The Rice Endosperm ADP-Glucose Pyrophosphorylase Large
Subunit is Essential for Optimal Catalysis and Allosteric
Regulation of the Heterotetrameric Enzyme
1
Institute of Biological Chemistry, Washington State University, Pullman, WA 99164, USA
Faculty of Agriculture, Kyushu University, Fukuoka, 812-8581 Japan
3
Department of Genetic Resources Technology, Kyushu University, Fukuoka, 812-8581 Japan
4
Kazusa DNA Research Institute, Department of Plant Genome Research, Kisarazu, Japan
5
Faculty of Bioresource Sciences, Akita Prefectural University, Akita City, 010-0195 Japan
6
These authors contributed equally to this work.
*Corresponding author: E-mail, [email protected]; Fax, +1-509-335-7643.
(Received December 30, 2013; Accepted April 13, 2014)
2
Although an alternative pathway has been suggested, the
prevailing view is that starch synthesis in cereal endosperm
is controlled by the activity of the cytosolic isoform of
ADPglucose pyrophosphorylase (AGPase). In rice, the cytosolic AGPase isoform is encoded by the OsAGPS2b and
OsAGPL2 genes, which code for the small (S2b) and large
(L2) subunits of the heterotetrameric enzyme, respectively.
In this study, we isolated several allelic missense and nonsense OsAGPL2 mutants by N-methyl-N-nitrosourea (MNU)
treatment of fertilized egg cells and by TILLING (Targeting
Induced Local Lesions in Genomes). Interestingly, seeds from
three of the missense mutants (two containing T139I and
A171V) were severely shriveled and had seed weight and
starch content comparable with the shriveled seeds from
OsAGPL2 null mutants. Results from kinetic analysis of the
purified recombinant enzymes revealed that the catalytic
and allosteric regulatory properties of these mutant
enzymes were significantly impaired. The missense heterotetramer enzymes and the S2b homotetramer had lower
specific (catalytic) activities and affinities for the activator
3-phosphoglycerate (3-PGA). The missense heterotetramer
enzymes showed more sensitivity to inhibition by the inhibitor inorganic phosphate (Pi) than the wild-type AGPase,
while the S2b homotetramer was profoundly tolerant to Pi
inhibition. Thus, our results provide definitive evidence that
starch biosynthesis during rice endosperm development is
controlled predominantly by the catalytic activity of the
cytoplasmic AGPase and its allosteric regulation by the effectors. Moreover, our results show that the L2 subunit is
essential for both catalysis and allosteric regulatory properties of the heterotetramer enzyme.
Regular Paper
Aytug Tuncel1,6, Joe Kawaguchi2,6, Yasuharu Ihara2, Hiroaki Matsusaka2, Aiko Nishi2,
Tetsuhiro Nakamura2, Satoru Kuhara3, Hideki Hirakawa4, Yasunori Nakamura5, Bilal Cakir1,
Ai Nagamine1,2, Thomas W. Okita1,*, Seon-Kap Hwang1 and Hikaru Satoh2
Keywords: ADP-glucose pyrophosphorylase Rice endosperm Starch metabolism.
Abbreviations: AGPase, ADPglucose pyrophosphorylase;
DTT, dithiothreitol; EM, endosperm mutant; Glc 1-P, glucose
1-phosphate; LS, large subunit; MNU, N-methyl-N-nitrosourea; OsAGP, Oryza sativa ADPglucose pyrophosphorylase;
3-PGA, 3-phosphoglycerate; shr, shrunken; SS, small subunit;
SuSy, sucrose synthase; TILLING, Targeting Induced Local
Lesions in Genomes; WT, wild type.
Introduction
ADPglucose pyrophosphorylase (AGPase) plays a pivotal role in
starch biosynthesis in higher plants by catalyzing the first committed step of the pathway. The enzyme converts glucose 1-phosphate (Glc 1-P) and ATP to ADPglucose, the activated form of
glucose utilized by starch synthases, and inorganic pyrophosphate
(PPi) (Ballicora et al. 2004, Lee et al. 2007, Hwang and Okita 2012).
The catalytic activity of AGPase is normally subjected to allosteric
regulation by the ratio of effectors 3-phosphoglycerate (3-PGA)
and Pi. The activator 3-PGA increases the net catalytic activity,
which is reversed by the inhibitor Pi (Ballicora et al. 2004, Hwang
and Okita 2012). Detailed kinetic studies on the maize endosperm
enzyme indicate very complex regulatory properties depending on
the levels of effectors and substrates (Boehlein et al. 2010b,
Boehlein et al. 2013a, Boehlein et al. 2013b). AGPase activity is
further enhanced by reduction of the enzyme in response to
increasing light and sugar levels (Geigenberger 2011).
Unlike the cyanobacterial and prokaryotic AGPases which
are homotetrameric enzymes comprised of identical subunit
Plant Cell Physiol. 55(6): 1169–1183 (2014) doi:10.1093/pcp/pcu057, available online at www.pcp.oxfordjournals.org
! The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
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Plant Cell Physiol. 55(6): 1169–1183 (2014) doi:10.1093/pcp/pcu057 ! The Author 2014.
1169
A. Tuncel et al.
type (a4), the higher plant enzymes consist of pairs of large
subunits (LSs) and small subunits (SSs) that collectively form
a heterotetrameric structure (a2b2) (Morell et al. 1987, Okita
et al. 1990). The SS and LS have non-identical, yet complementary, roles in AGPase function. The SS of potato tuber AGPase
was proposed to play a dominant role in catalysis as D160N
mutation in the LS, the equivalent of the SS D145N mutation
which almost completely abolishes the catalytic activity, results
in only a 2-fold decrease in enzyme activity (Frueauf et al. 2003).
The suggestion of the SS being the dominant partner in catalysis and LS having just a regulatory role was challenged, however,
in many studies where both subunits were shown to influence
catalytic and allosteric regulatory properties of the enzyme
(Cross et al. 2004, Hwang et al. 2005, Hwang et al. 2007, Kim
et al. 2007, Boehlein et al. 2010a). In fact, maize endosperm
heterotetramer AGPase activity was shown to be equally vulnerable to random non-synonymous mutations in either subunit when expressed in bacteria (Georgelis et al. 2007).
Although plant AGPases are most catalytically efficient in
heterotetrameric form, the SS is capable of forming a catalytically active homotetramer as first demonstrated for the potato
SS homotetrameric enzyme (Ballicora et al. 1995, Laughlin et al.
