Plant Cell Physiol.

41(11): 12351242 (2000)

JSPP 2000

Presence of Multiple cDNAs Encoding an Isoform of ADP-Glucose Pyrophosphorylase Large Subunit from Sweet Potato and Characterization of Expression Levels
Chee Hark Harn 1, 3, Jung Myung Bae 2, 4, Sang Sook Lee 2, Sung Ran Min 2 and Jang Ryol Liu 2, 5
1

Biotechnology Center, Nong Woo Bio, Ganam-myun, Yeoju-kun, Kyonggi-do, 469-880, Korea
Plant Cell and Molecular Biology Research Unit, Korea Research Institute of Bioscience and Biotechnology, P.O. Box 115, Yusong, Taejon, 305600, Korea
2

Key words: ADP-glucose pyrophosphorylase large subunit

AGPase activity Sweet potato Starch biosynthesis.

Introduction
ADP-glucose pyrophosphorylase (AGPase) plays a central role in starch biosynthesis in both photosynthetic and nonphotosynthetic plant tissues as a regulatory enzyme. It catalyzes the first step of starch biosynthesis generating ADP-glucose
3
4
5

and pyrophosphate from glucose 1-phosphate and ATP. ADPglucose functions as a glucosyl donor for =-glucan synthesis by
starch synthase. This enzyme is also present in bacteria for synthesis of glycogen. The catalytic activity of AGPase is allosterically regulated by small effector molecules whose levels control the rate of starch biosynthesis (Preiss 1982). Plant AGPase
is activated by 3-PGA and inhibited by Pi while the AGPase is
activated by fructose-1,6-bisphosphate and inhibited by AMP
in E. coli. However, barley endosperm AGPase has been reported to be relatively insensitive to 3-PGA/Pi regulation
(Kleczkowski et al. 1993) indicating some degree of heterogeneity among the plant enzymes.
Bacterial AGPase is expressed by a single gene forming a
homotetramer with a subunit mass of 48 kDa. By contrast,
plant AGPase is characterized as a heterotetramer composed of
two large subunits and two small subunits which are encoded
by different genes (Preiss 1991). Depending on the plant species, the small subunits range from 50 to 56 kDa, whereas the
large subunits range from 51 to 60 kDa in size. cDNA clones
encoding small and large subunits (SS and LS) have been isolated from several plants (corn SS: Bae et al. 1990; corn LS:
Bhave et al. 1990, Giroux et al. 1995; potato LS: Mller-Rber
et al. 1990, Nakata et al. 1991, La Cognata et al. 1995; potato
SS: Mller-Rber et al. 1990, Nakata et al. 1991; sugar beet SS
and LS: Mller-Rber et al. 1995; wheat LS: Olive et al. 1989,
Villand et al. 1992a; barley SS and LS: Villand et al. 1992b;
Arabidopsis SS and LS: Villand et al. 1993; bean SS: Weber et
al. 1995; barley LS: Elimert et al. 1997; sweet potato SS: Bae
and Liu 1997; tomato LS: Park and Chung 1998; oriental
melon LS and SS: Park et al. 1998). Analysis of the deduced
amino acid sequence identity among AGPases has revealed that
the small subunits are highly conserved while the large subunits are relatively divergent.
Sweet potato is the sixth largest agricultural crop in the
world, and its starch is used both as a food and as an industrial
resource. However, the biochemical and molecular aspects of
the biosynthetic machinery of starch formation have not been
well studied for the tuberous root of sweet potato. Herein, we
characterized one full (iAGPLI-1) and two partial cDNAs
(iAGPLI-2, 3) encoding an AGPase large subunit (AGPL) from

Corresponding author: E-mail, chharn@nongwoobio.co.kr; Fax, +82-31-884-7065; Phone, +82-31-883-7055.

Present address: Graduate School of Biotechnology, Korea University, Seoul, 136-701, Korea.
To whom the authorship of this paper belongs.
1235

Downloaded from http://pcp.oxfordjournals.org/ at Korea Research Institute of Bioscience & Biotechnology on June 6, 2015

Three cDNAs (iAGPLI-1, iAGPLI-2, and iAGPLI-3) encoding an isoform of AGPase large subunit were isolated
from a sweet potato cDNA library constructed from tuberous root tissue. iAGPLI-1 was 2,161 bp in length and contained an open reading frame of 517 amino acids with a calculated molecular mass of 57,689 Da. iAGPLI-2 and
iAGPLI-3 were 1,804 and 1,524 bp in length, respectively,
and contained partial open reading frames of 490 and 385
amino acids. Deduced amino acid sequence comparison
analysis showed that iAGPLI-1 has sequence identity with
iAGPLI-2 (97.9) and iAGPLI-3 (98.7%) while iAGPLI-2
and iAGPLI-3 have 96.8% sequence identity. iAGPLI-1
had the highest sequence identity of 77.8% with potato AGPase (sAGPL1). Steady-state levels of iAGPLI-1 transcripts
were expressed predominantly in the stem, and moderately
in the tuberous root, but not in either the roots or leaves.
However, AGPase activity was present in all tissues. The expression level in the stem declined dramatically after a 12 h
incubation in the dark to nearly 3% of the value under
light, although the activity under a dark condition remained at half the levels in light. The activity levels were
not correlated with the transcript levels. iAGPL transcripts
in leaves were induced by sucrose feeding but not by glucose or fructose. Therefore, the expression of iAGPLI-1 is
regulated in stem tissue preferentially and by sucrose.
Southern blot analysis showed that the sweet potato genome contained several copies of iAGPLI gene probably
due to polyploidy.

