Large Subunit from Sweet Potato

Mol. Cells , Vol. 10, No. I, pp. 108-112
Molecules
and
Communication
Cells
© Springer-Verlag 2000
Isolation and Characterization of Polymorphic cDNAs Partially
Encoding ADP-glucose Pyrophosphorylase (AGPase) Large
Subunit from Sweet Potato
Sang Sook Lee, Jung Myung Bae, Mi Sook Oh, Jang Ryol Liu, and Chee Hark Harn t ,""
Plant Cell and Molecular Biology Research Unit, Korea Research Institute of Bioscience and Biotechnology, Taejon 305-600, Korea.
(Received on October 23, 1999)
cDNA clones encoding sweet potato AGPase large
subunit (iAGPLI) from the cDNA library constructed
from the tuberous root were isolated. Two clones were
characterized and named iAGPLI-a and iAGPLI-b.
They were 1,661 bp and 1,277 bp in length and
contained partial open reading frames of 450 and 306
amino acids, respectively. Both nucleic acid and amino
acid se quence identities between iAGPLI-a and
iAGPLI-b were 83.8 % and 97.3 %, respectively. Based
on the amino acid sequence analysis, iAGPLI-a and
iAGPLI-b share the highest sequence identity (81%)
with potato AGPase large subunit. The iAGPLJ-a and
iAGPLI-b genes were expressed predominantly in the
stem and weakly in the tuberous root, and no transcript
was expressed in other tissues. The sweet potato
genome contains several copies of the iAGPLI gene.
Keywords: ADP-Glu cose Pyrophosphorylase Large
Subunit; Starch Biosynthesis; Sweet Potato.
Introduction
Biosynthesis of starch in all plants and glycogen in bacteria
requi ~e ADP-glucose as a main substrate for elongati ng the
polymers. ADP-glucose is generated by the catalytic
reaction of ADP-glucose pyrophosphorylase (AGPase)
which converts glucose I-phosphate and ATP to ADPglu cose and pyropho sphate . AGPa se in plants is a
heterotetramer composed of two large subunits and two
small subunits encoded by different genes (Preiss, 1991 ).
t Present address: Biotechnology Center Nongwoo Seed Research
Institute, Kyonggi-do 442-372, Korea.
* To whom correspondence should be addressed.
Tel: 82-337-883-7055 ; Fax: 82-337-883-1193
E-mai l: chharn @nongwooseeds.co. kr
Several isoforms exist in plants with the sizes of the small
subunits ranging between 50- 56 kDa, and the sizes of the
large subunits range between 51-60 kDa (Lin et at., 1988 ;
Morell et at., 1987; Sowokinos and Preiss, 1982). By
contrast, bacterial AGPase is expressed by a single gene
forming a homotetramer with a subunit mass of 48 kDa. As
an allosteric enzyme in enteric bacteria, AGPase is
activated by fructo se- I,6-bi sphosphate and inhibited by
AMP. The plant enzyme is activated by 3-PGA and
inhibited by Pi (Preiss, 1978), although the AGPase, in a
few plants such as barley, showed in sensitivity to the
all osteric regul ation by 3-PGA/Pi (Kleczkowski et at.,
1993).
cDNA clones encoding the large subunit (LS) have been
isolated from many pl ants (corn LS : Bhave et at., 1990,
Giroux et ai., 1995; potato LS : La Cognata et ai. , 1995 ,
MUller-Rober et ai., 1990, Nakata et ai. , 1991 ; sugar beet
LS : MUller-Rober et ai., 1995 ; wheat LS: Olive et at.,
1989; barley LS : Villand et ai. , 1992a, 1992b, Elimert et
ai., 1997; Arabidopsis LS : Villand et ai., 1993; tomato LS :
Park and Chung, 1998; oriental melon LS : Park et at. ,
1998) . Analysis of the deduced am ino acid sequence
identity among cDNA clones revealed that small subunits
are highly conserved (Bae and Liu, 1997) while the large
subunits are relatively divergent.
Sweet potato is an important agricu ltural crop used both
for food and for its derivaties. In order to investigate the
biochemical and molecular mechanisms of the biosynthetic
machinery of starch formation in sweet potato tubers, we
have cloned and characterized cDNA clones encoding a
large subunit of AGPase from sweet potato.
