Plant Physiol. (1984) 76, 1047-10540032-0889/84/76/1047/08/$0 1.00/0

Starch Degradation Metabolism towards Sucrose Synthesis inGerminating Araucaria araucana Seeds'

Received for publication February 16, 1984 and in revised form May 18, 1984

LILIANA CARDEMIL* AND JOSEPH E. VARNERDepartamento de Biologia, Facultad de Ciencias Basicas y Farnaceuticas, Universidad de Chile,Santiago, Chile (L.C.) and Department ofBiology, Washington University, St. Louis, Missouri 63130(J.E.V.)

ABSTRACT

As starch is the main seed reserve material in both species ofAraucariaof South America, A. araucana and A. angustifolia, it is important tounderstand starch breakdown in both embryo and megagametophytetissues of Araucaria seeds. Sugar analysis by thin layer chromatographyindicates that sucrose is the main sugar produced in both tissues. Enzymereactions coupled to benzidine oxidation indicate that sucrose is the mainsugar moved from the megagametophyte to the growing regions of theembryo via the cotyledons.

Phosphorylase was detected in both embryo and megagmetophytetissues by the formation of I2Pjglucose-I-P and by formation of 114qamylopectin from ['4Cjglucose-I-P. The enzyme activity increases 5-foldin both embryo and gametophyte to a peak 18 hours after the start ofimbibition. Debranching enzyme, a-glucosidase, and hexokinase are alsopresent in both embryonic and megapmetophytic tissues.

Branched glucan oligosaccharides accumulate during this time, reach-ing a maximum 40 hours after imbibition starts, and decline after ger-mination occurs.The pattern of activity of the enzymes studied in this work suggests

that starch degradation is initiated by a-amylase and phosphorylase inthe embryo and by phosphorylase mainly in the megagametophyte.Sucrose-P synthase seems to be the enzyme responsible for sucrosesynthesis in both tissues.

of special interest to Chile, Brazil, and Argentina (6), it is con-venient that the Araucaria seed are so very large. Thus, a com-plete range of analyses, with several replicates, can be done onembryo and megagametophyte tissues from a single seed. It isalso fortunate that the megagametophyte cells are large. Beingcoenocytes they are about 180 gm in diameter (5) and can beobserved under the stereomicroscope in thick sections of in vivotissue. This allows in situ cytochemical identification of mole-cules that are being secreted by the megagametophytic cells intothe intercellular spaces and transported from there to the cavitywhere the embryo is lying.The question which arises is: how is starch degraded by Arau-

caria seeds in the first 90 h after the start ofimbibition? Therefore,the purposes of this work were to investigate whether there areother enzymes beside a-amylase which may initiate starch deg-radation in the embryo and in the megagametophyte and toidentify the enzymes that might be involved in the degradationofstarch and formation ofsoluble sugars. Ultimately, the increasein the amount of soluble sugars and soluble metabolites in theembryo will increase osmotic pressure and the seed will germi-nate (17).

This research then indirectly addresses the question of howthe osmotic 'push' required for germination is developed. Thisis an important question because it is closely related to theproblems of understanding dormancy, triggers for germination,seedling growth, and seedling vigor (3). Most of what may belearned about these problems in Araucaria species could applyto the same problems in other species.

In the seeds of both Araucaria species of South America (A.araucana and A. angustifolia), the main reserve is starch (4).Starch accounts for 60% of the seeds and seems to be initiallydegraded by a-amylase during germination. a-Amylase is presentmainly in the embryo and in low levels in the megagametophyte(4, 19). a-Amylase shows a peak of activity in the embryo from12 to 24 h after the start of imbibition (4, 19). The seedsgerminate at 40 h of imbibition, apparently through the use ofembryo tissue reserves which provide the nutrients required forgermination (4). Later in seedling growth, megagametophyteamyloplasts disappear from the cells (4) and the product ofdegradation of megagametophyte reserves seems to be trans-ported to the cavity where the embryo cotyledons are lying.These products are transported to the growing regions of theembryo via the cotyledons which serve as haustoria (15).

In addition to the fact that South America Araucaria sp. are

' Supported by Comision Nacional de Investigaciones Cientificas yTechnologicas and National Science Foundation, Cooperative SciencePrograms in Latin America; and by grant number B-1580/8325 ofDepartamento de Desarrollo de la Investigacion, Universidad de Chile.

