solution was then added to 0.4% potato starchsolution dissolved in acetate buffer (pH 5.3) and incubated up to40 min. Aliquots withdrawn from the reaction mixture at 5-minintervals were subjected to the a- and f8-amylase assay methods.From the results of these two analytical methods, we calculatedthe amount of maltose formed during the period in which the Aat 620 nm decreased by one-half from its original value.

Isoelectric Focusing on Polyacrylamide Gel. To detect multipleforms of amylase, isoelectric focusing on the polyacrylamide gelwas employed, basically following the method of Tanaka et al.

(22). Following electrophoresis, zymograms were prepared byrinsing the gels thoroughly with 0.1 M acetate buffer (pH 5.3) andplacing them on a glass plate coated with 0.4% hydrolyzed starchcontaining acrylamide gel. After incubation at 30 C for 20 to 30min, the glass plate was immediately soaked in an I2-KI solutionacidified with acetic acid. Amylase-bearing bands were decolor-ized on a blue-brown background. For characterizing amylasebands A, B, and C, general properties of the enzyme preparationswere tested according to the method of Jacobsen et al. (11). Thecrude enzyme preparation was either dialyzed overnight against20 mm acetate buffer (pH 3.7) or heated at 70 C for 15 min in thepresence of 10 mm CaCl2. The treated enzyme samples were

applied to the gel isoelectric focusing to make zymograms. Toexamine the effect of metal ions on enzyme stability, the same

enzyme samples were applied to the gels after being incubatedwith 1 ,UM HgCl2 or 1 mm EDTA at 37 C for 1 hr. To examine thespecificity of the amylase bands against different substrates, 0.4%amylopectin or 0.4% f-limit dextrin-coated gel plates were used tomake zymograms.

Starch Film Method. Rice seeds at selected germination stageswere frozen with liquid N2, then sectioned with a Cryostat (Ames,Iowa). The sections of seeds obtained were placed directly on glassslides coated with a thin starch film and incubated at 25 C for 5

to 20 min (0.5 day, 20 min; I day, 10 min; 1.5-6 days, 5 min).Starch-coated microscope slides were prepared in a manner similarto that for making amylase zymogram (22). At the end of incu-bation, seed specimens were removed and the glass plate was

immersed in an acidic I2-KI solution. Areas having amylaseactivity were decolorized on a blue-brown background.

RESULTS AND DISCUSSION

We first determined the time sequence changes of a- and /B-amylase activities in each of the scutellum and endosperm samplesduring germination of rice seeds (Table I). On day 2, a-amylaseactivity in the endosperm, determined by the blue value method,was approximately seven times higher than that in the scutellum,whereas 83-amylase activity in the endosperm, assayed by thereducing value method, was four times higher than that in thescutellum. As germination proceeded, enzyme activities in theendosperm continually increased, while those in the scutellum didnot change greatly during germination; scutellum /8-amylase ac-

tivity showed a gradual decline.We then examined the specific localization of amylase activities

by means of the starch film technique. At an early stage ofgermination, about 12 hr after seed imbibition, amylase activitywas almost exclusively localized in the epithelium (Fig. 1); after1.5 days, the activity had spread progressively to the endosperm.Amylase activity in the aleurone layers was detectable only afterday 2, and became intense at the 4- to 6-day stage. Inasmuchas essentially the same reaction patterns were obtained using thefl-limit dextrin film as the substrate, it is likely that amylasemolecules present in the epithelium at the very initial stage are ofa-type.To characterize the nature of amylase molecules produced

during germination, it was necessary to compare the zymogrampatterns between endosperm and scutellum. Results of gel isoelec-tric focusing, using extracts prepared from either scutellum or

endosperm samples, clearly revealed nearly identical major bands(A, B, and C) in the two tissue extracts (Fig. 2). Mixing the twoextracts did not alter the band patterns (data not shown). Somefeatures merit description from the results of the zymogram anal-ysis. In contrast to a relatively uniform enzyme pattern in thescutellum during the 2- to 6-day incubation (except for one

additional band appearing below A on the 4th day) it was apparentthat the endosperm tissue band C, together with several additionalbands above band C, intensified in the late stages of germination.Our previous studies with endosperm tissues indicated that bandsA and B are a-amylase while band C is fi-amylase (18). It isevident from results shown in Table II that band C cannot attackfl-limit dextrin, showing characteristic f8-amylase property. Thelower yield of reducing sugar (maltose) in bands A and B, incomparison with that in band C, indicates that the former twoamylases are of the endo-type whereas the latter is of the exo-type.We previously reported (22) that amylase patterns on isoelectric

focusing gels were nearly the same between GA-treated embryo-less rice seeds and the embryo-attached half-seeds. The presentexperiments show that in the very initial germination stage (12 hrafter imbibition), amylase activities were almost exclusively local-ized in the epithelium, suggesting that enzyme biosynthesis in situ

Table I

Changes in amylase activity of germinating rice seeds

Germination Amylase activity(day) Tissue a-amylase (unit x 10-2) p-amylase (unit)

2 Scutellum 0.7 5.7Endosperm 4.7 21

4 Scutellum 1.2 4.3Endosperm 18 66

6 Scutellum 0.6 3.1Endosperm 21 73

Samples of 100 each of scutellum and endosperm tissues, dissected after2, 4, or 6 days of germination, were homogenized as described in the text.Eluates from a Sephadex G-25 column were assayed for a- and 0-amylase activi-ties; data presented in the table are the averages of duplicate analyses.