1236

cDNAs encoding an AGPase large subunit

sweet potato.

Materials and Methods

Screening library and DNA sequencing

A  lox cDNA library constructed from poly (A)+ RNA of tuberous root was screened using a 32P-labeled (1106 d.p.m. ml1) heterologous probe of a potato clone (sAGPL1, originally called AGPS, a courtesy of Dr. Bernd Mller-Rber). Plaque filters were hybridized in a
solution containing 6SSC, 5Denharts, 0.1% SDS and 50 g ml1
calf thymus DNA for 16 h at 60
C and washed with 1 SSC0.1%
SDS several times. The filters were exposed to X-ray film at 70
C
prior to autoradiography. Three clones (iAGPLI-1, iAGPLI-2 and iAGPLI-3) were subcloned into KpnI site of Bluescript vector and sequenced by the dideoxynucleotide chain termination method using Sequenase II (USB, Cleveland, OH, U.S.A.). All sequencing reactions
were performed with custom-synthesized oligonucleotide primers. To
obtain correct sequences, overlapping subclones were sequenced on
both strands and subjected to automated sequencing. Sequence identities were analyzed by MacDNASIS (Hitachi Software Engineering
America) and the BLAST network service (National Center for Biotechnology Information).
Assay of AGPase activity
Sweet potato tissues (0.3 g) were ground in a mortar and a pestle
in 80 mM HEPES buffer (pH 7.4) containing 10 mM DTT. The sample extracts were precipitated at 10,000g for 10 min and the supernatant was saved for activity measurement. The pyrophosphorylase of
ADP-Glc was coupled with phosphoglucomutase and Glc-6-P dehydrogenase and assayed by measuring NADH production at 340 nm and
37
C (Plaxton and Preiss 1987). A standard reaction mixture (1 ml)
contained 80 M of HEPES buffer (pH 7.4), 5 M of MgCl2, 10 M
of 3-phosphoglycerate, 0.2 mg of BSA, 1 M of ADP-Glc, 1 M of
sodium PPi, 0.6 M of NAD+, 10 M of Glc-1,6-bisphosphate, and 2
units each of rabbit muscle phosphoglucomutase and Leuconostoc mesenteroides Glc-6-P dehydrogenase. Assays were initiated by addition
of PPi. The AGPase activity showed linearity to both time and amount
of enzyme extract added. One unit of activity was defined as the
amount of enzyme required to produce 1 mol Glc-1-P min1 at 37
C.
RNA gel blot analysis
Total RNA was isolated from leaf, stem, root, and tuber tissues
by the method originally described by Chomczynski and Sacchi (1987)
with modification. Thirty g of total RNA was denatured and loaded
onto 1% agarose gel containing 1MOPS buffer and 0.66 M formaldehyde. After electrophoresis at 80 V for 2 h the gel was transferred to
a nylon membrane in blotting buffer (10SSC). The membranes were
hybridized with 32P-labeled (5106 d.p.m. ml1) probe of a 633 bp PCR
product. The primers used were 5-TTGGATTCTCACTTCTGGGTA3 at position 124 bp (sense) and 5-GCCAGCACCTCAACAAAT-3

Genomic DNA blot analysis

Thirty g of genomic DNA isolated from sweet potato tuber by
CsCl gradient ultracentrifugation (Sambrook et al. 1989) was digested
with HindIII and EcoRI. Neither of the restriction enzyme sites are
present within the cloned cDNA sequence. Restriction fragments were
separated on 0.8% agarose gel at 80 V for 6 h. The gel was soaked for
20 min in a denaturation solution (1.5 M NaCl and 0.5 M NaOH) and
neutralized for 40 min in a neutralization solution (1.5 M NaCl and
0.5 M Tris-HCl, pH 8.0). DNA was then blotted onto nylon membranes in 10 SSC and the membranes were hybridized under the
same conditions as used for RNA gel blot analysis (above). PCR fragments of 218 bp at 3 terminal end of iAGPLI-1 was used as a probe.
The primers used were 5-AAGTTTACCTGGACTTGTGA-3 at position 1,921 bp (sense) and 5-GAACTTTTTACCACCATAGCC-3 at
position 2,139 bp (antisense) of iAGPLI-1. This nontranslated region
(1,9212,139) shares partial homology with iAGPLI-2, 3 but not with
other AGPLs.