MATERIALS AND METHODS

Source of Materials and Germination Conditions. Seeds ofAraucaria araucana were obtained from Malalcahuello, ReservaForestal de Chile.The seeds were germinated as described by Cardemil and

Reinero (4). For time course experiments, seeds were taken at 0,12, 18, 24, 40, 48, 60, 72, and 90 h after start of imbibition.Sugar Composition Analysis. TLC was performed on micro-

crystalline cellulose thin layer plate squares, 20 x 20 cm, and100 ,m thick (Merck Chemical Company). The solvent mixturewas ethyl acetate, acetic acid, HCOOH, and water in a ratio of18:3:1:4 (v/v/v/v). The plate was run three times in this solvent.Sugars were detected on the plates by means of silver nitrate-

alcoholic NaOH (25) and by the resorcinol method described byRoe (20). Sucrose quantification was performed by the methoddescribed by Roe (20) and modified by Cardini et al. (7), remov-ing the corresponding area of the thin layer plate and eluting thesugar from the cellulose powder with 1 ml of 25% ethanol.

Identification of Sucrose In Vivo. The in situ identification ofsucrose was performed by coupling enzyme reactions to benzi-

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CARDEMIL AND VARNER

dine. The enzymes used were: peroxidase from horseradish(Sigma), glucose oxidase from Aspergillus niger (Sigma), andinvertase from Candida utilis (Sigma).Under a stereomicroscope, transverse sections about 1 mm

thick were wet with two or three drops of 33% benzidine solutionprepared in 25% ethanol. After 1 min, three drops of peroxidaseat a concentration of 2 units/ml of 0.1 M sodium acetate buffer(pH 5.5), three drops of glucose oxidase diluted 10 times in 0.1M sodium acetate buffer (pH 4), and three drops of invertase ina concentration of 4 units/ml sodium acetate buffer (0.1 M, pH4.5) were added successively within 30-s intervals. Two or 3 minat room temperature was sufficient to obtain positive colordevelopment.The rationale of the sucrose in situ identification is: benzidine

on tissue identifiies peroxidase and H202, by development of ablue to brownish color; benzidine + peroxidase identifies- H202,by development of a blue to brownish color; benzidine + per-oxidase + glucose oxidase identif-ie glucose, by development ofa blue color, benzidine + peroxidase + glucose oxidase + inver-tase identifies sucrose, by development of an intense blue color;benzidine + peroxidase + glucose oxidase + sucrose identifies

invertase, by development of an intense blue color.Starch Phosphorylase Assay. Starch phosphorylase was de-

tected by assaying the two reactions that the enzyme catalyzes invitro.1. (glucose), + 32Pi -_. [32P]glucose-l-P + (glucose)n-12. (glucose)n + ['4C]glucose-l-P -. (['4C]glucose)n+I + PiTwo embryos and the equivalent weight of megagametophyte

fresh tissue, taken at different imbibition times, were homoge-nized in mortars with 3 ml of the respective buffer for the assays.For the first assay, the buffer used was 0.1 M sodium citrate

(pH 6) containing 20 mm NaCl. The homogenized material was

centrifuged for 2 min in a microfuge and the supernatant wascollected and kept on ice until used. For the enzyme reaction,50 ,l of the above supernatant were incubated with 250 ,1 of a20 mm solution of soluble starch prepared in 0.1 M sodium citrate(pH 6) containing 1 mM EDTA and 20 mm sodium phosphate(10 nCi/nmol32Pi; New England Nuclear). The mix was incu-bated at 25C for 20 min and the reaction stopped by placingthe samples in a boiling water bath for 2 min. [32P]Glucose-l-Pwas identified in a two-dimensional TLC on a cellulose plate 20x 20 cm. The plate was run using the solvent mixture describedby Feige et al. (10) for sugar phosphates. The chromatographywas run until the solvent reached 2 cm from the upper edge ofthe plate. Two Ml of the reaction mix plus 2,ul of glucose-l-P ina concentration of 2 mg/ml were spotted in a corner of the plate,leaving 5 cm of the plate as a margin. The phosphate and sugar

phosphate spots were visualized by means of perchloric ammo-nium molybdate HCI reagent (2). Autoradiograms were obtainedby exposing the plate to X Omat Kodak film for 24 h.The second assay, which measures the formation of ['IC]

amylopectin, was performed using 50Il of the homogenizedmaterial and 250 ul of a 0.1 M sodium citrate buffer containing1 mM EDTA, amylopectin in a concentration of1%, and 50 mM['4C]glucose-1-P (294 mCi/mmol). The mix was incubated at25C for 20 min. The production of['4C]amylopectin was linearfor 30 min; therefore, this assay was used for enzyme activityquantification. The reaction was stopped by placing the samplesin a boiling water bath for 2 min. Detection of['4C]amylopectinwas achieved by TLC on cellulose plates. FiveM1 of each samplewere spotted on the plate, 2 cm from the lower edge. The solventsystems used were those described by Feige et al. in which solvent

