Amylase Occurs Concomitant with the Accumulation of Starch and

Plant Physiol. (1991) 96, 902-9090032-0889/91 /96/0902/08/$01 .00/0/0-

Received for publication December 10, 1990Accepted March 12, 1991

Sucrose-Induced Accumulation of ,-Amylase OccursConcomitant with the Accumulation of Starch andSporamin in Leaf-Petiole Cuttings of Sweet Potato1

Kenzo Nakamura*, Masa-aki Ohto, Nobumasa Yoshida2, and Kyoko Nakamura

Laboratory of Biochemistry, School of Agriculture, Nagoya University, Chikusa-ku, Nagoya 464-01, Japan

ABSTRACT

,8-Amylase of sweet potato (Ipomoea batatas L.), which consti-tutes about 5% of the total soluble protein of the tuberous root,is absent or is present in only small amounts in organs other thanthe tuberous roots of the normal, field-grown plants. However,when leaf-petiole cuttings from such plants were supplied with asolution that contained sucrose, the accumulation of 8-amylasewas induced in both leaf and petiole portions of the explants. Thesucrose-induced accumulation of fl-amylase in leaf-petiole cut-tings occurred concomitant with the accumulation of starch andof sporamin, the most abundant storage protein of the tuberousroot. The accumulation of fl-amylase, of sporamin and of starchin the petioles showed similar dependence on the concentrationof sucrose, and a 6% solution of sucrose gave the highest levelsof induction when assayed after 7 days of treatment. The induc-tion of mRNAs for f-amylase and sporamin in the petiole couldbe detected after 6 hours of treatment with sucrose, and theaccumulation of j6-amylase and sporamin polypeptides, as wellas that of starch, continued for a further 3 weeks. In addition tosucrose, glucose or fructose, but not mannitol or sorbitol, alsoinduced the accumulation of fl-amylase and sporamin, suggestingthat metabolic effects of sucrose are important in the mechanismof this induction. Treatment of leaf-petiole cuttings with waterunder continuous light, but not in darkness, also caused theaccumulation of small amounts of these components in the pe-tioles, probably as a result of the endogenous supply of sucroseby photosynthesis. These results suggest that the expression ofthe gene for f-amylase is under metabolic control which is cou-pled with the expression of sink function of cells in the sweet-otato.

p-Amylase (a- 1,4-glucan maltohydrolase, EC 3.2.1.2) isfound only in plants and certain species of bacteria. Seeds ofcereals and soybean contain a large amount ofB-amylase, andfl-amylase is also present in vegetative tissues of plants, forexample, in leaves, roots, and cotyledons (27, 28). Although#-amylase activity has been detected in chloroplasts fromspinach leaves (23), several studies with other plants have

'Supported in part by Grants-in-Aid for Scientific Research onPriority Areas ("Totipotency of Plant Cells" and "Metabolite Accu-mulation in Higher Plants") from the Ministry of Education, Scienceand Culture, Japan.

2 Present address: Bioscience Research Laboratories, Mitsui Petro-chemical Industries, Ltd., Waki-cho, Kuga-gun, Yamaguchi-ken 740,Japan.

indicated that most, if not all, ofthe cellular 3-amylase activityis extrachloroplastic (13, 14, 18, 25, 26) and may be localizedin the vacuole (15, 3 1). Precursors to the fl-amylase of barley(1 1) and soybean (19) seeds and a precursor to the subunitsof 3-amylase in sweet potato tuberous roots (30) do notcontain N-terminal transit peptide sequences that play a rolein the transport of proteins into plastids. No f-amylase hasbeen demonstrated to attack native starch granules that havenot already been partially degraded by other enzymes orsolubilized by boiling. Furthermore, varieties of soybean (1,9) and rye (4) that exhibit greatly reduced levels of f3-amylaseactivity in their seeds germinate normally. Thus, in spite ofextensive enzymological studies of its amylolytic activity invitro, the precise physiological role of fl-amylase in plants isnot known at present.Tuberous roots of the sweet potato are unusually rich in 1-