Results
Characterization of sweet potato AGPase cDNAs
Approximately 300,000 recombinant phages were
screened by using a 32P-labeled sAGPL1 cDNA. Three clones,
named iAGPLI-1 (accession number: AF068260), iAGPLI-2
(AJ249256) and iAGPLI-3 (AJ249257), were isolated. iAGPLI1 was the longest cDNA (2,161 bp in length) and contained an
open reading frame of 1,551 bp flanked by 5 and 3 untranslated regions of 250 and 360 bp, respectively. The open reading
frame began with an ATG codon at position 251 bp and ended
with a TAG termination codon at position 1,802 bp. It predicted a polypeptide of 517 amino acids with a calculated molecular mass of 57,689 Da. The N-terminus contained a transit peptide of 56 amino acids followed by a putative cleavage site of
SVLT at amino acid position 57. The polyadenylation region of
AATAAA is located at 2,155 bp. iAGPLI-2 and iAGPLI-3 were
1,804 and 1,524 bp in length, respectively, and contained open
reading frames of 490 and 385 amino acids. iAGPLI-1, 2, and 3
shared DNA base pair identity of ca. 95% (data not shown)
with the most divergence at the 3 terminal end. iAGPLI-2 and
3 were 330 and 646 bp shorter, respectively, than iAGPLI-1
(data not shown). Attempts to find 5 termini for the full length
of iAGPLI-2 and 3 were not successful.
The deduced amino acid sequence of three forms contained highly conserved Lys residues, which are allosteric activator binding sites for the large subunit of spinach AGPase
(Morell et al. 1988). iAGPLI-1 exhibited Lys residues at 180,
470, and 508 amino acids. iAGPLI-2 exhibited these residues
at 153, 443, and 481 while in iAGPLI-3 they were located at

Downloaded from http://pcp.oxfordjournals.org/ at Korea Research Institute of Bioscience & Biotechnology on June 6, 2015

Plant materials
Sweet potato (Ipomoea batatas cv. White Star) plants were raised
in a greenhouse. Leaves, stems, roots and tuberous roots were harvested from mature plants at the same time. Stems for developmental analysis were grouped into three stages based on size: 1 mm or less, 1
2 mm, and more than 2 mm in diameter. For dark treatment, stems
with attached leaves from several plants were cut, submerged in water, and kept in the dark at 25
C for 12 h. For light treatment, the stems
were kept under light at 400500 Em2 s1 at 25
C for 12 h. For sugar treatment, petioles with leaves were harvested from several plants
after 12 h in the dark in water and incubated in a 3% sucrose, glucose
or fructose solution for 12 h under light at 25
C.

at position 756 bp (antisense) of iAGPLI-1. The first 400 bp at 5 end

of the PCR product is specific only to iAGPLI and the rest shares some
nucleotide homology with other AGPLs. Hybridization was performed
in a solution containing 5Denhardts solution, 0.1% SDS, 100 g ml1
salmon sperm DNA and 5SSC for 20 h at 60
C. After hybridization,
the blots were washed three times in 1SSC0.1% SDS at 60
C prior
to exposure to X-ray film at 70
C. As a control to show equal loading, the same membrane was hybridized with 25S rDNA of petunia as
a probe.

cDNAs encoding an AGPase large subunit

1237

Table 1 Amino acid sequence identity (%) of AGPase large subunits calculated by MacDNASIS program (Hitachi Software
Engineering America) and the BLAST network service (National Center for Biotechnology Information)
bAGPL hAGPL sAGPL1 sAGPL2 sAGPL3 zAGPL iAGPLI-1 lAGPL1 1AGPL2 1AGPL3 cAGPL1 cAGPL2
12.3

12.0
47.8

9.5
63.8
57.6

12.0
63.9
52.0
62.5

12.0
55.2
43.0
53.4
49.4

10.6
44.3
75.4
53.1
48.1
38.3

11.1
57.0
52.9
77.8
55.1
50.5
49.5

9.3
57.0
52.0
97.8
57.2
51.9
48.3
72.7

12.3
67.5
50.9
65.3
92.8
53.0
48.0
57.0
59.6

13.3
54.4
42.0
56.5
49.2
92.5
36.6
49.8
51.1
51.7

9.7
54.9
48.0
69.1
56.4
47.6
44.9
62.2
63.1
57.7
45.9

11.6
63.1
48.4
63.4
60.6
52.5
45.5
56.4
58.3
63.7
51.9
53.8

b, sugar beet (Mller-Rber et al. 1995, X78900); c, oriental melon (Park et al. 1998, cAGPL1: AF030383; cAGPL2: AF030384); h, barley
(Elimert et al. 1997, U66876); l, tomato (Park and Chung 1998, lAGPL1: U88089; lAGPL2: U85496; lAGPL3: U85497); s, potato (La Cognata et
al. 1995, sAGPL2: P55242; sAGPL3: P55243; Nakata et al. 1991, Q00081); z, maize (Giroux et al. 1995, z38111); i, sweet potato (this work).