is a mixture of isobutyric acid, n-butanol, isopropanol, n-

propanol, water, NH3, and EDTA in a ratio of1000:30:140:380:40:0.5 (v/v/v/v/v/v). The origin area of theplate, where the sample wasapplied, was removed fromthe plate,

mixed with 5 ml of Amersham scintillation mix (AMS).The phosphorylase activity was expressed as mU2/mg of dry

weight. One mU of phosphorylase activity is defined as 1 nmolof glucose incorporated in amylopectin in 10 min per reactionmixture.Hexokinase Assay. This was performed by coupling hexoki-

nase activity to glucose-6-P dehydrogenase. The final concentra-tion of NADPH produced was read at 340 nm using a Gilsonspectrophotometer with an automatic recorder.Two embryos and an equal gametophyte fresh tissue weight

were homogenized separately in 3 ml of 10 mM K-phosphatebuffer (pH 7) containing 1 mM EDTA, 1 mm DTT, and 20 mmNaCl. The homogenized material was centrifuged at 10,000 rpmfor 20 min. The supernatant was collected and kept refrigerateduntil used. Fifty Ml of the above extract were mixed with 250 Ml

of the reaction mix containing 80 mm Tris, pH 8; 1 to 1.5 mMEDTA; 150 mm KCI; 6 mM MgC12; 2.5 mm DTT; 0.5 mmNADP; 0.2 unit glucose-6-P dehydrogenase. The extract waspreincubated at room temperature for 15 min to eliminateendogenous substrates and then 50 5 mM glucose were added.The absorption at 340 nm was read against a blank of the

same reaction mix, except that ATP was replaced by water. Thehexokinase activity is expressed as mU/mg of dry weight. OnemU of kinase activity is defined as 1 nmol of glucose-6-Pproduced in 10 min per reaction mixture.

a-Glucosidase Assay. The enzyme extract was prepared byhomogenizing two embryos and the equivalent gametophytefresh tissue weight, in 3 ml of 0.033 M K-phosphate buffer (pH6.9) containing 20 mM NaCl. The homogenized material wascentrifuged in a microfuge for 2 min, and the supernatant wasused for the assay.The assay was performed with p-nitrophenyl-a-glucopyrano-

side in a concentration of 0.1 mg/ml of the above phosphatebuffer. Fifty Ml of the tissue homogenate supernatant were incu-bated with 250 Ml of p-nitrophenyl-a-glucopyranoside solutionat 37°C, for 20 min. The reaction was stopped by placing themix in a boiling water bath. After cooling, the volume was madeup to final volume of 1 ml with 0.05 N KOH. The absorptionwas read at 400 nm in a Gilson spectrophotometer and activityexpressed as mU/mg of dry weight. One mU is defined as 1 Mugof p-nitrophenol released in 10 min per reaction mixture.

Debranching Enzyme Assay. Tissue homogenates were pre-pared as described before for the other enzyme assays. The bufferused for homogenization was 0.06M sodium phosphate (pH 6.8)having 20 mM NaCl. The homogenized tissues were centrifugedat 10,000 rpm and the supernatant dialyzed against the phos-phate buffer (pH 6.8). One hundred Ml of the supernatant wasmixed with 500 MAl of a 5% pullulan solution diluted 3.5-fold in0.02M sodium citrate buffer (pH 5). The above mix was incu-bated at 37°C for 20 min. The reaction was stopped by boilingthe extracts in a boiling water bath for 3 min. After cooling, 2ml of Nelson and Somogyi reagent (21) were added to each tubein order to quantify the amount of reducing sugars. The enzymeactivity was expressed as mU/mg of dry weight. One mU isdefined as 1 Mg of reducing sugars, measured as maltotriose,released by the enzyme in 10 min. The sugars released bydebranching enzyme activity from the commercial pullullan weredetected by TLC analysis on microcrystalline cellulose plates,using the same solvent system described for sugar analysis. Theplate was run three times in the solvent, and the sugars weredetected on the plate by silver nitrate alcoholic sodium hydrox-ide. A standard solution of maltotriose containing1 mg/ml wasspotted on the plate for RF determination.