Abbreviations: AGPase, ADP-glucose pyropho sphoryl ase;
AGPL, ADP-glucose pyrophosphorylase large subunit; AGPS ,
ADP-glu cose pyrophosphorylase small subuni t; LS , large
subunit; PGA, phosphoglycerate; SS, small subunit.
Sung Sook Lee et al.
109
Materials and Methods
Results
Plant materials Sweet potatoes (Ipomoea balatas cv. White
Star) were grown in a green house for one year. Leaves, stems,
roots and tuberou roots were harves ted at the same time from
mature plants.
Characterization of large subunit AGPase eDNA clones
Approximately 300,000 recombinant phages were screened
and two positive clones were isolated (iAGPLl-a, 1661 bp
and iAGPLI-b , 1277 bp) as shown in Figs. 1A and lB . The
respective open readin g frames ended with the TAG
termination codon at position 1,353 bp and 920 bp, flanked
by 3' untranslated regions of 308 and 357 bp. The
polyadenylation regions were located at 1652 (iAGPLI-a)
and 1267 bp (iAGPLl-b ). The deduc ed amino ac id
sequence of both forms contained highly conserved Lys
residues, which are allosteric activator binding sites for the
large subunit of spinach AGPase (Morell et aI., 1988).
iAGPLI-a exhibited Lys residues at 113 , 403 and 441
amino acids and iAGPLl-b exhibited two residues at 259
and 297 amino aci ds. The first Lys residue at 113 amino
acid of iAGPLI-a was not present in iAGPLl-b because the
iAGPLI-b is 144 amino acids shorter than iAGPLI-a. Both
4I3
clones also contained Aspartic acid 413 (D ) , a putative
Isolation of eDNA clones and sequencing A cDNA library
constructed from poly (A)+ RNA of tuberous root was screened
by using a 32P-labeled (1 X 106 dpm/ml ) hetero logous probe
from potato AGPase large subun.it cDNA (sA GPLl , co urtesy of
Dr. Bernd MUller-Rober). Approx imately 3.0 X 105 reco mbinant
phages were screened. Plaque fil ters were hybridi zed in a solution
co ntaining 6 X SSC, 5 X Denhart's solu tio n, 0. 1% SDS and
SO j..lg/rnJ calf thymus DNA for 16 h at 60°C and washed with 1X
SSC-O.l % SDS several times. T he filters were exposed to X-ray
film at -70°C prior to autoradiograph y. Two positive clones
(iAGPLl-a and iAGPLl-b) were subcloned into the KpnI site of
Bluescript vector and sequenced by the dideoxy nucleotide chain
termin ation method using Seq uenase II (USB , Cleveland, OH).
A ll sequ e nc in g rea c tion s we re pe rfo rm e d wi th c us tom
sy nth es ized o li go nu c leo ti de prim ers . To co nfirm co rrec t
seq uences, overlapp ing subc lones were sequen ced on both
strand s a nd s ubjected to a uto mated sequ e nc in g. Sequence
homologies were analyzed with MacDNASIS (Hitachi Software
Engineerin g America) and th e BLAST network servi ce (Nation al
Center for Biotechnology Information).
1
N
ACA
T
CCA
30 Q
L
F
P
L
T
N
R
A
A
T
P
A
V
P
135 CTT GGA GGA TGC TAT AGG TTG ATA GAC ATT CCG ATG AGe MC TGC
45
RNA gel blot analysis Total RNA was isolated from leaf, stem,
root, a nd tub e r ti ss ues of sweet potato as desc rib ed by
Chomczynski and Sacchi ( 1987) with modificati on. Thi rty j..lg of
tota l RNA was loaded on 1% agarose gel co ntaining 1 X MOPS
buffer and 0.66 M formaldehyde. After e lectrophoresis at 80 V
for 2 h, the gel was transferred to a nylon membrane in blotting
buffer ( lOX SSC). The membranes were hybridized by 32 Plabe led (l X 10 6 dpm/ ml ) probes of tota l cDNA frag ments
generated by KpnI restri ction di gestion of iAGPLl-a and iAGPLlb. Hybridizati on was performed in a solu tion contai ning 5 X
Den.hardt's soluti on, 0.1 % SDS, 100 mg/ml salmon sperm DNA,
and 5 X SSC for 20 h at 60°C. After hybridization, the blots were
was hed three times in I X SSC-O.l % SDS at 60°C pri or to
exposure to X-ray film at - 70°C.