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OKAMOTO AND AKAZAWA

0.5-day

1.5-day

4-day

1-day

2-day

6-day

FIG. 1. Amylase localization in germinating rice seeds, as detected by the starch tlm method. Germinating rice seeds at various stages of germinationwere freeze-sectioned and applied to acrylamide gel film containing soluble starch. After incubation, tissue sections were removed from the starch filmand the supporting glass plates were placed in the 12-KI solution for resolution. At stages day 0.5 and 1, sections were made as follows: A: medianlongitudinal section along the axis dividing seed into two mirror images; B: median longitudinal section along the axis separating the germinal side ofseed from the abgerminal side; C: transverse section through the scutellum. Other experimental details are described in the text.

is in this specific cell region. A massive amount of experimentaldata has accumulated that shows the production of a-amylase tobe induced by GA in the aleurone cells of barley seeds (5, 7, 9, 10,11, 12). There is doubt concerning whether or not similar mecha-nism(s) operate in other germinating cereal seeds. Although itappears that GA occupies a crucial role in amylase formation instarchy seeds in general, some circumstantial evidence indicatesthat starch digestion begins from the site adjacent to the embryo(3, 23).The previous research and our present results raise the following

questions: (a) Are the scutellar amylase(s) identical to those from

the endosperm? (b) Are the scutellar amylase(s) induced by GA?Concerning question (a), the present experiments strongly suggestthe epithelium to be the site of initial enzyme formation in theembryo, and also indicate that the epithelium has a more impor-tant role in the hydrolytic digestion of starch reserve than do thealeurone cells. It is established that in the later germination stagesamylase activities develop in the aleurone cells (Fig. 1). The newlyformed amylase bands detectable above band C after 4 days onthe gels (Fig. 2) may well reflect this additional enzyme activity.With regard to question (b), there is considerable experimentalevidence showing the primary production of GA to be in embry-

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AMYLASE FORMATION IN RICE SEED EPITHELIUM

Scutellum E ndosperm

2 4 6 2 4 6Germination Days

FIG. 2. Comparison of amylase zymograms of crude extracts obtained from scutellum and endosperm tissues of rice seeds at various stages ofgermination. Crude scutellar extracts (100 Al each) and crude endosperm extracts (20 Id each) prepared at various germination stages were applied togel for isoelectric focusing (pH 4-6). To make zymograms, gels were incubated with the starch gel-coated glass plates for 20 min. Other experimentaldetails are described in the text.

Table IIProperties of scutellar amylases of germinating rice seeds

Amylase band

A B C

Enzyme stability:acid treatment (pH 3.7) _ _ +heating (70 C, 15 min) + +HgC12 ( 1 M) + +ED'A (1 MM) _ _ +

Substrate susceptibility:amylopectin + + +P-limit dextrin + +

Reducing power (mg maltose) 0.03 0.04 0.18

Crude enzyme was prepared from scutellar tissue as described in the text.Enzyme properties in crude extracts were determined from the zymogram patterns ofthe gel electrofocused samples (cf. Fig. 2). In experiments testing enzymestability, the symbol (+) indicates that the enzyme was resistant to varioustreatments, whereas (-) signifies that the enzyme activity was inhibited. Inexperiments to test substrate susceptibility, (+) signifies that the enzymeband appeared on the zymograsm. Reducing power of amylase bands A, B and C, repre-sents the amount (mg) of maltose molecules produced during the assay period, inwhich one half of the original soluble starch was hydrolyzed (see text).

pH 6

C

B-'Ap-

pH 4

-B

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340 OKAMOTO At

onic tissues (14, 19, 20). It is likely that GA first takes part inamylase formation in the epithelium, later diffusing to aleuronecells where GA induces the de novo synthesis of additional amylasemolecules (after the 2nd day).

In contrast to very thorough investigations on a-amylase for-mation in germinating cereal seeds, the nature of,-amylasebiosynthesis and its role in the reserve starch digestion are rela-tively unexplored. In barley and wheat seeds, it has been demon-strated that ,B-amylase molecules exist in association with theprotein in the endosperm tissues (21, 24). It is now found that /-amylase (band C) exists in the scutellum of germinating rice seeds;it is presumably synthesized in the epithelium. Since 83-amylaseactivity was found to increase in the endosperm tissues as germi-nation proceeded, it is intriguing to explore further the mecha-nism(s) of /3-amylase formation and activation.

Acknowledgments-The authors wish to record their sincere thanks to L. Rappaport for carefulreading of the manuscript and kind help. They also thank Y. Kono for valuable advice.

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N]D AKAZAWA Plant Physiol. Vol. 63, 1979

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