Quantification of Endogenous Brnched Oligosacchandes. Acommercial pullulanase from Enterobacter and obtained from

and the cellulose power suspended in 0.5 ml of 50% ethanol and Abeato:U,mlin.

1048 Plant Physiol. Vol. 76, 1984

I Abbreviation: mU, milliunit.

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STARCH DEGRADATION IN A. ARAUCANA SEEDS

Sigma was used to quantify endogenous branched glucan oligo-saccharides (a-( l-)-glucose oligosaccharide having glucosebranches linked by a-( 1-*6) bonds). The enzyme was assayed ona crude tissue homogenate prepared in a 0.02 M sodium citrate(pH 5) having 20 mm NaCl. The homogenate suspension wasallowed to stand for 1.5 h at 370C with occasional shaking, tohydrolyze all the endogenous linear glucan chains, and thencentrifuged at 10,000 rpm. The supernatant was diluted 10-fold,heated in a boiling water bath for 1 min to denature the endog-enous enzymes. After cooling, 500 Ml of this extract were incu-bated with 0.33 unit of the pullulanase for 20 min at 25C. TheMg of reducing sugars, measured as glucose, released by theenzyme were quantified by Nelson's test.

Sucrose-P-Synthase. The enzyme activity was measured bythe method described by Leloir et al. (14) and modified by Amirand Preiss (1). Tissue of eight embroys and the equivalentmegagametophyte fresh tissue weight were homogenized in 3 mlof buffer containing 5 mm Tris-HCI, 5 mm EDTA, 10 mM KCN,and 20 mM NaCl (pH 8). The homogenized material was allowedto stand for I h with occasional shaking and the mix wascentrifuged for 15 min at 10,000 rpm (14). The supernatant wasdialyzed for I h at 5C against two changes of a buffer containing20 mm Tris-HCI, I mm EDTA, and I mM DTT (pH 7.5). SixtyMgl of the above extract were incubated for 20 min at 37°C witha mix of 80 Ml of 200 mm Hepes NaOH (pH 7.5), 96 ,l of 50mM fructose-6-P, 100 Ml of 8 mg/ml of BSA, 40 Ml of 100 mmMgCI2, and 120 Ml of 50 mm of UDP-glucose.The reaction was stopped by boiling the mix in a boiling water

bath. After cooling, 500 Ml of the mix were incubated at 37°C for20 min with 15 units of alkaline phosphatase from E. coli inorder to hydrolyze the sucrose-6-P to sucrose. The alkalinephosphatase catalyzed hydrolysis was stopped by boiling the mixin a water bath. Sucrose was quantified by Roe's method. Theenzyme activity was expressed as mU/mg of dry weight. OnemU is defined as 1 nmol of sucrose synthesized in 10 min.

For product analysis, the sucrose-P synthase assay was per-formed two other times as described before except that 20 nCi/mmol of ['4C]fructose-6-P were added to 96 ML of 50 mm fructose-6-P of the reaction mix. After hydrolysis of the sugar-P withalkaline phosphatase, detection of the ['4C]sucrose was per-formed by autoradiography of a TLC as it was described forsugar analysis. Autoradiograms were obtained by exposing theplate to X-Omat Kodak film for about 1 week.

Sucrose Synthase. This assay was identical to sucrose-P-syn-thase assay except that fructose-6-P was replaced by fructose.

RESULTSThe a-amylase activity, reported for A. araucana embryo

during germination and early seedling growth, peaks rapidly at20 h, declines until germination, and increases slowly thereafter(4, 19). These fluctuations in the levels of amylolytic activity ofthe embryo correlate with starch degradation rate which is de-graded 84% in 40 h after imbibition starts (4) and with the levelof soluble sugars in the same tissue (4). In the megagametophyte,a-amylase activity and soluble sugars do not change markedlyduring the first 90 h after the start of imbibition in starch isdegraded 25% during the first 40 h with almost no changesthereafter (4). However, in time, the amyloplasts of the megaga-metophyte disappear and, therefore, the starch is degraded (4).