48, 338, and 376. These clones also contained aspartic acid 413
(D413) (469 for iAGPLI-1, 442 for iAGPLI-2, 337 for iAGPLI3), a putative binding site of 3-PGA found in potato AGPase
(Greene et al. 1996). They also contained an allosteric activator site of SGITIIMEKATI at positions 500, 473, and 368 amino acids, respectively.
Amino acid sequence identity of AGPase large subunits
The three AGPLs showed highly homologous sequence
identity. iAGPLI-1 differed by 10 amino acids from iAGPLI-2
(97.9%) and by 12 amino acids from iAGPLI-3 (96.8%). iAGPLI-2 differed by only five amino acids from iAGPL-3
(98.7%). The deduced amino acid sequences among other AGPLs were aligned with iAGPLI-1 and sequence identity calculated (Table 1). The iAGPLI-1 of sweet potato shared sequence identity of 72.7 and 77.8% with tomato lAGPL1 and
potato sAGPL1, respectively, and 4962% with other AGPLs.
The amino acid sequences of potato and tomato shared the
highest sequence identity (92.597.8%).
Amino acid sequence homology between large and small
subunits in sweet potato
Figure 2 shows the amino acid sequence homology between large and small subunits of sweet potato AGPase. The
small subunits (iAGPS1 and iAGPS2, originally named sTL1
and sTL2, Bae and Liu 1997) shared 90.6% sequence identity
whereas the large subunit of iAGPLI-1 exhibited 30.9% and
28.8% homology with iAGPS1 and iAGPS2, respectively. Divergency appears along the sequences between large and small
subunits, but the N-terminal region shows the least homology.
However, several conserved amino acid motifs are present in
large subunits thus far characterized, and in two small subunits

of sweet potato, as well as E. coli AGPase. The motifs are: ILGG (at amino acid position 89 of iAGPLI-1); A_PAV (106);
SNC_NC (125); TADAV (184); F_EKP (263); A__G_YV
(296); YW_D_GT (344); AIIDKN_RIG (366).
Northern blot analysis
RNA gel blot analysis was performed using total RNA extracted from the leaf, stem, root and tuberous root. iAGPLI-1
transcript was detected predominantly in the stem and only
moderately in the tuber but not in either the root or leaf (Fig.
3A). The hybridization with probes obtained from both iAGPLI-2 and iAGPLI-3 generated the same results (data not
shown). Transcripts levels at different developmental stages of
the stem were also investigated (Fig. 3B). At an early stage of
development (S1) the transcript level was approximately 18
and 30% of the values at stages S2 and S3, respectively.
The iAGPLI-1 gene was not expressed in the leaf incubated
under light for 12 h (Fig. 4A). Further light irradiation for different times (6, 18 and 24 h) in a growth chamber or different
harvesting times after sunrise in the greenhouse did not affect
the expression level (data not shown). However, the transcripts
were produced in the stem after incubation under light for 12 h.
The majority of transcripts in the stem disappeared during the
12 h incubation in the dark.
Leaf petioles were treated with different sugars at 3% concentration after the 12 h incubation in the dark to examine if
iAGPLI-1 is specifically inducible by sucrose. iAGPLI-1 gene
expression was induced only by sucrose (Fig. 4B). Glucose and
fructose did not induce iAGPLI-1 gene expression (Fig. 4C).
Southern blot analysis
Genomic DNA was digested with HindIII and EcoRI.

Downloaded from http://pcp.oxfordjournals.org/ at Korea Research Institute of Bioscience & Biotechnology on June 6, 2015

eAGP
bAGPL
hAGPL
sAGPL1
sAGPL2
sAGPL3
zAGPL
iAGPLI-1
lAGPL1
lAGPL2
lAGPL3
cAGPL1

1238

cDNAs encoding an AGPase large subunit

Both yielded several fragments detected by 32P-labeled

(5106 d.p.m. ml1) probe of PCR product which is present at
the nontranslated region of 3 terminal end (Fig. 5). This restriction pattern suggested that the sweet potato genome contains several copies of iAGPLI gene.
AGPase activity assay
In order to detect changes in enzyme activity of AGPase
among tissues, the activity was measured by coupling phosphoglucomutase and glucose-6-P dehydrogenase. The in vitro
activity of AGPase was the highest in tuberous root
[455.4 nmol min1 (mg protein) 1]. It was three times higher

than the activity in the root [155.4 nmol min1 (mg protein) 1]
(Fig. 6). Considerable activity was also present in other tissues. The magnitude of difference in activity was authentic because the basal activity (measured without extracted sample,
data not shown) was 43 nmol min1 (mg protein) 1. Interestingly, the activity levels did not correlate with the transcript
levels. Although the iAGLI-1 gene was expressed predominantly in the stem, the activity level of the same tissue accounted
for only 71% of the activity level in the tuberous root. While
enzyme activities were present in the root, leaf and tuberous
root, the transcripts were not present in the same tissues to the
degree indicated by the enzyme activities.