Genomic DNA blot ana lysis Thirty j..lg of ge nomic DNA
isolated from sweet potato tub er by CsC I gradient
ultracentriguation (Sambrook et aI. , 1989) was digested with
XbaI and EcoRI. Neither of the restricti on enzy mes 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 soluti on ( 1.5 M NaCI and
0.5 M NaOH) and neutralized for 40 min in a neutrali zation
solution (l .S M NaCI and 0.5 M Tris-HCl, pH 8.0). DNA was
then blotted o nto nylon membranes in lO X SSC and th e
membranes were hybridi zed by the same co nditi ons used for
RNA gel blot analysis (above). The primers used for generating
PCR fragment s for hyb ridi zatio n probe s are 5 ' CGATTCGTGGGATGTTAT-3' (sense) and 5 ' -ACCATAGCCAAATGGCCA-3' (antisense) for 280 bp of AGPLl-a at 3'
terminus, and 5 '-AGTTTAC-CTGGACTIGTG-3 , (sense) and
S'-AACCATAGCCAA-ATGGCC-3' (a nti sense) for 177 bp of
AGPLl-b at 3' terminus.
CT TTG ACT GTA GAA GCA eCG ATA T TG GAG AGG CGT eGG GCA Me 44
1"
L
T
V
E
A
P
!
L
ERR
R
A ·
45 CCT AAG MT GTG GCT GeA ATC ATA CTG GGA GGG GGT GCA GGG
15 P
K
N
V
A
A
I
I
L
G
G
GAG
90 CM CTA TTC CCT CTC ACC AAC CGA GeT GCA ACC CCT GCT GTT
L
G
G
C
Y
R
LI
D
I
P
M
S
N
C
14
89
29
134
44
179
59
180 ATC Me AGe GGG GTT Me AAG ATC T TT GTG erG Ace GAG TTC AAT 22 4
60 I N S
G
V
N
KIF
V
L
T
Q
F
N 74
225 TCT GCT TCT CTT MC CGT CAC ATT TCC CGT ACC TAC TTT GGC MT 269
75
S
A
S
L
N
R
HIS
R
T
Y
F
G
N
89
270 GGT GTG AGC TTC GGA GAT GGA TTT GTT GAG GTG CTG GCT GCA ACC 3 14
90 G
v
S
F
GOG
F
V
E
V
L
A
A
T 10 4
315 CM ACA CAA GGG GM ACG GGG ATG ~G TGG TTT CAG GGA ACT GCA. 359
105 Q
T
Q
GET
G
M ~
W
F
Q
G
T
A 119
360 GAT GeT GTT AGA CAG TTT ACA TGG GTT T TT GAG GAG GCG MG Me 40 4
120
0
A
V
R
Q
F
T
W
V
FED
A
K
N
13 4
405 MG GAC ATT GA.T M T ATA GTT ATT CTG TCA GGA GAT CM CTT TAT 449
135 K
0
I
ON
I
V
I
L
S
G
0
Q
L
Y 14 9
450 eGC ATG GAT TAG ATG GAe TTA GTG CAG AAT CAe ATC GAA eGC AAT 49 4
R
M
0
Y
M
O
L
V
Q
N
HIE
R
N 164
150
495 TeT GAT ATT ACT CTT TCA TGT Gee ACA GTT GGG GAT AGe eGA GCA 539
1 65 S
D
I
T
L
S
CA
T
v
G
0
S
R
A 179
540 TCG GAC TTT GGG CTG GTG AM ATT GAC CGT AGA GGC CGA GTT GTC 584
180
S
0
F
G
L
V
KID
R
R
G
R
v
V
194
5 85 CAG TTT TGC GAG AM CC T AM GGC ACT GAT CTA MA GCA ATG CM 629
195 Q
F
C
E
K
P
K
G
T
0
L
K
A
M
Q 209
630 GTA GAT ACT ACT eTC TTG GGA TTG eGA . eCG CM GAT Gee AGA TTA 674
210 V
0
TT
L
L
G
L
P
P
Q
0
A
R
L 224
675 M1' CCT TAT ATT GCT TCG ATG GGG GTT TAT GTC TTT AAG ACG GAT 719
225
N
P
Y
I
A
5
N
G
V
Y
V
F
K
T
0
239
720 GTC CTT TTG CGG eTC CTG AGG TGG AGA TAT CCC ACA TCC MC GAC 764
240
v
L
L
R
L
L
R
WRY
?