1998, Hwang et al. 2008). The potato SS enzyme had about the
same catalytic activity as the heterotetrameric wild-type (WT)
enzyme although it required >30-fold higher amounts of
3-PGA for maximal catalytic activity than required by the WT
enzyme. Likewise, barley endosperm recombinant SS homotetramer is substantially activated by 3-PGA and possesses half
the catalytic activity of the heterotetrameric enzyme (Doan
et al. 1999). Similar to the potato and barley homotetrameric
enzymes, a recombinant form of the Arabidopsis SS (APS1)
homotetramer was also shown to be as active as the APS1–
APL1 heterotetramer but only in the presence of excess 3-PGA
(Crevillén et al. 2003). In contrast, homotetramers formed by
the potato AGPase LS are catalytically inefficient and insensitive
to 3-PGA (Hwang et al. 2008). While Arabidopsis APL3 and
APL4 homotetramers are catalytically defective, APL1 and
APL2 and tomato L3 homotetramers possess substantial catalytic activities, suggesting that the LSs from higher plants have
diverse catalytic properties (Ventriglia et al. 2008, Petreikov
et al. 2010).
In general, the SSs are encoded by one or two genes while
the LSs are encoded by many more paralogs. The variation in
number of isoforms depends on the plant species and tissue
type as well as the plant’s developmental stage (Akihiro et al.
2005, Crevillén et al. 2005, Ohdan et al. 2005, Lee et al. 2007). In
addition, the primary sequences of the SSs are more conserved
than those of LSs between different plant species (Georgelis
et al. 2008). The more conserved primary sequence of this subunit was proposed to be due to more evolutionary constraints
on the SS gene since it is less tissue specific and has fewer copies
than the LS gene (Georgelis et al. 2007).
AGPases are localized in chloroplasts of photosynthesizing
leaves and in the specialized starch-containing plastids,
amyloplasts, of non-photosynthetic sink organs. In addition
1170
to the amyloplast AGPase, cereal endosperms of maize
(Denyer et al. 1996), rice (Sikka et al. 2001), barley
(Thorbjørnsen et al. 1996) and wheat (Burton et al. 2002,
Tetlow et al. 2003) possess a second cytosolic form of the
enzyme which constitutes the major activity during grain filling.
The importance of the cytosolic isoform in starch accumulation
is corroborated by several genetic studies where mutations in
either the cytosolic SS or LS resulted in significant reduction in
endosperm AGPase activity and seed starch content. The maize
endosperm AGPase LS mutant, shrunken-2 (sh2), had 25–30%
of the starch content of the WT kernels (Tsai and Nelson 1966).
Mature seeds of the risø 16 mutant of barley, which lacks the
functional cytosolic SS, had 44% and 72% of the starch content
and seed weight of the WT seeds, respectively (Tester et al.
1993). Consistent with this result, endosperm AGPase activity
in the risø 16 mutant was shown to be 15–25% of that observed
for the WT (Johnson et al. 2003). Similar results were also obtained in the rice mutants osagps2-1 and osagpl2-1, which
lacked the S2b and L2 isoforms, respectively (Lee et al. 2007).
Seed AGPase activities in both mutants were only about 20%
that of the WT. In parallel, osagps2-1 and osagpl2-1 mutants
accumulated 31% and 23% of WT seed starch, respectively. In
comparison with the decreases in AGPase activity and starch
content observed in these mutants, expression of cytosolic
AGPases with up-regulatory allosteric properties increased
seed yields and plant biomass in maize (Giroux et al. 1996,
Wang et al. 2007, Hannah et al. 2012), wheat (Smidansky
et al. 2002, Smidansky et al. 2007) and rice (Smidansky et al.
2003, Sakulsingharoj et al. 2004).
In rice, two genes were identified to encode three different
SS isoforms (Akihiro et al. 2005, Ohdan et al. 2005, Lee et al.
2007). OsAGPS1 encodes the plastidial isoform in endosperm.
OsAGPS2 undergoes alternative splicing to produce two different transcripts, OsAGPS2a and OsAGPS2b, which, in turn, code
for the chloroplast isoform (S2a) in leaves and the cytosolic
isoform (S2b) in endosperm, respectively. In contrast,
OsAGPL1, OsAGPL2, OsAGPL3 and OsAGPL4 encode four separate LS isoforms (Akihiro et al. 2005, Ohdan et al. 2005, Lee
et al. 2007, Cook et al. 2012, Hwang and Okita 2012). OsAGPL3
codes for the chloroplast isoform in leaves, whereas OsAGPL1
and OsAGPL2 code for the amyloplast and cytosolic isoforms in
seed endosperm, respectively. OsAGPL4 transcripts were found
to be present in seeds and leaves, but at very low levels (Akihiro
et al. 2005, Ohdan et al. 2005). Interestingly, OsAGPS2b and
OsAGPL2 transcript levels show similar trends during seed development. RNA levels of both genes peak at 5 d after flowering
and remain substantially high during the mid to late development stages, the period when maximum starch accumulation
occurs. OsAGPL1 and OsAGPS1 transcript levels, however, peak
at 3 and 5 d, respectively, after flowering, but decline rapidly
afterwards (Ohdan et al. 2005). The relationship between the
spatial and temporal expression patterns of OsAGPS2b/
OsAGPL2 and OsAGPS1/OsAGPL1 gene groups further supports
the importance of the cytosolic AGPase isoform in endosperm
starch synthesis.
Plant Cell Physiol. 55(6): 1169–1183 (2014) doi:10.1093/pcp/pcu057 ! The Author 2014.
Allosteric regulation of rice endosperm AGPase
The phenotype of shr1 shriveled seeds indicated a drastic reduction in starch synthesis and probably a defect in a major
biosynthetic enzyme (Fig. 1). In fact, this phenotype was similar
to that seen for endosperm mutant 22 (EM22; shr2) seeds
which are also severely shriveled due to the loss of the
AGPase S2b small subunit (Kawagoe et al. 2005). Thus, we
first attempted to examine if the phenotypic changes observed
among the EM lines were due to mutations in the S2b subunit.
Seed proteins were extracted from developing seeds of a total of
250 EM lines (194 lines of Taichung and 56 lines of Kinmaze)
and subjected to immunoblot analysis using anti-S2b antibody.