Downloaded from http://pcp.oxfordjournals.org/ at Korea Research Institute of Bioscience & Biotechnology on June 6, 2015

Fig. 1 Comparison of amino acid sequences of iAGPLI-1 (accession number: AF068260), 2 (AJ249256), and 3 (AJ249257). Dots (.) indicate
identity. Sequence identity was analyzed by MacDNASIS program (Hitachi Software Engineering America) and the BLAST network service
(National Center for Biotechnology Information).

cDNAs encoding an AGPase large subunit

1239

Fig. 2 Amino acid sequence alignment of AGPase large subunit

(iAGPLI-1) and small subunits (iAGPSI: Z79635 and II: Z79636).
Amino acids in white on black are identical.

AGPase activity levels under dark and light conditions

differed significantly. Under light conditions the activities of
AGPase from leaf and stem were 245.2 and 296.1 nmol min
1
(mg protein) 1, respectively (Fig. 7). However, the activities
in the dark were ca. 102.9 and 113.3 nmol min1 (mg protein)
1
, respectively (2.5 and 3 times lower than those under light
conditions). These values were higher than the basal activity of
2.22.1 nmol min1 (mg protein) 1, indicating that the AGPase
was moderately active in leaf and stem even under dark conditions. The activity levels under light and dark conditions did
not correlate with the transcript levels. Although 97% of the
transcript level in stem disappeared in the dark, the activity level decreased to only one third.

Discussion
The AGPLs in dicot plants were distinctly classified based
on the amino acid sequence identity into three large subgroups,
designated as 1, 2 and 3 (Table 2). The sweet potato clone belongs to group 1 because of the highest sequence identity with
potato AGPL1. The sequence identity among potato AGPL iso-

forms 1, 2, and 3 is approximately 5062%. Therefore, AGPL

isoforms in the same species belong to different groups and exhibit much lower identity than tomato and potato AGPL (92
98%) in each group. This indicates that each isoform in the
same species plays a unique role in synthesizing ADP glucose.
Like other plants thus far characterized, sweet potato may have
several distinct isoforms and obviously iAGPLI is one of them.
In contrast with the distinct isoforms, the amino acid sequence
identity between iAGPLI-1, 2, and 3 is 96.898.7%. The nucleotide sequence identity is also almost identical except for 3

Table 2 Distinct groups of AGPase large subunits classified

by dendrogram (MacDNASIS program) based on the amino
acid sequence identity
Group 1

Dicot
Group 2

lAGPL1
sAGPL1
cAGPL1
iAGPLI-1

lAGPL2
sAGPL2
cAGPL2
bAGPL

Group 3
lAGPL3
sAGPL3

Monocot
hAGPL
zAGPL

b, sugar beet; c, oriental melon; h, barley; l, tomato; s, potato; z, maize;

i, sweet potato.

Downloaded from http://pcp.oxfordjournals.org/ at Korea Research Institute of Bioscience & Biotechnology on June 6, 2015

Fig. 3 Northern analysis of iAGPLI-1 gene expression in various tissues (A) and developmental stages of stem (B). Thirty g of total RNA
was loaded in 1% agarose gel and blotted into nylon membrane. The
membranes were hybridized by 32P-labeled (5106 dpm ml1) probes
of a PCR product from iAGPLI-1. L, leaf; S, stem; R, root; T, tuber.
S1, 1 mm or less in diameter; S2, 12 mm; S3, over 2 mm.

1240

cDNAs encoding an AGPase large subunit

Fig. 4 Northern analysis of iAGPLI-1 gene expression in stem under

light and dark conditions (A), and sugars (B and C). The stems with
attached leaves from several plants were cut, submerged in water and
kept in the dark at 25
C for 12 h and in the light (400500 Em2 s1)
at 25
C for 12 h. For sugar treatment, the petioles with leaves were
detached after 12 h in the dark and incubated in 3% sucrose, glucose or
fructose for 12 h under light at 25
C. Thirty g of total RNA was
loaded in 1% agarose gel and blotted into nylon membrane. The membranes were hybridized with 32P-labeled (5106 dpm ml1) probes of a
PCR product relatively specific to iAGPLI-1. LL, leaf/light; LD, leaf/
dark; SL, stem/light; SD, stem/dark; C, control; S, sucrose; G, glucose; F, fructose; M, mannitol.