amylase (2), and it represents one of the major proteins of theorgan accounting for about 5% of the total soluble proteins.Only sporamin (17), which accounts for about 60 to 80% ofthe soluble proteins, is present at higher levels in these organs.Physicochemical and enzymological properties of the f3-amy-lase from sweet potato have been studied extensively (forreview, see ref. 28). Although the ,B-amylases from other plantand bacterial sources are monomeric enzymes with mol wt ofabout 50,000 to 60,000, the fl-amylase from sweet potato isunique in that it is a tetramer of identical subunits, each witha mol wt of about 50,000 (28). In addition, the 3-amylasefrom sweet potato has been shown to be identical to the starchphosphorylase-inhibitor protein that has been found in tub-erous roots (22). However, the primary structure of the sub-unit of the f,-amylase from sweet potato, as deduced from thenucleotide sequence of the corresponding cDNA, is not sig-nificantly different from those of the ,3-amylases of barley andsoybean seeds (30).The sweetness of tuberous roots of the sweet potato has

been ascribed to the hydrolysis of starch to maltose by 13-amylase during cooking, and the taste of varieties of sweetpotato that have no or only low #-amylase activities is onlyslightly sweet when tuberous roots of these varieties arecooked (12). #-Amylase cannot be detected by immunologicalmethods in some of the "nonsweet" varieties of sweet potato(K Oba, personal communication), yet these tuberous rootsaccumulate normal amounts of starch and support normalsprouting of the next generation, suggesting that f,-amylase isnot essential for the accumulation and mobilization of starchin the tuberous root.

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SUCROSE INDUCES SWEET POTATO #-AMYLASE

In this report, we describe our finding that, while fl-amylasepolypeptides are not detectable or are present in only smallamounts, in organs other than the tuberous roots of normal,field-grown sweet potato plants, the accumulation of a largeamount of fl-amylase occurs concomitant with the accumu-lation of starch and sporamin in leaf-petiole cuttings after theexogenous supply of sucrose or other metabolizable sugars.These observations suggest that the expression of the gene forfl-amylase is under metabolic control which is coupled withthe expression of storage function of the cells in the sweetpotato.

MATERIALS AND METHODS

Plant Materials

Sweet potatoes (Ipomoea batatas Lam. var Kokei No. 14)were grown at the Nagoya University Experimental Farm.Petioles from plants with the intact leaf still attached were cutwith a sharp razor blade. Five to ten of these leaf-petiolecuttings were combined and dipped in a solution of sucrosein water at the cut edge of the petioles, and they were thenincubated under continuous light, unless otherwise indicated,at 25TC. Samples were either analyzed immediately or frozenin liquid nitrogen and stored at -80'C.

Extraction of Proteins and Analysis of fl-Amylase andSporamin

Proteins were extracted from tissues (7) and the proteincontent of each extract was determined, after precipitationwith 10% TCA, by the method of Lowry et al. (16) withbovine serum albumin as a standard. SDS-PAGE and im-munoblotting with sporamin-specific or f3-amylase-specificantisera were carried out as described previously (7). Quanti-tation of the subunit of fl-amylase in the extract was achievedby analysis of densitometric tracings of the immunoblots andamounts of sporamin were estimated by a dot-blot immu-noassay. For activity staining of j3-amylase, proteins in theextract were separated on 7% polyacrylamide gel containing0.2% soluble starch. After electrophoresis, the gel was soakedin 0.2% soluble starch in 0.1 M sodium phosphate (pH 6.5) at370C for 1 h, stained for starch in 10 mM 12-14 mM KI in 1%acetic acid, and postfixed in 5% TCA-50% ethanol.

Determination of Starch

Tissues were pulverized in liquid nitrogen with the aid of amortar and pestle, and the powder was suspended in 3 vol-umes (v/w) of ice-cold 10 mM Hepes-NaOH (pH 7.0). Aftergrinding with a mortar and pestle, the homogenate was cen-trifuged at 20,000g for 15 min at 4°C. To remove solublecarbohydrates, the resultant precipitate was washed two timeswith cold 80% methanol and resuspended in water. Theamount of starch was determined, after solubilization byheating in a boiling water bath for 2 h, with a starch-deter-mination kit from Boehringer Mannheim.