The results showed that the S2b protein was present in all EM
lines examined (see Fig. 2 for examples), indicating that the
shriveled phenotypes are not due to the lack of S2b subunit.
This raised the question of whether the phenotypes are due to
the lack of the L2 subunit of AGPase. Indeed, additional
immunoblot analysis using anti-L2 antibody confirmed this suspicion. Unlike the WT, which shows a prominent polypeptide
at about 57 kDa, seed extracts from EM541 (as well as EM123
and EM1033; results not shown) were devoid of this LS band
(Fig. 2A), indicating that shr1 might be the gene locus for
OsAGPL2.
Further genetic studies supported this hypothesis. First, in
order to show that the shr1 locus is genetically unrelated to the
shr2 locus (encoding S2a or S2b), we crossed the EM541 line
with EM22 (the marker line for the shr2 locus) (Kawagoe et al.
2005). F1 seeds derived from the cross had a WT-like grain
phenotype, while the segregation mode of F2 seeds for the
shr1 phenotype (chalky/shriveled grain) and shr2 phenotype
(shrunken more severely shriveled grain) fitted well to an expected ratio of independent inheritance, i.e. normal : opaque/
shriveled :shrunken = 9 : 3 : 4 (Table 1A). This F2 segregation
pattern confirms that the shr 1 gene locus of EM541 is independent from the shr2 locus and that the mutation in shr2 is
epistatic to the null mutation in shr1 (Table 1A).
EM541 was also crossed reciprocally with WT TC65 (parental cultivar), and the F2 population was analyzed for seed
phenotype and the presence or absence of the L2 subunit.
Normal to shrunken seed phenotypes segregated in the
expected 3 : 1 (116 : 32) ratio, indicating a single recessive inheritance. Moreover, the 57 kDa L2 polypeptide band was present
only in normal seeds and not in shrunken seeds (Table 1B).
These results are consistent with the loss of the L2 polypeptide
band as being under the control of a single gene locus.
The genomic sequence of the OsAGPL2 gene from EM541
was analyzed by DNA sequencing and compared with that
(GenBank accession No. NC_008394; chromosome 1) in the
rice genomic database. It had a G to A nucleotide substitution
at the 50 border end of the ninth intron. Although the L2 gene
was not examined at the transcript level, it is likely that the
substitution disrupts the splicing of the gene and results in
read-through in the intron to a termination stop 19–21 nucleotides downstream of the normal splice site (Fig. 3). Such an
incorrect and premature transcript would generate a truncated
Fig. 1 Morphology of wild-type, shr1, shr1a and shr2 de-hulled seeds.
A cross-section of each seed is also shown. The shr1 and shr1a lines
harbor nonsense (null) and missense mutations in the L2 subunit,
respectively. The shr2 line contains a null mutation in the S2b subunit.
Fig. 2 Immunoblot analysis of seed proteins extracted from the
mature seeds of wild-type (TC65) and endosperm mutant (EM)
lines. (A) The EM541 line was used to represent the shr1 EM lines.
(B) The shr1a lines EM540, EM715 and EM817 were analyzed to detect
AGPase S2b and L2 proteins by using antibodies raised against rice
endosperm AGPase S2b and L2. The S2b and L2 protein bands were
detected slightly below and above the 55 kDa molecular weight
marker.
Of various allelic mutants of starch biosynthesis in rice endosperm (Satoh and Omura 1979, Satoh and Omura 1981), three
types of starch-deficient mutants, shrunken-1 (shr1), shrunken1altered (shr1a) and shrunken-2 (shr2), were isolated (Yano
et al. 1984), with shr1a seeds exhibiting a more severely shriveled phenotype than shr1 seeds. shr2 has been previously
demonstrated to be the structural gene for the S2b subunit
of endosperm AGPase (Yano et al. 1984, Kawagoe et al. 2005).
Here, we present evidence that shr1 and shr1a contain null and
missense mutations, respectively, in the structural gene for the
rice endosperm cytosolic AGPase L2 subunit. Our results with
the purified recombinant AGPases indicate that the AGPase L2
subunit is essential for both the catalytic and allosteric regulatory properties of the WT enzyme and that the cytosolic enzyme’s Pi sensitivity is a major determinant of controlling
AGPase activity in starch synthesis during rice grain filling.
Results
The shr1 mutations are caused by a genetic lesion
in the OsAGPL2 structural gene
Plant Cell Physiol. 55(6): 1169–1183 (2014) doi:10.1093/pcp/pcu057 ! The Author 2014.
1171
A. Tuncel et al.
polypeptide (344 amino acids) missing its C-terminal sequences
(174 amino acids). In bacteria, C-terminally truncated LS polypeptides rapidly turn over (Laughlin et al. 1998), a condition
that may account for its absence in rice seed extracts.
Alternatively, loss of the splice site can activate a cryptic
splice site, a mechanism which was observed in maize
AGPase mutants (Lal et al. 1999), that would also generate a
truncated enzyme or a non-functional protein targeted for degradation. Collectively, the results from immunoblot analysis,
crossing experiments and sequencing data confirm that the
shr1 is the gene locus for OsAGPL2.
Additional shr1 alleles were identified by analyzing plant
lines of the EM library by immunoblot analysis. This effort resulted in identification of two other OsAGPL2 null mutants,
EM123 of the Kinmaze cultivar and EM1033 of the Taichung
cultivar. Crossing experiments using EM123 and EM1033 lines
with the WTs showed an inheritance mode for the OsAGPL2
gene similar to that observed in WT EM541, indicating that
the shrunken phenotype of these lines is due to the absence of
the L2 subunit (data not shown). DNA sequence analysis revealed that both mutants contained a G to A replacement at
the 21st nucleotide of the 10th exon, resulting in the formation
of a nonsense codon instead of Trp346 (Fig. 3).
Identification of missense mutations among the
endosperm mutants
lines were found to contain missense mutations resulting in
amino acid replacements in the L2 primary sequences. Of this
group, we chose three mutant lines, EM540, EM715 and EM817,
for further study as these lines displayed severely shrunken
(shr1a) seed phenotypes (Figs. 1, 2). EM540 and EM817 had
C to T substitutions at the same position in the third exon,
leading to the replacement of T139I. EM715 had a C to T substitution in the fourth exon, resulting in the A171V mutation
(Fig. 3).