T
S
N
D
254
765 TTT GGA Tec GAG ATT CTT CCT GeT GCT GTG ATG GAG CAC AAT GTT 809
255
r
G
S
ElL
P
A
A
V
f.f
E
H
N
V
269
810 CM GCT TAC ATT TTC AGA GAC TAC TGG GAG GAC ATC GGG ACA ATA 854
270 Q
A
Y
I
FRO
Y
WE
D
I
G
T
r
284
. 855 AM TCT TTC TAC GAC GeT MC TTG GCC CTA ACT GM GAG TTC CCG 899
285 K
S
F
Y
D
1\
N
L
A
L
TEE
F
P
900 MG TTT GAG TTC TAT GAT CCA MG ACA CCT TTC TAC ACG TCC CCA
300
K
F
E
F
'i
D
P
K
T
P
F
Y
T
S
P
945 AGA TTC CTA CCG CCA ACC AM ATT GAT MC T GC MG ATC MG GAT
315 R
F
L
P
P
T
KID
N
C
K
I
K
0
990 GCA ATA ATC TCC CAC GGG TGT TTC TTA CGA GAG TGC AGT GTG GM
330 A
I
I
SH
G
C
F
L
R
E
C
S
V
E
1035 CAC TCC ATC ATT GGT GAA AGG TCA CGG CTA GAT TGT GGT GTT GM
345
H
S
I
I
G
E
R
S
R
L
D
C
G
v
E
299
9 44
3 14
989
329
1034
344
1079
359
1080 CTG AAG GAT ACT 'l'TA ATG ATG GGA GCA GAT ACC TAT GM ACA GM 1124
360 L
K
D
T
L
H
M
GAD
T
YET
E 374
1125 TCT GM ATT GCC TCG CTT CTG GeA GAT GGA AM GTC CCG ATT GGG 1169
375
S
E
I
A
S
L
1
A
D
G
K
v
PIG
389
1170 GTT GGA GAA MC ACA AAA ATT AGG AAT GCT ATC ATC GAC P;A..G AAT 1214
390 v
G
E
N
T
K
I
RNA
I
I
Oil W N 404
1215 GTA AGG ATT GGG AM GAC GTT GTC ATC ATG AAT AAA GA.T GGT GTT 1259
405 v
R
I
G
K
D
V
V
I
M
N
K
D
G
v 419
1260 CM GAC TCA GAC CGC CCC GAT GM GGT TTC TAC AT C AGA TCA GGG 1304
420 Q
D
S
0
R
P
OE
G
FYI
R
S
G 4 34
1305 ATT ACT ATT AT A ATG GAG ~ GCA Ace ATT ceT GAT GGA ACC GTe 1349
435 I
T
I
I
MEW
A
TIP
D
G
T
v 449
1350 ATA TAA TAT CAA eGA AGA ACG ATT CGT GGG ATG TTA TGT TTC GGT 1394
450 I
•
CTAGGATGAATGCGGTTTTCCTTCTCCCGGAGAAACAAAAGTGGCAACCCAA.CCAAGAT
GTTTTGTTGAAGTTTACCTGGACTTGTGAMAGAGGMGCGGAGGGCAGTTGMGCAGA
GGGMTATGAATAAAACTG.~GGGATGCAT CCA T ATCTGGTGeATTC TTGCTATTGT ACA
ATATCATP.TCATATCATATGTAAGATATAGTTATMTTTCGATGATMTMTGTMGGT
TGGCCATTTGGCTATGGT'rGTAMAAAAAM 1661
Fig. lA. Nucleotide sequ ence a nd its derived a mino ac id
sequence of iAGPL/-a (accession number: AJ2S2316).