Isolation and Analysis of RNAs

Total RNA was prepared from the tissue as describedpreviously (6). RNA samples were denatured with formalde-

hyde and formamide, and then they were subjected to electro-phoresis on 1.2% agarose gels that contained formaldehyde.Gels were then blotted onto Zeta probe nylon membranes(Bio-Rad). The membranes were hybridized with a 32P-labeledcDNA probe and washed essentially as described previously(6). The cDNA inserts from the following plasmids were usedfor hybridization: pIMO23 (8), coding for A-type sporamin;pSPBA6, coding for the subunit of f3-amylase from sweetpotato (30); pF1S'A 1, coding for the 5'-subunit of mitochon-drial FIATPase from sweet potato (unpublished results). Afterwashing, membranes were either exposed to x-ray film (KodakX-Omat) for autoradiography or each bands was subjected toa determination of radioactivity by a radioanalytical imagingsystem (Ambis, San Diego).

RESULTS

Localization of j8-Amylase in the Sweet Potato

Proteins were extracted from leaves, petioles, stems, tuber-ous roots, and nontuberous roots of sweet potato plants. Asshown in Figure 1 (top), the extract from the tuberous rootcontained several major proteins that cannot be seen inextracts of the other organs. The most heavily stained band,corresponding to a protein with an apparent mol wt of25,000,represents sporamin (17). A protein with an apparent mol wtof 48,000 corresponds to the subunit of f,-amylase. Immu-noblot analysis of proteins from these organs showed thatpolypeptides that cross-reacted with g-amylase-specific anti-serum were detectable only in the extract from the tuberousroot (Fig. 1, center). However, some preparations of extractsfrom nontuberous roots, stems and leaves contained verysmall amounts of p-amylase that was detectable by immuno-blotting (data not shown).

f3-Amylase in the extracts could be detected with greatersensitivity by separating proteins on nondenaturing polyacryl-amide gels that contained soluble starch, with a subsequentassay for amylolytic activity in situ (activity staining; Fig. 1,bottom). The extract from the tuberous root showed a broadband of strong amylolytic activity around the region thatcorresponded to proteins with apparent mol wt of about200,000. This band of amylolytic activity corresponds to theactivity of f,-amylase since the ,B-amylase purified from tub-erous roots migrated to the same position and this band ofamylolytic activity was absent in the extract from tuberousroots of the f3-amylase-null varieties of the sweet potato (datanot shown). By contrast, only very weak amylolytic activitywas detected in this region of the gel in extracts from otherorgans examined. These results indicate that in the normal,field-grown sweet potato plants, fl-amylase is present in sig-nificant amounts only in tuberous roots, as is the case forsporamin (17). However, we have not examined the presenceof f3-amylase in other organs of the sweet potato such asflowers and seeds where relatively high f3-amylase activitiesare present in some plant species (25, 27).

Induction of Accumulation of fi-Amylase by SucroseOccurs Concomitant with the Accumulation of Starch andSporamin in Leaf-Petiole CuttingsWe showed previously that the accumulation of sporamin

can be induced in leaves and petioles when leaf-petiole cut-

903

Plant Physiol. Vol. 96, 1991

from petioles treated with 6% sucrose. The amount of thesubunit of fl-amylase in each extract was estimated by densi-tometric tracing of the immunoblot of the gel, and theamounts relative to the value obtained with extracts ofpetiolestreated with 6% sucrose were plotted (Fig. 2). The relativeamount of sporamin in the same extracts was also determinedby dot-blot immunoassay of the extract with sporamin-spe-cific antiserum. The greatest accumulation of sporamin wasalso obtained with 6% sucrose (Fig. 2).We determined the amount of starch in the petiole samples

used for the extraction ofproteins. The accumulation ofstarchwas also induced by sucrose, and the dependence of theaccumulation on the concentration ofsucrose was also similarto that in the case of f3-amylase and sporamin (Fig. 2). Theaccumulation of these components was also observed in theleaf parts of the sucrose-treated leaf-petiole cuttings (data notshown; for sporamin, see ref. 6). These results indicate thatall of the major biochemical markers of the tuberous root,namely, starch, sporamin, and f3-amylase, can be induced toaccumulate in leaf-petiole cuttings by an exogenous supply ofsucrose, with similar responses to variations in the concentra-tion of sucrose.