UTR, suggesting that a single iAGPL gene may have diverged

into iAGPLI-1, 2, and 3. Since sweet potato is hexaploidy (6;
2n = 90), the heterogeneity of the original gene may have occurred during evolution. The small subunits of AGPase from
sweet potato (Bae and Liu 1996) showed a 1% heterogeneic
difference in amino acid sequences, indicating that the sweet

potato genome also contains extra gene copies for each AGP
small subunit gene. Therefore, both large and small subunit
genes consist of isoforms and each isoform is polymorphic in
the genome due to polyploidy. The presence of several copies
of each gene shown in Southern blots (Fig. 5 as well as in the
data of Bae and Liu 1996) supports this idea. Although no evidence has been reported yet, AGPase genes of other polyploid
plants are probably polymorphic in the genome.
Potential binding sites for allosteric effectors in AGPase
have been revealed (Greene et al. 1996, Ball and Preiss 1994).
Aspartic acid 413 (D413), a putative binding site of 3-PGA
which is an allosteric activator of AGPase, is present in iAGPLI-1, 2, and 3 (Fig. 1). This site is also present in the small subunits at 474 (iAGPSI) and 475 (iAGPSII) amino acids. Three
highly conserved Lys residues, as allosteric activator binding
sites of spinach AGPase (Morell et al. 1988), are present in
iAGPLI-1, 2, and 3. The Lys residue at 470 of iAGPLI-1 is preserved in the small subunits neighboring the putative binding
site D. Additionally these polypeptides contain an allosteric
activator site of SGITIIMEKATI which is also present in the
AGPase small subunit as SGIVTVIKDAL, with moderate
sequence identity (Mller-Rber et al. 1990). These potential
binding sites are present in all the AGPLs thus far characterized. Further studies on the substrate binding sites and structure-function analysis will help elucidate the regulation mechanism of AGPase with two distinct large and small subunits.
The iAGPLI-1 gene was expressed predominantly in the
stem and moderately in the tuberous root (Fig. 3). It is unique
that the full sequence of a cDNA of the AGPase subunit is ex-

Downloaded from http://pcp.oxfordjournals.org/ at Korea Research Institute of Bioscience & Biotechnology on June 6, 2015

Fig. 5 Southern blot analysis of genomic DNA from sweet potato.

Thirty g of genomic DNA was digested with H: HindIII and E:
EcoRI. PCR fragments at 3 terminal end of iAGPLI-1 was used as a
probe for hybridization. Numbers indicate molecular size markers.

cDNAs encoding an AGPase large subunit

1241

Fig. 7 Comparison between signal intensity of transcript levels and

enzyme activity obtained from light-and dark-treated leaves. Data
points of AGPase are means of three replicates with SE. Basal activity
measured without the sample was 2.22.1 nmol min1 (mg protein)1.
The transcript bands were scanned by an Image Analyzer (Fujifilm,
BAS-1500) and the peak areas were calculated as a percentage based
on the band intensity. The signal intensities detected by iAGPLI-1
hybridization were 0.0% (L/L), 0.0% (L/D), and 3.0% (S/D) of 100%
(S/L). L/L, leaf/light; L/D, leaf/dark; S/L, stem/light; S/D, stem/dark.

pressed mainly in the stem. Potato sAGPL1 is predominantly

expressed in tubers (La Cognata et al. 1995). Since the cDNA
library was constructed with mRNA isolated from the tuberous
root, the predominant expression in stems was unexpected.
However, minute levels of transcripts, representing a stem iAGPLI, might have been expressed in tuberous root and picked up
when the cDNAs were synthesized. iAGPLI-1 may not be a
highly expressed form in the tuberous root. The transcript levels of iAGPLI-1 in the early stage of stem were much lower
than the levels at later stages, indicating developmental regulation. Conversely, expression of the potato AGPL gene
(sAGPL2) in the tuber declined when tuber size increased at
older stages (La Cognata et al. 1995).
Sugars are regulators of gene expression in plants (Koch
1996). mRNA expression of two potato AGPL genes (sAGPL1,
Mller-Rber et al. 1990; sAGPL2, La Cognata et al. 1995)
was induced by sucrose while the other AGPL (sAGPL3, La
Cognata et al. 1995) was not induced by sucrose. Only one
small subunit gene of sweet potato was sucrose inducible (Bae
and Liu 1997). Gene expression of the AGPase isoforms is
selectively regulated by sucrose. The sweet potato large subunit
gene (iAGPLI-1) was also induced by sucrose but it was not induced by either glucose or fructose (Fig. 4B, C), indicating that
only sucrose is a specific inducer for iAGPLI-1 gene expression.
Transcript levels under light and dark conditions differed
significantly (Fig. 4A). The level after a 12 h incubation in the
dark was reduced to 3% of the level under light. The gene expression of sweet potato small subunits (iAGPSI and iAGPSII,