Cloning of cDNAs Encoding AGPase Large Subuni t from Sweet Potato
110
1
1
44
15
89
30
13 4
45
179
60
224
75
269
90
3 14
1 05
359
1 20
4 04
1 35
4 49
1 50
494
165
539
lBO
5B4
1 95
629
210
674
225
719
240
764
255
809
270
854
2B 5
899
30 0
A GGA GAT CAA CTT TAT CGC ATG GAT TAC ATG GAC T TA GTG CAG 43
GOQLYRMOYMOLVQ14
MT CAC ATC GM CGC MT TCT GAT ATT ACT CTT TC A TGT GCC ACA BB
N
H
IE
R
N
SOL
T
L
S
C A T 29
GTT GGG GAT AGe CGA GCA TCG GAC TTT GGG CTG GTG AAA ATT GAC 133
v G 0 S R A S 0 F G L v KID 44
CGA AGA GGC CGA GTT GTC CAG TTT TGC GAG AAA CCT MA GGC ACT 178
R
R
G
R
V
V
Q
F
C
E
K
P
K
G
T
59
GAT CTA AAA GCA ATG CAA GTA GAT ACT ACT CTC TT G GGA TTG CCA 223
E
H
K
A
M
Q
v
0
TTL
L
G
L
P 74
CCG CM GAT GCC AGA TTA MT CCT TAT ATT GCT TCG ATG GGG GTT 26B
P
Q
0
A
R
L
N
P
Y
I
A
S
14
G
V 89
TAT GTC TTT MG ACG GAT GTC CTT TTC CGG CTC CTG AGG TGG AGA 313
Y
V
F
K
T
0
V
L
F
R
L
L
R
Ii
R
10 4
TAT CCC ACA TCC MC GAC TTT GGA TCC GAG ATT CTT CCT GCT GCT 358
Y
P
T
S
N
0
F
G
S
ELL
P
A
A
119
GTG AT G GAG CAC MT GTT CAA GCT TAC ATT TTC AGA GAC TAC TGG 4 03
v
M
E
H
N
V
Q
II
Y
I
FRO
Y
w 13 4
GAG GAC ATC GGG ACA ATA AAA TCT TTC TAC GAC GCT MC TTG GCC 4 4 8
E
0
I
G
T
I
K
S
F
Y
DA
N
L
A 149
CTA ACT GM GAG TTC CCG AIIG TTT GAG TTC T AT GAT CCA MG ACA 493
L
TEE
F
P
K
F
E
F
.Y
0
P
K
T 16 4
CCT TTC TAC ACA TCC CCC AGA TTC CTC CCG CCA ACC AAA ATT GAT 538
P
F
Y
T
S
P
R
F
L
P
P
T
KID
1 79
MC TGC MG ATC MG GAT GCA ATT ATC TCC CAC GGG TGT TTC TTA 5B3
N
C
K
I
K
D
A
I
IS
H
G
C
F
L 194
CGA GAG T GC ACT GTG GM CAC TCC ATC ATT GGT GAA AGG TC~. CGG 62B
R
E
C
T
V
E
H
S
I
I
G
E
R
S
R 209
CTA GAC TGT GGT GTT GAIl CTG AAG GAT ACT TTA AT G AT G GGA GCA 673
L
0
C
G
V
EL
K
0
T
L
M
~1
G
A 224
GAT MC TAT GIlA ACA GAA TCT GAA ATT GeC TCG CT T CT G GCA GAT 718
0
N
YET
ESE
I
A
S
L
L
A
D 239
GGA AAA GTC CCG ATT GGG GTT GGA GAA MC ACA AAA AT'!' AGG MT 763
G
K
V
PIG
V
G
E
N
T
K
I
R
N 25 4
GeT ATC ATT ~ FJI!!f MC GTA AGG ATT GGG AAA GAC GT T GTC ATC 808
A
I
I
l.JlJ ~ N V R I G
K
0
v
V
I
269
ACG AAT AAA GAT GGT GTT CAA GM TCA GAC CGC CCC GAT G~.A GGC 853
T
N
K
0
G
V
Q
E
S
0
R
PO
E
G 2B4
TTC TAC ATC AGA TCA GGG ATT ACT ATT ATA ATG GAG W GCA ACC 898
FYI
R
S
G
I
T
I
I
M
E
(J$) A
T 299
ATT CGT GAT GGA ACC GTC ATA TAG TAT CM CGG AGC AGG CIIC CGG 943
I
R
D
G
T
V
I
NKTKPGVl\YSVT 'T'j'F',NOTQTVFVDl-1 PRLF,RRRAN PKDVAAVrI.GGGEGTK
-------------- -- - --- LT. EA . I . . . •. .. . . N . . • I ..••• A .• Q
50
30
LFPLTSRTATPAVPVGGCYRLIDI PMSNCINSAINKIFVLTQYNSAPLNR
. . . .• N . A ....•• L •.• . .•. . . ... . .. . . GV • . .•. . .. F ... S . ..