In order to establish if there are other enzymes responsible forthe initiation of starch degradation, starch phosphorylase activitywas measured during the same imbibition time as a-amylaseactivity was measured.When the enzyme is assayed by the starch degradation reaction

using soluble starch and 32Pi as substrates, and product analysisby radioautography of a TLC, the results show two radioactivespots for the embryo extracts and one radioactive sugar for the

megagametophyte extracts. In the embryo, the sugars were iden-tified as radioactive glucose- I -P and radioactive glucose-6-P, withappropriate sugar phosphate standards. The glucose- 1-P spotshows 3 times more radioactivity than the glucose-6-P spot after10 min of incubation; the radioactivity is similar in both sugarphosphates after 20 min of incubation. The radioactive spotobtained with the megagametophyte homogenate was identifiedas glucose-1-P; using glucose-I-P as standard (Fig. 1, A and B).When starch phosphorylase was assayed in the direction of

starch synthesis, it was found that the enzyme increases 5-fold in

I

23

FIG. 1. Radioautograph of a TLC analysis of sugar phosphates, theproducts of the starch phosphorylase reaction. A, Embryo extract asenzyme source: (1), "2Pi; (2), glucose-1-P; (3), glucose-6-P. B, Megaga-metophyte as enzyme source.

1049

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CARDEMIL AND VARNER

-C4.

0

E0x

E

10 30 60 70 90 110 130 150 170Hours after imbibition starts

FIG. 2. Total starch phosphorylase activity in the embryo and mega-gametophyte over 90 h after imbibition starts. Points are the average (+SD) of three independent experiments. (0), Embryo; (U), megagameto-phyte.

7

6

3: 5

0 4

'-3

Ca

72

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0

0

10 20 30 40 50 60 70 80 90

Hours after imbibition startsFIG. 3. Total branched oligosaccharides accumulated over 90 h after

imbibition starts. Points are the average (+ SD) of three independentexperiments. (0), Embryo; (U), megagametophyte.

the embryo as well as in the megagametophyte tissues during thefirst 24 h after imbibition, and declines rapidly after that. So, atgermination, approximately 40 h after the start of imbibition,the enzyme again reaches the initial level which is maintainedwith almost no fluctuation after germination (Fig. 2).a-Amylase and starch phosphorylase cleave only a-(1-*4)

glucosidic linkages (9, 12, 16, 23). This means that during thefirst 40 h of seed imbibition a-( l-4), glucan oligosaccharideshaving a-(1-*6) glucose branches may accumulate in embryo

and megagametophyte cells. Quantification of branched glucanoligosaccharides show that they increase in level reaching a peakat 40 h of imbibition, 6.0 x I0-' ng ofbranched glucan oligosac-charides/mg of dry weight in the megagametophyte. The oligo-saccharides decline slowly after that (Fig. 3). The level ofbranched glucan oligosaccharides accumulated is greater in theembryo than in the megagametophyte (Fig. 3).The enzyme which hydrolyzes a-(1-46) glucosidic bonds is the

debranching enzyme. The presence of the enzyme in tissueextracts was detected and quantified using a commercial pullulanas substrate. This pullulan has a repeated unit of maltotrioselinked by a- 1l-*6) glucosidic bonds. Therefore, the expectedproducts of pullulan hydrolysis will be mainly glucose and mal-totriose if the enzyme is present in A. araucana tissues. Analysesofthe products of the enzyme reaction by TLC demonstrate thatafter 10 min ofincubation ofthe tissue extracts with the substrate,glucose and maltotriose are the main sugars released. Theyincrease linearly over incubation time.The debranching enzyme activity, measured over imbibition

time, shows that, from quiescent seeds up to 18 h, there is nodetectable enzyme activity in the embryo and megagametophyte.After 24 h, debranching enzyme activity is detected in embryoand it rises to 10-fold the initial activity by 90 h after the start ofimbibition. In the megagametophyte, debranching enzyme activ-ity begins to increase after 24 h, becoming 5-fold the initialactivity after 90 h of imbibition (Fig. 4).The linear a-(l-4) glucan oligosaccharides released by the

debranching enzyme, such as maltopentaose, maltotetraose, mal-totriose, and maltose, will be ultimately degraded to glucose bythe action of a-glucosidase.The levels of a-glucosidase during 90 h of seed imbibition, are

shown in Figure 5. As in the a-amylase and the debranchingenzyme cases, a-glucosidase levels are significantly higher in theembryo than in the megagametophyte tissues for all imbibitiontimes recorded. In the quiescent seeds, a-glucosidase activity inthe embryo is 8-fold the activity found in the megagametophyte:0.08 mU/mg of dry weight in the embryo and 0.01 mU/mg ofdry weight in the megagametophyte.

4-

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x

E

26

24

22

20

18

16

14

12

10

8

6.

4.

2

0 10 20 30 40 50 60 70 80 90

Hours after imbibition starts.FIG. 4. Total debranching enzyme activity in the embryo and mega-

gametophyte over 90 h after imbibition starts. Points are the average (±SD) of three independent experiments. (0), Embryo; (U), megagameto-phyte.