Bae and Liu 1997) and potato large subunit (sAGPL1) were not
induced in the dark, which is in agreement with the pattern of
iAGPLI-1 expression. Although how light affects gene regulation is not clear yet, three possibilities can be considered. Firstly, mRNA is degraded in the dark although the process is unknown. For example, transcripts of cytosolic fructose-1,6bisphosphatase involved in sucrose biosynthesis were synthesized in the light and disappeared rapidly in the dark (Khayat et
al. 1993). Secondly, since dark-incubated leaves are unable to
perform photosynthesis, the mRNA expression of AGPLI-1
would be largely dependent upon photosynthetic assimilates.
This idea was supported by DCMU treatment, an inhibitor of
photosynthesis, which causes a much reduced expression of
potato AGPL (Mller-Rber et al. 1990). Finally, light, per se,
is not a direct controlling factor for expression of the AGPL
gene since exogenously added sucrose in the dark induced transcript levels (Mller-Rber et al. 1990, Bae and Liu 1997).
Dark-incubated leaves and stems exhibited AGPase activity, although 2.5 and 3 times lower than that observed under
light conditions (Fig. 7). Light-induced activity was more stable than activity in the dark, regardless of the length of incubation. The pattern was similar for shorter (6 h) and longer (18
and 24 h) incubation times (data not shown). The levels of enzyme activity of AGPase under light and dark conditions were
not correlated with the transcript levels of iAGPLI-1 in the
same conditions. This is supported by the results illustrated in
Fig. 6 where the enzyme activity in the stem accounts for ca.
70% of that in the tuberous root while the transcript levels in
the stem are 20 times higher than that in the tuberous root.

Downloaded from http://pcp.oxfordjournals.org/ at Korea Research Institute of Bioscience & Biotechnology on June 6, 2015

Fig. 6 Comparison between signal intensities of transcript levels and

enzyme activities obtained from different tissues. Data points of
AGPase are means of three replicates with SE. Basal activity measured without the sample was 43 nmol min1 (mg protein)1. The transcript bands were scanned by an Image Analyzer (Fujifilm, BAS-1500)
and the peak areas were calculated as a percentage based on the band
intensity. The signal intensities detected by iAGPLI-1 hybridization
were 1.0% (L), 5% (TR), and 1.0% (R) of 100% (S). TR, tuberous
root; S, stem; R, root; L, leaf.

1242

cDNAs encoding an AGPase large subunit

Acknowledgements
We would like to thank Dr. Hyun Sook Pai for critical review.
This work was sponsored by a grant from Korea Ministry of Science
and Technology (KG1111) to J. R. L.

References
Bae, J.M., Giroux, M. and Hannah, L.C. (1990) Cloning and characterization of
the brittle-2 gene of maize. Maydica 35: 317322.
Bae, J.M. and Liu, J.R. (1996) Molecular heterogeneity of small subunit ADPglucose pyrophosphorylase cDNAs isolated from tuberous roots of sweet
potato. Mol. Cells 6: 521527.
Bae, J.M. and Liu, J.R. (1997) Molecular cloning and characterization of two
novel isoforms of the small subunit of ADP-glucose pyrophosphorylase from
sweet potato. Mol. Gen. Genet. 254: 179185.
Ball, K. and Preiss, J. (1994) Allosteric sites of the large subunit of the spinach
leaf ADP glucose pyrophosphorylase. J. Biol. Chem. 269: 2470624711.
Bhave, M.R., Lawrence, S., Barton, C. and Hannah, L.C. (1990) Identification
and molecular characterization of shrunken-2 cDNA clones of maize. Plant
Cell 2: 581588.
Chomczynski, N. and Sacchi, N. (1987) Single step method of RNA isolation by
acid guanidinium thiocyanate phenol chloroform extraction. Anal. Biochem.
161: 156159.
Elimert, K., Luo, C., Dejardin, A., Villand, P., Thorbjornsen, T. and Kleczkowski, L. (1997) Molecular cloning and expression of the large subunit of
ADP-glucose pyrophosphorylase from barley (Hordeum vulgare) leaves.
Gene 189: 7982.
Giroux, M., Smith-White, B., Gilmore, V., Hannah, L.C. and Preiss, J. (1995)
The large subunit of the embryo isoform of ADP-glucose pyrophosphorylase
from maize. Plant Physiol. 108: 13331334.
Greene, T.W., Woodbury, R.L. and Okita, W. (1996) Aspartic acid 413 is important for the normal allosteric functioning of ADP glucose pyrophosphorylase.
Plant Physiol. 112: 13151320.
Khayat, E., Harn, C. and Daie, J. (1993) Purification and light-dependent molecular modulation of the cytosolic fructose-1, 6-bisphosphatase in sugarbeet

leaves. Plant Physiol. 101: 5764.