The allelic relationship between the missense EM540 (shr1a)
and the null EM541 (shr1) was studied by crossing experiments
(Table 2). Although seeds from both lines share a common
shriveled, wrinkled phenotype, the EM540 (shr1a) seeds have
glassy, vitreous endosperm while the EM541 (shr1) seeds are
opaque. The F1 seeds of EM540 EM541 had a phenotype that
is similar to that of the EM540 (glassy/vitreous), while the F2
seeds showed a ratio of vitreous to opaque endosperm that
fitted very well to the expected 3 : 1 ratio of single inheritance
mode (Table 2). Moreover, vitreous endosperm always co-segregated with the presence of the L2 polypeptide (Fig. 2B).
Missense and nonsense mutations in the OsAGPL2
gene result in similar decreases in seed weight
Average seed weights of rice lines harboring nonsense or shr1a
missense mutations in the OsAGPL2 gene were significantly
lower compared with that of the WT (Fig. 4). The WT
As immunoblot analysis is incapable of identifying missense
mutations in the OsAGPL2 coding sequence, the EM collection
of endosperm-defective lines was screened by TILLING
(Targeting Induced Local Lesions in Genomes). Sixteen EM
Table 1 Segregation analysis of F2 seeds derived from the cross
between EM541 (shr1) and EM22 (shr2) lines (A) and between
EM541 (shr1) and TC65 (wild type) lines (B)
A
Cross combination
Total 2 (9 : 3 : 4)
Segregation
in F2
F1
seeds
WT shr1 shr2
EM541 (shr1a) EM22 Normal 143 38
(shr2a)
B
Cross combination
Segregation
in F2
L2(–)
EM541 (shr1a) TC65
(Shr1a)
49
230
Total
3.29
2 (1 : 3)
L2(+)
shr1
Shr1
shr1
Shr1
32
0
0
116
148
3.29
L2(–) and L2(+) indicate the absence and presence of the AGPase L2 subunit in
rice seed based on immunochemical detection using anti-L2 antibody.
The segregation pattern for EM541 EM22 is well fitted to the ratio of
WT : shr1 : shr2 = 9 : 3 : 4 (2 = 3.29, P < 0.05) and that for EM541 TC65 is also
well fitted to shr1 : Shr1 = 1 : 3 (2 = 0.90, P < 0.05).
a
Shr1, normal (WT) seed; shr1, shriveled seed; shr2, severely shriveled
(shrunken) seed.
1172
Fig. 3 Location of nonsense (EM541, EM123 and EM1033) and missense mutations (EM540, EM187 and EM715) in the OsAGPL2 gene
which codes for the L2 subunit. G to A mutation at the ninth exon–
intron border causes read-through to a nonsense TAA codon 19–21
nucleotides downstream. A similar G to A mutation in the tenth exon
created a termination codon in EM123 and EM1033. These premature
stops resulted in a truncated polypeptide, which is probably very
prone to proteolysis. A mutation of C416 to T resulted in threonine
(T) at position 139 being replaced by isoleucine (T139I) in both EM540
and EM817. A similar mutation at nucleotide 512 resulted in A171V
substitution.
Plant Cell Physiol. 55(6): 1169–1183 (2014) doi:10.1093/pcp/pcu057 ! The Author 2014.
Allosteric regulation of rice endosperm AGPase
(TC65) plant had an average seed weight of 23.4 mg. In contrast,
the EM541 and EM1033 lines, which are null for the L2 subunit,
had seed weights of 12.4 and 9.2 mg, respectively, demonstrating the importance of the cytosolic AGPase isoform in starch
biosynthesis. Likewise, seed weight of the EM123 (L2 null
mutant of Kinmaze) was 45% that of the WT, with an average
of 10.0 mg. In contrast, loss of the S2b subunit resulted in a
more severe effect as the EM22 seeds were only 7.2 mg, a decrease in seed weight similar to that reported by Kawagoe et al.
(2005). Average seed weights of rice lines harboring the
shr1a missense mutants EM540, EM817 and EM715 were
about 27–39% those of the WT. Interestingly, seed weights of
Table 2 Segregation analysis of F2 seeds derived from the cross
between EM541 (shr1) and EM540 (shr1a) lines
Cross combination
Total 2 (1 : 3)
Segregation
in F2
L2(–)
L2(+)
shr1 shr1a shr1 shr1a
EM541 (shr1a) EM540 61
(shr1aa)
0
0
146
207
2.2
L2(–) and L2(+) indicate the absence and presence of the AGPase L2 subunit in
rice seed based on immunochemical detection using anti-L2 antibody.
The segregation pattern for EM541 EM540 is well fitted to the ratio of
shr1 : shr1a = 1 : 3 (2 = 2.2, P < 0.05).
a
shr1: shriveled (chalky and wrinkled) seed; shr1a: severely shriveled (glassy and
wrinkled) seed.
these shr1a missense lines (ranging from 6.3 to 9.1 mg) were
comparable (or even lower in some cases) with seed weights of
the null EM541 and EM1033, indicating that the T139I and
A171V mutations in the L2 subunit were as deleterious as the
null mutation with respect to AGPase catalytic and/or allosteric
regulatory properties. In parallel, starch contents of the shr1a
missense mutant seeds were as low as those of the nonsense
mutant seeds. For example, EM541 seeds had 43% and the
missense mutants had 30–50% starch content of the WT
seeds, while EM22 had 27% (data not shown). As the seed
weights (mainly dictated by starch content) are directly impacted by AGPase activity which, in turn, are primarily dictated
by the cytosolic isoform in the endosperm, these results indicate that the endosperm AGPase activities are similar in the null
and missense mutant lines.
In the absence of the LS, Arabidopsis leaf, barley endosperm
and potato tuber SSs are capable of assembling into a catalytically active homotetrameric enzyme (Li and Preiss 1992, Doan
et al. 1999, Salamone et al. 2000, Hwang et al. 2008). In view of
the strong conservation of the SS primary sequences among
higher plants, it is highly likely that the null EM541 line is expressing a SS homotetramer AGPase in the endosperm cytosol.