Plant Physiol. Vol. 76, 19841050

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STARCH DEGRADATION IN A. ARAUCANA SEEDS

-

.r_

0

E

c7E

a

24

22

20

18

16

14

12

108

8..

6..

4-

21 1T

0 10 20 30 40 50 60 70 80 90

Hours after imbibition startsFIG. 6. Total hexokinase activity in the embryo and megagameto-

phyte over 90 h after imbibition starts. Points are the average (± SD) ofthree independent experiments. (0), Embryo; (U), megagametophyte.

0 10 20 30 40 50 60 70 80 90

Hours after imbibition starts

FIG. 5. Total a-glucosidase activity in the embryo and megagameto-phyte over 90 h after imbibition starts. Points are the average (± SD) ofthree independent experiments. (0), Embryo; (U), megagametophyte.

The a-glucosidase activity increases rapidly in the embryotissue to 2.5-fold after 18 h from the start of imbibition, declinesrapidly after this time to 0.10 mU/mg of dry weight, and in-creases again after 30 h from the start of imbibition. In themegagametophyte tissue, the initial activity of the enzyme doesnot change significantly during 90 h of seed imbibition.The level of hexokinase, is also higher in embryo tissue than

in megagametophyte tissue. However, the initial activity in quies-cent seeds was the same for both tissues (32 mU/mg of dryweight in the embryo and 31 mU/mg of dry weight in themegagametophyte). The total activity of the enzyme shows apeak at 18 h after the start of imbibition. The peak is 5-fold theinitial activity: 150 mU/mg of dry weight; it decreases to 100mU/mg of dry weight after 40 h of imbibition and begins toincrease again after germination, to become 182 mU/mg of dryweight 90 h after imbibition started. In megagametophyte tissue,maximum activity registered is at 60 h after the start of imbibi-tion, when the activity becomes about 2-fold the activity of thequiescent seed: from 34 to 66 mU/mg of dry weight. After 60 hof seed imbibition, the activity declines to the initial level(Fig. 6).Gas chromatographic analysis of sugars previously reported

(4) indicates that there are no free monosaccharides present inthe seeds during imbibition. When the samples are hydrolyzed,glucose, fructose, and mannose are the only hexoses detected,glucose being 10 times more abundant than mannose. Othercommon sugars such as galactose were not detected (4). TLC ofsoluble sugars extracted from embryo and megagametophytetissues shows the sucrose is the main soluble sugar present inboth tissues at all times during the 90 h that the seed has beenimbibed, and the total amount of sucrose accounts for most ofthe soluble sugars reported before. In embryonic tissues, the levelof sucrose is about 2-fold the level of sucrose present in themegagametophyte tissue for all the sample times. In both tissues,

0%

0%

E

E

0 20 40 60 80

Hours after inbibition starts

FIG. 7. Total sucrose levels over 90 h after imbibition starts. Pointsare the average (± SD) of three independent experiments. (0), Embryo;(a), megagametophyte.

sucrose level shows a peak 18 h after imbibition starts. In theembryo, sucrose declines to the initial level and increases againsteadily after germination. In megagametophytic tissue, sucroselevel increases slightly after the seed germinates (Fig. 7).

Sucrose synthase enzyme is absent in both tissues of the seedduring the 90 h of imbibition. However, sucrose-P synthase ispresent. The levels of total enzyme activity are shown in Figure8. The activity of the enzyme in embryo tissue is about twice theactivity in the megagametophyte tissue for all times recorded. Inboth tissues, activity shows a discrete peak at 18 h from start ofimbibition. In the embryo tissue, the peak is 1.5-fold the initialactivity, going from 8 mU/mg of dry weight to 12.2 mU/mg ordry weight. The peaks decrease to about the initial activity inboth tissues at 24 h from start of imbibition and increase afterthat. In embryo tissue, the sucrose-P synthase activity showsanother peak at 60 h of the start of imbibition.The sucrose-P synthase was assayed by coupling the alkaline-

phosphatase reaction to the main enzyme reaction.

fructose-6-P + UDP-glucose sucrse-P sucrose-6-P+UDP

sucrose-6-P aklne phospha sucrose+Pi

Analysis of the products of these reactions by TLC shows that

35S

34-

28

26

24

22

20

18

16

14

1 2

10

8

6 .

4 -

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.-

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1051

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CARDEMIL AND VARNER

12

10.

8

6.