Kinetics of Accumulation of fl-Amylase, Sporamin, andStarch in Petioles after Treatment with Sucrose

The kinetics of the accumulation of fl-amylase, sporamin,and starch after treatment of leaf-petiole cuttings with various

1.00

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tings from field-grown sweet potato plants are supplied witha 3% solution of sucrose (6). In addition to the accumulationof sporamin, qualitative and quantitative changes in the poly-peptide composition of leaf and petiole tissues were observedafter such treatment with sucrose (6). To examine whetherthe accumulation of fl-amylase is also induced by sucrose,leaf-petiole cuttings from field-grown plants were treated withvarious concentrations of sucrose for 7 d under continuouslight. Extracts were prepared from the petiole portions ofmore than six cuttings and analyzed for fl-amylase protein byimmunoblotting of SDS-polyacrylamide gels with j3-amylase-specific antiserum.A band corresponding to the subunit of fl-amylase was

detected in extracts of petioles treated with sucrose, and theamount of cross-reactive material was highest, when compar-isons were based on equal amounts of proteins, in the extract

0

Sucrose (%)Figure 2. Dependence of the accumulation of fl-amylase, sporaminand starch in the petiole portions of leaf-petiole cuttings on theconcentration of sucrose. Leaf-petiole cuttings were treated with asolution of sucrose at various concentrations for 7 d under continuouslight. Proteins were extracted from the petiole portions of the cuttingsand the relative amounts of fl-amylase (0) and sporamin (0) polypep-tides were determined. The relative amount of starch (El) was alsodetermined with a portion of the same petioles as were used for theextraction of proteins. For f3-amylase and sporamin, the relativeamounts on the basis of equal amounts of proteins are plotted withthe value obtained with an extract of petioles treated with 6% sucrosetaken as 1. For starch, the relative amounts on the basis of equalfresh weights of tissue are plotted.

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904 NAKAMURA ET AL.

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SporaminFigure 3. Time course of accumulation of ,3-amylase, sporamin and starch in the petiole por-tions of leaf-petiole cuttings after treatment withvarious concentrations of sucrose. Petiole por-tions of the cuttings were pulverized in liquidnitrogen and used for the extraction of proteinsand the extraction of starch. The relative amountof fl-amylase was determined by densitometrictracing of the immunoblot of an SDS-gel, andthe relative amount of sporamin was determinedby a dot-blot immunoassay. The amount ofstarch was expressed as mg of starch per g offresh tissue.

Days Days

concentrations of sucrose were compared. The response ofleaf-petiole cuttings to various concentrations of sucrose withrespect to the kinetics and the extent ofaccumulation of thesecomponents fluctuated slightly from experiment to experi-ment, probably reflecting differences in the age and physio-logical condition of the explants. Figure 3 shows the result ofone such experiment. Because leaves of cuttings treated withhigh concentrations of sucrose for a long time wilted severely,the experiment with a 9% solution of sucrose was carried outonly once. The accumulation of $-amylase, sporamin, andstarch in petioles treated with a 9% solution of sucrose for upto 7 d was similar to that in petioles treated with a 6% solutionof sucrose (data not shown).The patterns of accumulation of the three components in

the petioles were not identical. In contrast to the accumulationof sporamin and starch, the amount of ,B-amylase accumu-

lated in the petioles seemed to reach a plateau after about 1

week of treatment in most of the experiments. In contrast tothe increase in the amount of sporamin, which required a lagperiod of a few days before significant increase to occur, theincrease in the levels of f3-amylase and starch did not showsuch a significant lag period. The accumulation of smallamounts of each of these three components in petioles oc-

curred when the cuttings were treated with water, and the

3% Sucrose

(Days) 0 1 3 5 7

Sporamin mRNA

P-Amylase mRNA

F1 ATPase

b-Subunit mRNA

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induction obtained with a 1% solution of sucrose was notsignificantly different from that obtained with plain water(Figs. 2 and 3).

Sucrose-Induced Accumulation of fl-Amylase andSporamin mRNAs

The induction of the accumulation of sporamin in petiolesafter treatment with sucrose occurs at the level ofmRNA (6).Total RNAs were prepared from the petiole portions of leaf-petiole cuttings treated with a 3% solution of sucrose or withwater for 0, 1, 3, 5, and 7 d and analyzed for the levels of fi-

amylase and sporamin mRNAs by Northern blot hybridiza-tion. Unlike the case for sporamin mRNAs, we often detectedsmall amounts of 3-amylase mRNA in the RNA preparedfrom fresh, untreated petioles (Fig. 4). However, treatment ofleaf-petiole cuttings with sucrose caused a dramatic increasein mRNAs for both f3-amylase and sporamin. Accumulationof lower levels of f3-amylase and sporamin mRNAs occurredin petioles treated with water. By contrast, neither electropho-retic patterns of RNAs after staining with ethidium bromide(data not shown) nor the level of mRNA for the nuclear-encoded V'-subunit of mitochondrial FATPase (Fig. 4) dif-fered significantly among these preparations of RNA.