In contrast, EM540, EM817 and EM715 contain a heterotetrameric enzyme composed of the WT SS and missense mutant
forms of the LS (Fig. 5). The similar seed weights of the missense
mutants compared with the null mutants suggest that the net
AGPase catalytic activities of the mutant heterotetramer
25
WT
Null L2/S2b homotetramer
20
Missense L2
Seed weight (mg)
Null S2b/no enzyme
15
*
10
*
*
5
0
WT
EM541 EM1033 EM540 EM715 EM817
Taichung
WT
EM123
EM6
EM22
Kinmaze
Fig. 4 Seed weights (mg) of the wild type and endosperm mutant lines. n = 10 for each line. Seed weights of the analogous mutants from the
Kinmaze cultivar are also shown. Each line contains the following mutation in the L2 subunit: EM123, EM541 and EM1033, nonsense mutations;
EM6, T139A; EM540 and EM817, T139I; EM715, A171V. Seed weight of EM22 which is null for the S2b subunit is also shown. Average seed weights
were statistically different based on one-way ANOVA with Tukey’s multiple comparison test (P < 0.05). *No significant difference was found
between the average seed weights of EM1033, EM540 and EM715 lines. Error bars represent the standard error.
Plant Cell Physiol. 55(6): 1169–1183 (2014) doi:10.1093/pcp/pcu057 ! The Author 2014.
1173
A. Tuncel et al.
Mutations
WT
shr1
(nLS)
shr1a
(mLS)
L2
L2
Cytosolic
S2b
S2b
S2b
S2b
S2b
S2b
S2b
L2
L2
L2
L2
S2b
L2
L2
Plastidial
shr2
(nSS)
+
+
+
+
L1
L1
L1
L1
S1
S1
L1
S1
S1
S1
L1
S1
S1
L1
S1
L1
Fig. 5 The AGPase composition of the wild type (WT), LS null (nLS), LS missense (mLS) and SS null (nSS) mutants in rice endosperm. WT
endosperm has both a major cytoplasmic AGPase and a minor plastidial enzyme. In null shr1 mutants, the S2b small subunits can assemble into a
cytoplasmic homotetramer enzyme in the absence of the L2 large subunits, while missense L2 subunits will assemble with S2b to form a mutant
AGPase. In shr2 null mutants, the L2 subunits can assemble into a homotetramer which shows little if any catalytic activity.
Native AGPase activities in the
mutant endosperms
Fig. 6 summarizes the amount of AGPase activities, measured
under optimal activated enzyme conditions, observed in developing seed extracts from WT, EM541 (L2 null), EM540
(T139I) and EM22 (S2b null mutation). The S2b null mutant
of EM22 seeds had the lowest AGPase activity (15.8 nmol min–
1
mg–1) with only 5% of that of the WT. The EM541 null mutant
seeds had 40% (133.4 nmol min–1 mg–1) of the catalytic activity
of the WT endosperm AGPase (329.8 nmol min–1 mg–1), while
the EM540 missense mutant seeds retained about 60% of the
activity (206.6 nmol min–1 mg–1) (Fig. 6). Although both L2
mutant seeds had significantly lower AGPase activity than
the WT, EM540 missense mutant seeds had >50% enzyme activity than that measured in EM541 null seeds. These observed
enzyme activity levels of the null and missense AGPase seed
extracts were unexpected as they were inversely correlated with
their seed weights. While EM541 exhibited larger seeds than
EM540 (Fig. 4), it contained less AGPase activity. One possible
explanation for this unanticipated result is that although the
S2b homotetramer present in the EM541 null mutant has less
catalytic activity than the missense mutant heterotetramer
(SWTLT139I) in EM540 in the presence of high activator 3-PGA
levels, the homotetramer enzyme may have superior allosteric
regulatory properties with regard to Pi inhibition. Such allosteric regulatory properties of the S2b homotetramer could in
fact help the enzyme compensate for the low activity under
physiological conditions.
1174
350
WT
300
AGPase activity
(nmol/min/mg)
enzymes are similar to those of the S2b homotetramer enzyme
present in the null L2 mutant. If true, the T139I and A171V mutations have a severe effect on AGPase activity and, in turn, starch
biosynthesis. Therefore, we measured the native AGPase activities
from developing endosperm of the WT and selected mutants.
Null L2/S2b homotetramer
Missense L2
250
Null S2b/no enzyme
200
150
100
50
0
WT
EM540
Taichung
EM541
WT
EM22
Kinmaze
Fig. 6 Activities of native AGPases from developing wild-type and
mutant endosperms. Assays were performed at 37 C for 10 min and
at 10 mM 3-PGA, 1.5 mM ATP and 1.0 mM Glc 1-P concentrations.
n = 2 for Taichung and n = 5 for Kinmaze data.
Expression and purification of the
recombinant AGPases
The inverse relationship between maximum AGPase activity
levels assayed under excess activator 3-PGA (Fig. 6) and seed
weight (Fig. 4) suggests that the allosteric regulatory properties
may be distinct for the heterotetrameric enzyme containing a
missense mutation in the L2 subunit and the homotetrameric
S2b enzyme. Therefore, efforts were directed at assessing the
relative enzyme activities of the mutant heterotetramer and
homotetramer enzymes by studying the kinetic properties of
the recombinant forms.
The cDNA sequences of the rice endosperm OsAGPS2b and
OsAGPSL2 genes were amplified, cloned and subjected to mutagenesis as described in the Materials and Methods. Our earlier
attempts to express the recombinant rice AGPase using protein
expression vectors, pSH208 for the S2b subunit and pSH476 for
Plant Cell Physiol. 55(6): 1169–1183 (2014) doi:10.1093/pcp/pcu057 ! The Author 2014.
Allosteric regulation of rice endosperm AGPase
A139I or T171V mutations affect both catalytic
and allosteric regulatory properties of the AGPase
The purified recombinant WT and mutant AGPases were
assayed to determine the effects of mutations on the enzyme’s
catalytic activity. Both the T139I and A171V mutations in L2
resulted in a >3-fold decrease in the specific activity of the
enzyme (Fig. 7). Under saturating concentrations of Glc 1-P,
ATP and 3-PGA, the WT (S2bWTL2WT) heterotetramer had a
specific activity of 31.8 U mg–1. In contrast, the missense
S2bWTL2T139I and S2bWTL2A171V mutants had specific activities
of 8.5 and 9.3 U mg–1, respectively. Surprisingly, the recombinant S2bWT homotetramer had even lower specific activity,
1.7 U mg–1, than the missense mutants. However, this measured catalytic S2bWT activity may be an underestimate since
the purified enzyme fraction exhibited noticeable proteolysis
(Supplementary Figs. S2, S3).