100
3:'
sAGPLl
iAGPLI-a
iAGPLI -b
sAGPLl
iAGPLI - a
iAGPLI-b
51
80
10:'
HIA..~TYFGNGVSFGDGFVEVLAATQT PGEAGKKW FQGTADAVRKFlWVrS
150
iAGPLI - a
iAGPLI - b
B:
.. 5 .. . . . . . . . . . . . . . . . . . .... Q . . T . M.. . . . .. . . . • Q .T... .
130
sAGPLl
iAGPLI - a
iAGPLI - b
15:
13:
OAKNKNIENIVVLSGOHLYRMOYHELVQNHIORNAOITLSCAPAEOSRAS
. • . .. 0 . 0 . . . 1. ... Q ••.• . • . 0 . . .... E . . S . •.. . .. TVG . . . . .
--------- - -- -- .• Q .• . . . . . 0 . . . .. . E . . S . •.•• . . TVG . . . . .
200
180
36
20 1
L)i-'GLVKl OSHGKvvQrAF:x P K GfDr~KAMQ v n1'T:'VGLSPQDAKKSPY J AS
.... R .•. . • . . C . . .•. T . .•...••... I •. • P • .•. I{LN . . • . .
••••••. • R .••.•. • C • •.• • T • . .
.. . L . • P .•.. RLN ..•.
250
18:
37
25:
231
87
MGVYVFK':'D'ILLKLLKWSY PTSNDFGSE I I PMI DDYNVQAYl FK DY~"EC
.. . . . R . . R. R . . . . . . . . . . . L ... VMEH . . . . .. . R . . . . .
.. . .. FR •. R.R. . • .
. . . L • . . VMP..H ..•.•.• R ..•..
300
280
iAGPLI-b
sAGPL1
iAGPLI - a
iAGPLI - b
30:
28:
137
IGT I KS FYNASLALTQE FPE FQFYOPKTPFYTS PRFLPPTK: DNCKIKOA
. . . . . D.N . . . . f. .. . K.F: . . . . . . . . .. .. .
. . .. . ..• O.N .... E . . . K.E . . . . . ...... . .... . . . . . . . . . . .
350
330
s AGPLl
352
1 I SHGCFLRDC SVEHSIVGERSRLDCGVELKDT:MMGADYYOTESEIASL
400
iAGPLI - a
iAGPLI - b
33:
187
.... . .... E ....... r. . .. . ... . . . .. . . L . .... T . E. ...
.... . ... . E.T .. . .. 1. . .. . . . . .. . .. . . L . . . . . N. E . .. .. . . .
3BO
236
sAGPLl
~O:
LAEGKV PI G I GENTK I RKCI I DKNAK I GKNVS I IN;{DGVQE.ADRPEEG FY
450
iAGPLI-n
iAGPLI - b
38 :
237
.. 0 .... .. V . . • . . .. Kl\ . •. .. VR . • . D.V.M . . . . DS ••. D ••..
. . D .. . .•. V .. • ... . NA . . . .. VR ... D . V.T . ...• . . S •.• D ...•
430
286
sAGPLl
iAGPLI -a
iAGPLI - b
45:"
01
2B7
sAGPLl
.sAGPLl
iA[';PLI"- a
iAGPLI - b
sAGPLl
iAGPLJ - a
~30
86
136
J 86
;.
TGGGATCACAGGAGAACGATT CATGGACGTTATGTTTCAGTCTAGGATGATCGCGGTTT
TGMGAAACAAAAGT GGCAGGCCMGATGTTTTGTTGAAGTTTACCTGGACTTGTGAAA
MGAGGAAGAGGGCAGTTGAAGCAGAGGGAATAAAACTGAGGGATGCATCCATATCTGG
TGCATTCTTGCTATTGTACAATATCATATCA T ATCATATGTAAGAT ATAGTATAATTTC
GATGATAATMTGTMGGTTGGCCATTTGGCTATGGT TGTAAAAAGTTCTTGATGCTTA
MAGT TAAAAACATGTTTCTTATT CATTCMAAAAAAAA
1277
Fig. lB. Nucleotid e seq uence and its derived amino ac id
seq uence of iAGPLI- b (accession number: AJ245392). The
asterisk indicates the stop codon. Circles indicate amino acids
corresponding to allosteric activator binding sites, and the box is
a putative binding site of 3-PGA.