-

3

I-,Z

4-

0 20 40

Hours after imbibition starts

60 80

FIG. 8. Total sucrose-P synthase activity in the embryo and megaga-metophyte over 90 h after imbibition starts. Points are the average (±

SD) of three independent experiments. (0), Embryo; (e), megagameto-phyte.

FIG. 9. Radioautograph of a TLC analysis of the sucrose-P synthaseproduct, after incubation of the reaction mix of the embryo extract andof the gametophyte extract with alkaline phosphatase. (S), Standardsucrose; (F), standard fructose; (E), embryo extract; (G), megagameto-phyte extract.

sucrose appeared after the extracts were incubated for 30 minwith alkaline phosphatase. Figure 9 shows the results of a ra-dioautograph of a thin layer plate with two radioactive sugars,sucrose and fructose, when ['4C]fructose-6-P was used as sub-strate for the coupled enzyme reaction to sucrose-P synthasewith alkaline phosphatase.Enzyme reactions coupled to benzidine oxidation demonstrate

that sucrose fills the cytoplasm and intercellular spaces of me-gagametophyte tissue. Indeed, broken cells and intercellularspaces immediately turn intense blue when drops of benzidine,peroxidase, glucose oxidase, and invertase are added to the seedsection. The cavity occupied by the cotyledons also develops anintense blue color. If invertase is omitted in this test, a faint bluestain develops, after a longer incubation time, demonstratingthat there is glucose and/or invertase present in megagameto-phyte intercellular spaces and in the cavity.When invertase is omitted and sucrose is added to the seed

section, an intense blue color develops in the periphery of thecotyledons, probably because invertase is on the surface of thecotyledons. Table I shows the qualitative results of the coupledenzyme reactions to benzidine which were assayed on seed

sections and the tissue areas which were positive to the differenttests.

DISCUSSIONWhen crude extracts from embryo and megagametophyte are

assayed for the presence of phosphorylase working as a starchdegradative enzyme, glucose-l-P is the only sugar phosphateproduct if the enzyme source is the megagametophyte. Glucose-1-P and glucose-6-P are the products if the enzyme source is theembryo, very probably, because phosphoglucomutase is presentin the embryo extract and may form glucose-6-P from glucose-1-P.Analysis of products and quantification of phosphorylase ac-

tivity show that the enzyme is present in both embryo andmegagametophyte. Starch phosphorylase coexists with a-amylasein plant tissues (8, 9, 11, 13, 22, 23) and both are responsible forinitiation of starch degradation in plants. Therefore, both en-zymes must initiate starch degradation in the embryo. In themegagametophyte, with very low a-amylase activity (4, 19) phos-phorylase is certainly the main initiator.

In vitro the enzyme also catalyzes starch synthesis; therefore,the enzyme may catalyze the reaction in both directions in theembryo since the starch level increases slightly in the embryoafter germination (4). However, it is improbable that the enzymewill catalyze starch synthesis in the megagametophyte becausethe end product of the overall carbohydrate reserve digestion inthe megagametophyte is sucrose, and this final product is re-moved from the system by the cotyledons which act as haustoria(15).

In both tissues, the enzyme peaks 18 h after the start ofimbibition, with a 5-fold increase in activity. Since A. araucanaseeds germinate around 40 h after imbibition starts, starch phos-phorylase seems to contribute to the increase of solutes in thecells before the seed germinates. This is corroborated by theincrease in branched glucan oligosaccharides during the first 40h ofimbibition and by increase ofsucrose which peaks 18 h afterimbibition starts.The debranching enzyme is not present at all in quiescent

seeds. Its activity is detectable at the time when the branchedglucan oligosaccharides begin to accumulate in the tissues, sug-gesting that enzyme activity is induced by the presence of theseoligosaccharides.

Product analysis of the enzyme reaction shows that glucoseand maltotriose are detectable as products of the reaction. Bythis test, the enzyme is then identified more as a pullulanasethan as a debranching enzyme. However, in the cells, pullulan isnot the natural substrate for the enzyme, and maltriose will notbe the main oligosaccharide product of hydrolysis. Althoughcritical tests have not been performed to distinguish betweenthese two enzymes (16, 24), the probable hydrolytic enzyme forbranched glucan oligosaccharides should be the debranchingenzyme.

In the embryo, a-glucosidase and hexokinase have similaractivity fluctuation patterns during seed imbibition. Both en-zymes are present before the seed is imbibed, both peak at 18 h,decline to the basal level at 24 h, and increase thereafter.