H20

O13 5 7 Figure 4. Analysis by Northern blot hybridiza-tion of mRNAs for sporamin and 4-amylase inpetiole portions of leaf-petiole cuttings. The gelswere loaded with 20 jg each of total RNA fromthe petiole portions of leaf-petiole cuttings thathad been treated with a 3% solution of sucroseor with water for 0,1 3 5, and 7 d undercontinuous light. Filters were hybridized withcDNA probes specific for sporamin, 13-amylaseand FATPase b'-subunit. Exposure of filterswas continued for 25 h for sporamin mRNA, 45h for d-amylase mRNA, and 6 d for FjATPase(5i-subunit mRNA.

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Radioactivity of32P-labeled probes hybridized in each band,shown in Figure 4, was determined and the relative radioac-tivity of individual bands was plotted (Fig. 5; top panels).Lower panels of Figure 5 shows the results of a similarexperiment with leaf-petiole cuttings treated with a 6% solu-tion of sucrose for a shorter period. Both of the #-amylaseand sporamin mRNAs could be detected as early as 6 h afterthe dipping of explants in the solution of sucrose, and theaccumulation of fl-amylase mRNA seemed to cease earlierthan that of sporamin mRNAs.

Effects of Various Sugars on Induction

Leaf-petiole cuttings were treated with 3% or 6% solutionsof various sugars for 4 d, and the accumulation of ,3-amylasein the petiole portions of the explants was analyzed by im-munoblotting of SDS-polyacrylamide gels and by activitystaining of nondenaturing polyacrylamide gels that containedsoluble starch (Fig. 6). The accumulation of f3-amylase was

Sporamin mRNA P-Amylas

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induced by treatment with 3% and 6% solutions of glucoseor fructose. Mannitol did not cause an accumulation of fi-amylase and sporamin, and it seemed actually to repress theaccumulation ofthe small amount off3-amylase and sporaminthat normally occurred during treatment with water alone.Sorbitol also did not cause any significant accumulation of fl-amylase or sporamin above the accumulation observed withwater. Minor amylolytic activities in the petioles other thanthe fl-amylase that were detected in activity-staining gels werenot affected significantly by treatment of petioles with sucrose(data not shown).

Effect of Light on the Accumulation of .8-AmylaseThe sucrose-induced accumulation of f3-amylase, sporamin

and starch occurred in the absence of light (Fig. 7). Althoughtreatment ofleaf-petiole cuttings with water under continuouslight induced the accumulation of small amouts of f3-amylase,sporamin and starch (Fig. 3), the accumulation of these

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Figure 5. Changes in steady-state levels ofmRNAs for sporamin and f3-amylase in petioleportions of leaf-petiole cuttings after treatmentwith sucrose. Steady-state levels of mRNAs forsporamin and ,B-amylase were estimated by thedetermination of radioactivity of 32P-labeledprobes hybridized after Northern blot hybridiza-tion of total RNA isolated from the tissue. Therelative radioactivities on the basis of equalamounts of RNAs are plotted with the valuewhich gave the highest level of radioactivitytaken as 1. The top two panels show changesin the levels of mRNAs for sporamin and f-

amylase in petioles after treatment with a 3%solution of sucrose (see Fig. 4), while the bottomtwo panels show those in petioles after treat-ment with a 6% solution of sucrose.

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906 NAKAMURA ET AL.