The affinity values for the activator 3-PGA were substantially
lower for the missense L2 heterotetramer mutants and S2bWT
homotetramer than for the WT (Table 3). The S2bWTL2WT had
an A0.5 value of 0.59 mM. The 3-PGA affinity of the missense
S2bWTL2T139I mutant was 6-fold lower, with an A0.5 value of
3.54 mM. The S2bWTL2A171V mutant and the S2bWT homotetramer showed similar affinities towards 3-PGA, with A0.5 values
35
30
Speciffic activvity
(µmoll/min/m
mg)
the L2 subunit (Hwang et al. 2007), resulted in sufficient expression of the S2b subunit, but very little induction of L2. Sequence
analysis revealed that the OsAGPSL2 gene contains 52 rare
codons (10% of the total) which are not frequently used for
translation in Escherichia coli. Therefore, we introduced the
fragment of genes coding for the rare tRNAs from the pRARE
plasmid (Novagen) into pSH208 to construct pSH558. The new
pair of expression plasmids, pAT28 and pAT16, carrying S2b
and L2 cDNA sequences (Supplementary Fig. S1), respectively,
significantly improved expression of the rice endosperm
AGPase. The various AGPase enzyme forms were purified to
near homogeneity and examined by SDS–PAGE to ensure that
the subunits were present at stoichiometric levels
(Supplementary Fig. S2). Immunoblot analysis of the purified
AGPases using anti-potato SS and anti-maize LS antibodies was
also performed to check for possible protein degradation. As
shown in Supplementary Fig. S3, no degradation was detected
in the heterotetramers. However, the S2b homotetramer was
partially degraded, indicating that the homotetramer enzyme is
less stable than the heterotetramer form and that the L2
subunit enhances the stability of the S2b subunit. Similar
degradation of partially purified recombinant SWT homotetrameric potato AGPase was previously eliminated by using a high
concentration of EDTA (5 mM) (Salamone et al. 2000),
suggesting that the origin of degradation is metalloprotease
based. Since our purification protocol involves IMAC, we
used an EDTA-free protease inhibitor cocktail to purify the
rice SWT AGPase. Use of a more robust protease inhibitor
cocktail reduced the degree of SWT degradation but did not
completely eliminate it.
25
20
15
10
5
0
SWTLWT
SWTLT139I
SWTLA171V
SWT
Fig. 7 Specific activities (mmol min–1 mg–1) of the purified recombinant AGPases. Assays were performed in replicates at 37 C for 10 min
and at 10 mM 3-PGA, 2 mM ATP and Glc 1-P concentrations. n = 2 for
each set of data.
Table 3 Kinetic parameters of the recombinant wild-type and
mutant AGPases
Enzyme
S2bWTL2WT S2bWTL2T139I S2bWTL2A171V S2bWT
Kinetic parameter
A0.5 (mM)
0.59 ± 0.05a 3.54 ± 0.76
2.14 ± 0.48
2.54 ± 0.53
Fold activation
25
6
13
At 0.6 mM 3-PGA 1.48 ± 0.05 0.71 ± 0.09
1.33 ± 0.16
24.67 ± 1.72
At 6.0 mM 3-PGA 9.47 ± 0.06 1.77 ± 0.01
1.86 ± 0.16
16.04 ± 1.20
6
I0.5 (mM)
S0.5 (mM)
ATP
0.58 ± 0.03 0.49 ± 0.03
0.51 ± 0.03
0.5 ± 0.01
Glc 1-P
0.41 ± 0.05 1.43 ± 0.01
0.72 ± 0.09
1.32 ± 0.15
Mg2+
2.76 ± 0.11 3.81 ± 0.22
4.34 ± 0.41
ND
n = 2 for each set of data.
a
Standard error.
ND, not determined.
of 2.14 and 2.54 mM, respectively. In addition, an increase in the
3-PGA concentration from 0.0 to 10 mM resulted in 25-fold
activation of the WT enzyme, whereas it was only about 6fold for the mutant enzymes. Interestingly, the SWT homotetramer had 13-fold activation in specific activity, twice that of the
mutants. Parallel differences in 3-PGA affinities were observed
between the native enzymes from the developing seeds of the
WT and mutants (Supplementary Fig. S4).
In addition to activator analysis, the effects of the inhibitor Pi
were also investigated under low (0.6 mM) and high (6.0 mM)
3-PGA concentration. At 0.6 mM 3-PGA, the SWTLT139I mutant
was twice as sensitive to Pi inhibition (I0.5 = 0.71 mM) as the WT
enzyme (I0.5 = 1.48 mM) while the SWTLA171V mutant was
almost equally resistant, with an I0.5 of 1.33 mM. Pi inhibition
was also measured at 6 mM 3-PGA. The S2bWTL2WT enzyme
was much more tolerant to Pi inhibition, with an I0.5 of
Plant Cell Physiol. 55(6): 1169–1183 (2014) doi:10.1093/pcp/pcu057 ! The Author 2014.
1175
A. Tuncel et al.
9.47 mM, while S2bWTL2T139I and S2bWTL2A17V mutants were
more sensitive to Pi inhibition, with I0.5 values of 1.77 and
1.86 mM, respectively. Interestingly, the S2bWT homotetramer
was more resistant to Pi inhibition than the other enzymes at
both low and high 3-PGA concentrations. The S2bWT
homotetramer had a very high I0.5 (16.04 mM) at 6 mM
3-PGA. It was even more resistant to Pi inhibition at 0.6 mM
3-PGA, with an I0.5 of 24.67 mM, indicating that the enzyme is
virtually insensitive to the inhibitor Pi at lower activator
concentration.
Analyses of the substrate binding affinities of the AGPases
revealed that there are no major differences in ATP S0.5 values
between the AGPases. The measured S0.5 values for ATP were
between 0.49 and 0.58 mM. The mutant enzymes, however,
showed slight decreases (30–40%) in Mg2+ affinity. In contrast,
affinity towards the other substrate, Glc 1-P, showed more variation among the enzymes. The S2bWTL2WT enzyme had an S0.5
of 0.41 mM for this substrate while the S2bWTL2A139I and
S2bWTL2T171V mutants had S0.5 values of 1.43 and 0.72 mM,
respectively. Similar to the mutants, the S2bWT homotetramer
had lower affinity for Glc 1-P, with an S0.5 of 1.32 mM.