In the megagametophyte, a-glucosidase is not detectable at allin the quiescent seed and its activity is very low during the first90 h of imbibition. Hexokinase activity is detectable, but is lessin this tissue than in the embryo for all times recorded; therefore,as in the embryonic tissue, a-glucosidase and hexokinase areenzymes which correlate well with a-amylase level fluctuations(4, 19).The peak of sucrose shown in the embryonic tissue at 18 h of

imbibition, seems to be explained by the peak of activity of a-amylase, starch phosphorylase, a-glucosidase, and hexokinase.Therefore, the four enzymes seem to be involved in starch

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STARCH DEGRADATION IN A. ARAUCANA SEEDS

Table I. Qualitative Results ofCoupled Enzyme Reactions to Benzidine

GametohytentercelularCotyledonsReagent Test for Gametophyte Intercellular Groove VascularPeripherySpaces Bundles

1. Benzidine + peroxi-dase H202 +a +

2. Benzidine + H202 Peroxidase + +3. Benzidine + glucose

oxidase Glucose + peroxidase + ++ +4. Benzidine + glucose

oxidase + peroxidase Glucose + +++ +++5. Benzidine + glucose

oxidase + peroxidaseinvertase Sucrose +++ +++

6. Benzidine + glucoseoxidase + peroxidase +sucrose Invertasea +, Slight positive reaction (light blue); ++, positive reaction (medium blue); +++, strong positive reaction

(intense blue); -, negative reaction.

metabolism leading to the sucrose.In the megagametophyte, the glucose-l-P which is an inter-

mediate for sucrose synthesis, may be formed mainly by starchphosphorylase activity and the low hexokinase activity detectedin this tissue may be required to form glucose-6-P from theglucose released by the debranching enzyme.When starch degradation is initiated by a-amylase, the flow of

carbon would be from glucose-6-P to glucose- I -P; when initiatedby starch phosphorylase, the flow of carbon would have to bemainly from glucose- 1-P to glucose-6-P with little requirementfor hexokinase. Therefore, in the megagametophyte the mainoperating pathway for starch degradation will be that initiatedby phosphorylase which would explain the low hexokinase activ-ity detected here as compared with embryo tissue.The two enzymes responsible for sucrose synthesis in plants

are sucrose synthase and sucrose-P synthase. This last enzymehas to be coupled to a phosphatase to give sucrose. The sucroselevel itself in plant tissues will depend on the following fourenzyme reactions ( 18):UDP-glucose + fructose-6-P sucrose-P synthase__ UDP

+ sucrose-6-P (1)sucrose-6-P sucrow-P phosphatasew_ sucrose + Pi (2)UDP-glucose + fructose suc'S sy'thase UDP + sucrose (3)

sucrose invel--* fructose + glucose (4)During the onset of germination in seed having starch as

reserves, sucrose synthesis might be much greater than sucrosedegradation. Therefore, one could imagine that the first threeenzymes will be present in the tissues, with sucrose synthasecatalyzing the reaction mainly towards sucrose synthesis. Aftergermination, when seedling growth starts, sucrose breakdownwill be the predominant process in the embryo and it might beadvantageous for this tissue to have an excess of invertase, aswell as sucrose synthase, catalyzing the reaction in both direc-tions. In the megagametophyte, where sucrose synthesis shouldbe much greater than sucrose degradation, one could imaginethat sucrose-P would serve to catalyze sucrose synthesis.The data show that sucrose-P synthase and not sucrose syn-

thase is the predominant enzyme for sucrose synthesis in bothtissues during the first 90 h of seed imbibition. This agrees withthe fact that in the embryo starch reserves are still being degradedto sucrose 90 h after the start of imbibition.The synthesized sucrose in the megagametophyte tissue is

moved through intercellular spaces towards the cavity as wasdemonstrated by color reactions of enzymes coupled to benzi-

dine, although small amounts of glucose may also be moved.Finally, it is necessary to point out that 18 h after the start of

seed imbibition seems to be a critical time in the physiology ofthe germination process of A. araucana seeds. a-Amylase, a-glucosidase, hexokinase, and starch phosphorylase all show apeak of activity in the embryo, which correlates with maximumactivity of sucrose-P synthase, with a maximum in sucrose level,and with the linear increase in the branched glucan oligosaccha-rides. All these events occur before germination. Therefore, allthese correlated processes would seem to play an important rolein the increase of the osmotic pressure required for germination.

Acknowledgments-The authors thank Juan Carlos Villablanca for performingthe hexokinase assay, Angelica Vega and Margarita Cortes for technical assistance,and Lilio YUafiez for help in photography.

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