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SUCROSE INDUCES SWEET POTATO j3-AMYLASE

Days

Sugar

P-Amylase

Sporamin

0 4 Figure 6. Induction of the accumulation of spor-

Su G F M SO amin and d-amylase in petiole portions of leaf-=G F M So

petiole cuttings after treatment with various sug-0 3 6 3 6 3 6 3 6 3 6 - ars. Leaf-petiole cuttings were treated with so-

lutions of various sugars and incubated undercontinuous light for 4 d. Su, sucrose; G, glucose;F, fructose; M, mannitol; So, sorbitol. Concentra-tions of the sugars are expressed as percent-ages (w/v). Molar concentrations of the sugarsused

were: 3% sucrose, 87.6 mM; 6% sucrose,I I I l ~~~175 mm; 3% glucose, 166 mm; 6% glucose, 333

mM; 3% fructose, 166 mM; 6% fructose, 333mM; 3% mannitol, 165 mM; 6% mannitol, 329mM; 30/o sorbitol, 165 mM; 6% sorbitol, 329 mM.For the detection of f3-amylase, 30 pg of proteinsor 3 mg of proteins from the petioles were usedfor immunoblotting with 3-amylase-specific anti-serum or for activity staining, respectively. Pro-teins from the tuberous root (TR) were used asa standard, 30 pg for immunoblotting and 0.5 pgfor activity staining. For the detection of spora-min by immunoblotting, 5 pg of proteins from thepetioles and 1 pg of proteins from the tuberoroot were used.

components after treatment with water was greatly reduced ifthe incubations were performed in darkness (data not shown;see Fig. 7 lane H20). These results suggest that the endogenoussupply of sucrose as a result of photosynthesis can induce theaccumulation of small amounts of fl-amylase, sporamin andstarch in petioles, and that the sucrose-dependent inductionof these components does not require light.

N H20 S6 S0, G3_ :Mo| llP-Amylase

DISCUSSION

In contrast to the many studies on seed f3-amylases, only afew studies on the regulation of the amount of fl-amylase invegetative tissues have been reported. Pongratz and Beck (23)reported that f3-amylase activity in chloroplasts of spinachleaves shows a diurnal oscillation, with low activity duringthe day and high activity at night. In cotyledons of mustard,a phytochrome-mediated, light-induced increase in the syn-thesis of extra-chloroplastic f3-amylase was observed (26).Caspar et al. (3) found that the amount of extra-chloroplasticf3-amylase in leaves of Arabidopsis thaliana could be veryhigh in mutants with altered starch metabolism, and theyimplied that f3-amylase may be induced in response to thehigh levels of soluble sugars that accumulate during the pho-toperiod in the starchless mutants.The results presented in this paper indicate that the expres-

sion of the gene for f3-amylase is inducible in leaf-petiolecuttings of sweet potato plants by an exogenous supply ofsucrose. Analysis of the nuclear gene for the subunit of fi-amylase of sweet potato suggests that there may be only onecopy per haploid genome of the gene that can hybridize withthe cDNA (unpublished results). It is likely, therefore, thatthe f-amylase gene whose expression is induced in leaf-petiolecuttings by sucrose is identical to the one that is expressed inthe tuberous root. In the case of sporamin, which is encodedby a multigene family, we showed previously that the sameset of sporamin genes seems to be expressed in tuberous roots

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10 -

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Figure 7. Sucrose-induced accumulation of 3-amylase, sporamin andstarch occurs in darkness. Leaf-petiole cuttings were treated withwater (H20), a 6% solution of sucrose (S,), a 0.5% solution of sucrose(SO 5), or a 3% solution of glucose (G3) for 4 days in darkness. N,nontreated petioles. The amount of proteins used for activity stainingof 3-amylase and immunoblotting of sporamin were 3 pg and 5 mg,respectively.

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907

F.et I


and in petiole portions of sucrose-treated leaf-petiole cuttings(6).The induction of the accumulation of fl-amylase in leaf-

petiole cuttings by an exogenous supply of sucrose occurredconcomitant with the accumulation of sporamin and starch,and the accumulation of these three major components ofthetuberous root in the leaf-petiole cuttings showed similar de-pendence on the concentration of sucrose (Fig. 2). Concomi-tant accumulation of f3-amylase and sporamin was also in-duced by glucose or by fructose, but not by mannitol (Fig. 6).fl-Amylase, sporamin and starch are accumulated within thesame parenchymatous cells in the tuberous root offield-grownsweet potato plants (S. Takeda, Y. Kowyama, K. Nakamura,manuscript in preparation). Accumulation of large amountsof sporamin also occurs in stems of sweet potato plantletscultured axenically on sucrose medium (7). The stems oftheseplantlets also contain large amounts of starch and f3-amylaseas well (our unpublished results). Taken together, these resultssuggest that the induction of the accumulation of f3-amylasein leaf-petiole cuttings by sucrose occurs as part ofthe cellularexpression of the vegetative storage functions, as normallyexhibited by parenchymatous cells of the tuberous root. Thefact that variable amounts of sporamin and ,3-amylase canoccasionally be detected in organs other than the tuberousroot in the field-grown plants suggests that, under certainphysiological conditions, the temporal and simultaneous ac-cumulation of sporamin, fl-amylase, and starch occurs incertain cells of restricted area. Transgenic tobacco plants witha sporamin promoter-,B-glucuronidase fusion gene express ,B-glucuronidase activity in cells that presumably function as atemporary sink (20).A similar phenomenon to that discussed herein has been