binding site of 3-PGA fo und in potato AGPase (Greene
et ai., 1996), at 402 (iAGPLI-a) and 258 (iAGPLI-b)
amino acid. iAGPLI-a and iAGPLI-b share a DNA base
pair identity of 83 .8% (data not shown). The nucleotide
di vergency lies on only noncoding region at 3' terminus
after stop codo n. B oth clo nes co ntained partial ope n
reading fra mes of 450 and 306 amino acid, respectively
(Fig. 2). iAGPLI-a and -b share 81 % amino acid sequence
identity with potato AGPase large subuni t whil e the amino
acid sequence identity between a and b is 97.3% due to a
d iffe rence of 6 amino ac ids. T hey also contained an
allosteric acti vator site of SGITIIMEKATI at position 433
and 289 amino aci ds, respecti vely.
Amin o acid sequen ce a lignmen t of AGPase la r ge
subu nits The deduced amino acid sequences fro m cDNA
clones encoding AGPase large subunits were aligned and
sequence identities were calculated. Several distinct groups
of AGPase large subuni t were classified (Fig. 3). Barley
(hAGPL) and maize (zAGPLE) represent a group (I) of
monocot pl ants with 75 % homology. Most AGPLs in dicot
plants can be grouped into three large subunits fro m potato
and tomato AGPLs, designated by the numbers of 1 (group
II), 2 (III) and 3 (IV). Bean AGPase large subunit (vAGPL,
group V) is a di stinct isoform showing the least identity
470
450
306
Fig. 2. Amino acid sequence alignment of AGPase large subunits
fro m sweet potato (iAGPLI-a and b) and potato (sAGPLl).
Dashes (-) and dots (.) indicate gaps and identity, respectively.
The allosteric activator site at the C-terminal end of the protei ns
is marked by a box.
hAGPL
7 .4
zAGPL
lAGPLJ
92 . 5%
sAGPL3
lAGPL 1
sAGPLl
52.6%
iAGPLI-a
iAGPLI -b
33.5%
cAGPL l
lAGPL2
62.0
sAGPL2
bAGPL
cAGPL2
v AGPL
Fig. 3. Dendrogram was generated by the MacDNASIS software
program . h, barley; z, maize; I, tomato; s, potato; i, sweet potato;
b, sugar beet; v, bean; c, oriental melon.
compa red to all AGPases. Th e amin o acid sequ e nce
identiti es of tomato and potato iAGPL in each group share
92-98% homology. Thi s degree of homology is due to
their belonging in the same family. By contrast, isoforms in
the same species (for example, sAGPL1 , 2, and 3) are
grouped di fferently and exhibit lower identities. Sweet
potato large subunits (iAGPLI-a and -b) belong to group II.
Sung Soak Lee et at.
Northern and Southern blot analysis RNA gel
blot
analysis was performed using total RNA extracted from
leaf, stem, root and tuberous root. The iAGPLl-a transcript
of 1.9 kb was detected predominantly in the stem but also
weakly in the tuberou s root (Fig. 4). Northern blot
hybridized with the probe from iAGPLl-b generated the
same results (data not shown). For Southern blot analysis,
genomic DNA was digested with EcoRl and KbaI which
did not cleave the internal area of coding region. For
hybridizaton , PCR probes obtai ned at 3' terminus from
iAGPLJ-a was used. The same results were generated by
PCR probes at iAGPLl-b and/or a total size of iAGPLl-a
and -b (data not shown) . EcoRI digestion yielded 1.0 kb, (a
doublet), 2.5 kb, 4.0 kb and 9.0 kb bands (Fig. 5). KbaI
digestion generated 2.7, 3.4 and 10.0 kb (more than two
bands) bands indicating that the sweet potato genome
contained several copies of the iAGPLI gene.
T
S
L
R
f-
1.9 kb
25S rDNA
Fig. 4. Northern analysis of iAGPLI-a gene expression in various
tissues. Total 30 Ilg was loaded in 1% agarose gel and blotted in
nylon membrane. The membranes were hybridized by 32 Plabeled (1 x 106 dpm/mJ) probes of total cDNA from iAGPLJ-a.
L, leaf; S, stem; R, root; T, tuberous root.
E
9.0~
4.0~
2.5
~
X
f-
10.0
ff-
3.0
2.7
1.0 ~
Fig. S. Southern blot analysis of genomic DNA from sweet
potato. Thirty Ilg of genomic DNA was digested with X, XbaI
and E, EcoRI . A PCR fragment (1359-1648 bp) of iAGPLI-a was
used as a probe for Southern hybridi zation. Hybridization with
the PCR frag ment (104 1-1217 bp) of iAGPLI-b generated the
same results (data not shown).