The relative in vivo AGPase activities as estimated by their
capacity to restore glycogen levels were also in good agreement
with the kinetic results. No glycogen accumulation was detected
in cells expressing the S2bWTL2A139I and S2bWTL2T171V mutant
heterotetramers or the S2bWT homotetramer, indicating that
the reduction in catalytic activities and down-regulatory allosteric properties were insufficient to synthesize adequate
ADPglucose for glycogen production (Supplementary Fig. S5).
Collectively, these results show that the differences between
the WT and the SWTLT139I, SWTLA171V and SWT enzymes are due
to changes in (i) catalytic activities, which are 3- and 18-fold
lower for the missense L2 heterotetramers and SWT homotetramer, respectively; (ii) Glc 1-P binding affinities, which are
about 3-fold lower for SWTLT139I and SWT and 2-fold lower for
SWTLA171V; (iii) 3-PGA affinity, which is 3.5- to 6-fold lower for
the missense L2 heterotetramers and SWT homotetramer; and
(iv) decreased tolerance of SWTLT139I and SWTLA171V to Pi
inhibition.
Discussion
The AGPase-catalyzed rate-limiting reaction of starch biosynthesis occurs mainly in the cytosol of cereal endosperms. Null
mutations that cause loss of either subunit of the cytosolic
AGPase result in significant decreases in seed starch content
and weight. We have identified three endosperm null (shr1)
mutants, EM541, EM123 and EM1033, which lack the L2 subunit but retain the S2b subunit of the cytosolic isoform of
endosperm AGPase (Figs. 2A, 3, 5). The mature seeds from
the shr1 mutants had 39–53% of the WT seed weight. The
decreases in seed weights of EM541, EM123 and EM1033 are
similar to those observed in the LS nonsense mutants of maize
(Tsai and Nelson 1966) and rice (Lee et al. 2007).
1176
In addition to the null mutants, we identified several missense mutants of the L2 subunit that also showed reduced seed
weight. Interestingly, the three missense (shr1a) mutants,
EM540, EM817 and EM715, had seed weights (27–39% of the
WT) as low as those of the L2 null mutants. To rule out the
possibility that a mutation(s) on a gene(s) other than OsAGPL2
may be responsible for the shrunken phenotypes and decreases
in seed weight, we performed genetic crossing experiments.
Extensive segregation analyses confirmed that the shriveled
phenotypes are probably only due to the null or missense mutations in the OsAGPL2 gene (Tables 1, 2). Although the genetic backgrounds of EM715 and EM817 were not examined by
crossing experiments, it is likely that the major changes in grain
appearance (glassy/vitreous, shrunken seeds) are contributed
by the mutation in the OsAGPL2 gene. However, we do not
exclude the possibility of additional mutations in the genomes
of EM715 and EM817. Such a possibility remains, especially for
the EM817 line which contains the same T139I mutation as
EM540, but displays lower seed weight.
Measurement of the native AGPase activities from developing endosperms of the mutant seeds showed that the lack of
the S2b subunit reduces the total enzyme activity more than
loss of the L2 subunit (Fig. 6), indicating that the activity of the
S2b homotetramer is higher than that of the L2 homotetramer.
The EM541 L2 null mutant, however, had less endosperm
AGPase activity than the EM540 missense mutant when
assayed in the presence of excess 3-PGA. These results indicated
differences in allosteric regulatory properties between the missense heterotetramers and the S2b homotetramer which were
revealed by characterization of the recombinant enzymes.
Although we cannot rule out the possibility that the native
enzyme activity measurements also include AGPase activity
coming from the amyloplast isoform, it is safe to assume that
the contribution from the plastid isoform, composed of the S1
and L1 subunits, is negligible. The rationale behind this assumption is the fact that the native enzyme assays were performed
using seeds at the mid development stage and the S1 and L1
subunit are mostly active during the early stages of seed development (Ohdan et al. 2005, Lee et al. 2007).
Kinetic characterization of the recombinant WT AGPase
revealed that the endosperm cytosolic enzyme is allosterically
regulated by the effectors 3-PGA and Pi. In fact, the kinetic
values of the recombinant enzyme are in good agreement
with those of the native enzyme partially purified from seed
extracts (Sikka et al. 2001). The A0.5 value for the endosperm
native AGPase was measured to be 0.65 mM, with the enzyme
having >40-fold activation in the presence of 5 mM 3-PGA
(Sikka et al. 2001). These values are very similar to the A0.5
value, 0.59 mM, and 25-fold activation of the recombinant
WT enzyme found in this study. The Km values for ATP
(0.58 mM) and Glc 1-P (0.41 mM) of the recombinant
enzyme also showed a good correlation with those of the partially purified native enzyme, which are 0.18 and 0.17 mM for
ATP and Glc 1-P, respectively.
Plant Cell Physiol. 55(6): 1169–1183 (2014) doi:10.1093/pcp/pcu057 ! The Author 2014.
Allosteric regulation of rice endosperm AGPase
The AGPase missense and null mutants exhibited lower affinity for 3-PGA than the WT enzyme. In addition to having 3fold less catalytic activity, the SWTLT139I and SWTLA171V mutants
had 3.5- to 6-fold lower affinity for 3-PGA and was activated
only 6-fold by this activator compared with the 25-fold activation of the WT enzyme. Moreover, these mutants were more
sensitive to Pi inhibition. At lower 3-PGA (0.6 mM), the
SWTLT139I mutant was already 2-fold less tolerant to the inhibitor Pi. The inhibitory effects of Pi were more prominent at
higher 3-PGA, with the mutants exhibiting 5-fold more sensitivity to Pi inhibition. Similar to the missense L2 heterotetramer
mutants, the S2bWT homotetramer also showed lower affinity
for 3-PGA and the substrate Glc 1-P. In addition, it had less
catalytic activity (about 5-fold) than the mutants. However, this
enzyme was extremely resistant to Pi inhibition compared with
the other enzymes at both low and high 3-PGA concentration.
These results show that the L2 subunit is essential for optimal
allosteric regulation and catalytic activity of the rice endosperm
AGPase.