reported previously in potato plants by Paiva et al. (21), whoobserved that the accumulation of large amounts of the tuberstorage protein, patatin, could be induced in stems and pe-tioles of single-leaf stem cuttings concomitant with the accu-mulation of large amounts of other major tuber proteins, aswell as starch. Results from several laboratories indicate thatthe expression of class I patatin genes of potato is also regu-lated by sucrose and that induction of these genes by sucroseaccompanies the accumulation of starch (10, 24, 29). How-ever, the concentrations of sucrose required to induce theaccumulation ofpatatin in potato plants and that off,-amylaseand sporamin in sweet potato plants are different. The maxi-mum accumulation ofpatatin in leaf sections of potato plantsrequires sucrose at a concentration of 10% (10, 29), which issignificantly higher than the concentration of sucrose thatresults in the maximum accumulation of,3-amylase, sporaminand starch in the explants of sweet potato (Fig. 2).The physiological role of the fl-amylase that accumulates

in the temporary and storage sink cells is not known. Theprecursor to the subunit of j3-amylase in sweet potato doesnot contain a presequence at its N terminus (30), suggestingthat j3-amylase is localized outside of the plastids as are the fi-amylases in vegetative tissues of other plants (13-15, 26, 30).Furthermore, ,B-amylase is unable to attack native starchgranules without their prior digestion by other enzymes orsolubilization by boiling (15, 27). Therefore, it is unlikely that3-amylase plays an essential role in the metabolism of tran-sitorily accumulated and reserve starch within the plastid.

Extrachloroplastic malto-dextrins have been suggested to bethe substrates ofB-amylase in vivo (15). However, the presenceof such a-glucans needs to be established (3, 27), and theamount of j3-amylase that is synthesized seems to be morethan sufficient for such a role. As an alternative explanation,Giese and Hejgaard (5) suggested that f3-amylase may have anitrogen-storage function in barley seeds, since the profile ofaccumulation of ,B-amylase in developing seed resembles tothat of the storage protein, hordein, and the synthesis of 3-amylase responds to increased supply of nitrogen. Although,B-amylase in the sweet potato may also participate in thetemporary and long-term storage of nitrogen, this possibilitydoes not rule out a possible role for ,B-amylase in the metab-olism of glucans. Further analysis of the regulation of expres-sion of the gene for f3-amylase in the sweet potato should shedlight on the physiological role of this enzyme.

ACKNOWLEDGMENTS

We thank Mr. Tomiji Izuhara and Dr. Shigekata Yoshida ofNagoya University Experimental Farm for growing the sweet potatoplants, and Dr. Yasunori Nakamura of the National Institute ofAgrobiological Resources for instructions on the determination ofstarch.

LITERATURE CITED

1. Adams CA, Broman TH, Rinne RW (1981) Starch metabolismin developing and germinating soya bean seeds is independentof f3-amylase activity. Ann Bot 48: 433-439

2. Balls AK, Walden MK, Thompson RR (1948) A crystalline f3-amylase from sweet potatoes. J Biol Chem 173: 9-19

3. Caspar T, Lin TP, Monroe J, Bernhard W, Spilatro SR, PreissJ, Sommerville C (1989) Altered regulation of fl-amylase activ-ity in mutants ofArabidopsis with lesions in starch metabolism.Proc Natl Acad Sci USA 86: 5830-5833

4. Daussant J, Zbaszyniak B, Sadowski J, Wiatroszak I (1981)Cereal ,B-amylase: immunochemical study on two enzyme-deficient inbred lines of rye. Planta 151: 176-179

5. Giese H, Hejgaard J (1984) Synthesis of salt-soluble proteins inbarley. Pulse-labeling study of grain filling in liquid- cultureddetached spikes. Planta 161: 172-177

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