Kleczkowski, L.A., Villand, P., Luthi, E., Olsen, O.A. and Preiss, J. (1993)
Insensitivity of barley endosperm ADP-glucose pyrophosphorylase to 3-phosphoglycerate and orthophosphate regulation. Plant Physiol. 101: 179186.
Koch, K.E. (1996) Carbohydrate-modulated gene expression in plants. Annu.
Rev. Plant Physiol. Plant Mol. Biol. 47: 509540.
La Cognata, U., Willmitzer, L. and Muller-Rober, B. (1995) Molecular cloning
and characterization of novel isoforms of potato ADP-glucose pyrophosphorylase. Mol. Gen. Genet. 246: 538548.
Morell, M., Bloom, M. and Preiss, J. (1988) Affinity labeling of the allosteric
activator sites of spinach leaf ADP glucose pyrophosphorylase. Biol. Chem.
263: 633637.
Mller-Rber, B., Kossman, J., Hannah, L.C., Willmitzer, L. and Sonnewald, U.
(1990) Only one of two different ADP-glucose pyrophosphorylase genes
from potato responds strongly to elevated levels of sucrose. Mol. Gen. Genet.
224: 136143.
Mller-Rber, B., Nast, G. and Willmitzer, L. (1995) Isolation and expression
analysis of cDNA clones encoding a small and a large subunit of ADP-glucose phrophosphorylase from sugar beet. Plant Mol. Biol. 27: 191197.
Nakata, P.A., Greene, T.W., Anderson, J.M., Smith-White, B.J., Okita, T.W. and
Preiss, J. (1991) Comparison of the primary sequences of two potato tuber
ADP-glucose phryophosphorylase subunits. Plant Mol. Biol. 17: 10891093.
Olive, M.R., Ellis, R.J. and Schuch, W.W. (1989) Isolation and nucleotide
sequences of cDNA clones encoding ADP-glucose pyrophosphorylase
polypeptides from wheat leaf and endosperm. Plant Mol. Biol. 12: 525538.
Park, S.W. and Chung, W.I. (1998) Molecular cloning and organ-specific
expression of three isoforms of tomato ADP-glucose phrophosphorylase
gene. Gene 206: 215221.
Park, S.W., Kahng, H.Y., Kim, I.J., Park, J.O. and Chung, W.I. (1998) Molecular cloning and characterization of small and large subunits of ADP-glucose
pyrophosphorylase from oriental melon. J. Plant Res. 111: 5963.
Plaxton, W.C. and Preiss, J. (1987) Purification and properties of nonproteolytic
degraded ADP-glucose pyrophosphorylase from maize endosperm. Plant
Physiol. 83: 105112.
Preiss, J. (1982) Regularion of the biosynthesis and degradation of starch. Annu.
Rev. Plant Physiol. Plant Mol. Biol. 33: 431454.
Preiss, J. (1991) Biology and molecular biology of starch synthesis and its regulation. Oxford Surv. Plant Mol. Cell. Biol. 7: 59114.
Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: a Laboratory Manual (2nd edition). Cold Spring Harbor Laboratory, Cold Spring
Harbor, New York.
Villand, P., Aalen, R., Olsen, O.A., Lthi, E., Lnneberg, A. and Kleczkowski,
L.A. (1992a) PCR amplication and sequence of cDNA clones for the small
and large subunits of ADP-glucose pyrophosphorylase from barley tissues.
Plant Mol. Biol. 19: 381389.
Villand, P., Olsen, O.A., Kilian, A. and Kleczkowski, L.A. (1992b) ADP-glucose pyrophosphorylase large subunit cDNA from barley endosperm. Plant
Physiol. 100: 16171618.
Villand, P., Olsen, O.A. and Kleczkowski, L.A. (1993) Molecular characterization of multiple cDNA clones for ADP-glucose pyrophosphorylase from Arabidopsis thaliana. Plant Mol. Biol. 23: 12791284.
Weber, H., Heim, U., Borisjuk, L. and Wobus, U. (1995) Cell-type specific,
coordinated expression of two ADP-glucose pyrophosphorylase genes in relation to starch biosynthesis during seed development of Vicia faba L. Planta
195: 352361.

(Received March 6, 2000; Accepted August 21, 2000)

Downloaded from http://pcp.oxfordjournals.org/ at Korea Research Institute of Bioscience & Biotechnology on June 6, 2015

AGPase as a holoenzyme in higher plants is a heterotetramer consisting of two small and two large subunits. Several
AGPase isoforms are present in a plant and all contribute to the
total activity possibly at different levels depending on their
functional roles. The discrepancy between transcript levels of
iAGPLI-1 and AGPase enzyme activity levels implies that the
transcript level of iAGPLI-1 in the stem is regulated post-transcriptionally. Identification of AGPase protein corresponding
to iAGPLI-1 will help understand the mechanism of gene regulation. For sweet potato, more AGPase subunits are yet to be
identified in different organs to investigate the difference in the
functional roles of AGPases for synthesizing starch.