11 1
Discussion
We have isolated two cDNA clon es (iAG PLl- a a nd
iAGPLl-b) which encode the AGPase large subunit fro m
swee t pot ato. Based on the amino ac id sequ e nc e
comparison with the full length clone of potato AGPase
large subunit (sAGPLl), these two clones contained partial
seq uences of the sweet potato AGPase large subunit.
Attempts to find the 5' terminus for the full length of each
clone were not successful (data not shown).
The seq uence identities among potato AGPL isoform 1,
2 and 3 are approximately 50-62%. This indi cates that
potato has at least three distinct isoforms of the AGPase
large subunit and each isoform would playa unique role in
generating ADP glucose. Sweet potato may have several
distinct isoforms like other plants thus far characterized. In
contrast with the distinct isoforms, the seq uence identity
between iAGPLI-a and iAGPLI-b is 97.3%. Besides, the
nucleotide sequences are almost identical along the DNA
sequence except for the 3' UTR region. A single gene of
iAGPLI may have been di verged into iAGPLI-a and
iAGPLI-b in the same manner that tomato and potato
AGPase large subunits have been differentiated fro m the
same gene through evolution. The heterogeneity of the
orig in al ge ne (iAGPLl) may have occurred during
evolution especiall y in sweet potato (Ipomoea batatas)
plant, which is hexaploidy (6x; 2n = 90). The cDNAs
encoding small subunits of AGPase fro m sweet potato,
which were previously isolated, also showed heterogeneity
of 1% difference in amino aci d sequences (Bae and Liu ,
1996), indicating that sweet potato genome contains the
extra gene copi es for eac h AGP small subunit gene.
Therefore, each gene encoding AGPase large and small
subunit isoform seem polymorphic in sweet potato genome
because of the pol yploidy nature. The presence of several
copies of each gene shown in Southern bl ots (Fig. 5 as well
as in the data of Bae and Liu , 1996) support this idea.
Taken together, our data suggest that each AGPase large
and small subunit gene of polyploid plants such as potato
(4x; 2n = 48) would also be polymorphic, although no
evidence has been reported yet.
Potential binding sites fo r sub strates and allosteric
effectors in the AGPase has been revealed by chemical
modification studies (Ball and Preiss, 1994; Greene et al. ,
1996a; 1996b). Aspartic acid 4 13 (D41 3) is a putative
binding site of 3-PGA, an allosteric activator of AGPase.
This res idue is pre se nt in a ll AGPase thu s far
characterized, as well as in iAGPLI-a and iAGPLI-b (Figs.
1A and IB ). Pyridoxal phosphate, an analog of 3-PGA,
interacts with three highly conserved Lys residues in large
subunits. These are cons idered as allosteric activator
binding site of spinach AGPase (Morell et al., 1988). Both
clo nes contain th e Lys resi du es K II3, K403, K 44 1 for
iAGPLI-a and K 263 and K 30 1 for iAGPLI-b. These clones
also contain an allosteric acti vator site of SGITIIMEKATI
112
Cloning of cDNAs Encoding AGPase Large Subunit from Sweet Potato
which is also present in the AGPase small subunit as
SGIVTVIKDAL where the sequence identity is moderate
(MUller-Rober et a l., 1990). A further study of structurefunction analysis will help to understand the regulation
mechanism of AGPase with two distinct large and small
subunits .
The expression levels of iAGPLI-a and iAGPLI-b were
predominant in stems, whereas the potato sAGPLl were
mainly expressed in tubers (La Cognata et al., 1995). Since
the eDNA library was constructed with the mRNA isolated
from the tuberous root, the predominant expression in
stems is unusual. However, minute levels of transcripts,
representing a stem iAGPLI, must have been expressed in
the tuberous root and picked up when the cDNAs were
synthesized. Considering that the iAGPLI and sAGPLl
were differenti ally expressed in different tissues, each AGP
large subunit gene would have preferentially expression in
ti ssues depending on its role, although each gene belongs
to the same group based on sequence identity.
Acknowledgments We would like to thank Young Ock Ahn
for technical help on manuscript preparation . Thi s work was
sponsored by a grant from Korea Mini stry of Science and
Technology (KGl111 ).
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