Sequence analysis of the L2 subunit shows that the T139 and
A171 residues are highly conserved among the LS sequences
from diverse plant species (Fig. 8). These two residues are also
conserved in the S2b partner, supporting the importance of
T139 and A171 in enzyme function. In fact, our homology
modeling of the AGPase L2 revealed that the two residues,
which are spatially very close to each other, are located near
the potential substrate and inhibitor binding sites, with T139
being closer to the sites than A171 (Fig. 9). Thus, the substitution of T139 by isoleucine having a non-polar and bulkier side
chain might result in a disorder of the local structure that alters,
albeit indirectly, the substrate (Glc 1-P) and/or effector (3-PGA,
Pi) binding to L2. The A171V substitution also results in a
bulkier side chain, but could be less direct, as seen in the modeled structure. Our kinetic results show that both substitutions
impair the regulatory and catalytic properties of the enzyme.
The correct topology of the loop structures, including the two
residues near the potential substrate and effector binding sites,
is essential for proper enzyme functioning.
The residual starch contents of the L2 null and missense
mutant seeds can be attributed, partly, to the activity of
amyloplast AGPase composed of the S1 and L1 subunits and
mostly active during early stages of development (0–5 d after
flowering) (Ohdan et al. 2005), and to the activity of sucrose
synthase (SuSy), which can provide an alternative source of
ADPglucose (Li et al. 2013). In addition, the missense mutants
contain the heterotetramers (SWTLT139I or SWTLA171V) in the
cytosol, while the nonsense mutants have only the S2bWT
homotetramer in this compartment (Fig. 5). The differences
in the seed weight and starch content between the WT and
missense mutant plants (shr1a) can easily be explained by the
differences in both the allosteric regulatory and catalytic properties of the AGPases that these plants are expressing. Likewise,
the kinetic properties of the missense AGPases, SWTLT139I and
SWTLA171V, and S2bWT can account for the similar levels of
starch being accumulated. Although the S2bWT has substantially lower levels of catalytic activity than the missense enzymes (though some of this difference may be due to
proteolysis), the homotetramer enzyme is highly resistant to
Pi inhibition (Table 3). Hence, the sensitivity of the missense
AGPases to Pi inhibition lowers the net catalytic activity to a
level comparable with that of the less active S2bWT.
The indirect role of the Pi levels in controlling the endosperm AGPase activity and, hence, the net flow of carbon
into starch is also evident from previous transgenic studies.
Seed weights of the maize kernels expressing the rev6 mutant
of the endosperm cytosolic AGPase increased 18% due to the
insertional mutations in the LS which made the enzyme more
resistant to Pi inhibition (Giroux et al. 1996). Likewise, rice seeds
overexpressing the glgC-TM, which encodes a Pi-insensitive
form of the E. coli AGPase, accumulated 11% more starch compared with the WT seeds. In addition, the [Pi]/[3-PGA] ratio was
estimated to be about 56 from the calculated [Pi] = 27 mM and
[3-PGA] = 0.48 mM levels in cytosol of developing barley seeds
harvested at 14 d after flowering (Tiessen et al. 2012). Our kinetic results show that the WT AGPase in cytosol is readily inhibited by a relatively low concentration of Pi at 0.6 mM 3-PGA.
These lines of evidence suggest that the Pi levels have an indirect, yet an important, role in endosperm starch biosynthesis
and that the rice endosperm AGPase operates at low efficiency
due to a probably high [Pi]/[3-PGA] ratio.
Fig. 8 Sequence alignment of AGPase LSs from different plant species. Residues that are conserved among all species are shaded in gray. T139
and A171 residues of the rice endosperm AGPase L2 are indicated in boxes. GenBank accession numbers of the sequences are: O. sativa,
BAG92523; Z. mays, P55241; H. vulgare, CAA47626; T. aestivum, CAA79980; S. bicolor, AAB94012; S. tuberosum, Q00081; S. lycopersicum,
AAC49943; A. thaliana, AAB58475.
Plant Cell Physiol. 55(6): 1169–1183 (2014) doi:10.1093/pcp/pcu057 ! The Author 2014.
1177
A. Tuncel et al.
Unlike the accepted role of allosteric regulation in governing
the catalytic activity of the leaf and potato tuber AGPases
(Hwang and Okita 2012) and, in turn, carbon flow into
starch, the involvement of this control process does not
extend uniformly to the cereal endosperm AGPases as they
exhibit varying responses to the activator 3-PGA and inhibitor
Pi. For example, endosperm AGPase partially purified from developing wheat seeds does not respond to the activator 3-PGA.
However, 3-PGA does reverse the inhibitory effect of Pi (GomezCasati and Iglesias 2002). Likewise, the activity of the partially
purified enzyme from developing barley endosperm increases
only 30% with 3-PGA, a weak activation which is reversed by Pi
(Kleczkowski et al. 1993). Similar to the native enzyme, the
recombinant form of the barley endosperm AGPase was also
not affected by 3-PGA or Pi (Rudi et al. 1997) but was inhibited
46% at 20 mM Pi in another study (Doan et al. 1999). In contrast
to the wheat and barley enzymes, the maize endosperm
AGPase is moderately or highly activated by 3-PGA whose
effect is reversed by Pi. The partially purified native enzyme is
substantially activated (20-fold) in the presence of 3-PGA
(Plaxton and Preiss 1987) while the recombinant forms show
varying moderate activation (5- to 10-fold) depending on the
study (Burger et al. 2003, Cross et al. 2004, Boehlein et al. 2008,
Boehlein et al. 2013a). Interestingly, the maize endosperm
enzyme is activated by low amounts of Pi in the absence of 3PGA (Boehlein et al. 2010a).
The reason(s) for the lack of a common allosteric regulatory
mechanism among the cereal endosperm AGPases is totally
unclear. It could have arisen during evolution when some of
the cereal species (e.g. barley and wheat) lost response to the
effectors 3-PGA and Pi while the others (e.g. maize and rice.)
retained this regulatory mechanism. In terms of allosteric regulation, the rice endosperm AGPase (both the native and the
recombinant form) aligns closer to the maize enzyme.
Considering the fact that primary sequences of SSs from different species are more conserved (Georgelis et al. 2008), the
differences in allosteric regulatory properties are most probably
due to the differences in LSs. Indeed, alignment of the LS primary sequences shows that the rice L2 subunit shares 77%
identity with maize LS while it shares lower (71%) identity
with wheat and barley LSs. This hypothesis is further supported
by very high sequence identity (99%) between the wheat and
barley LSs and the similarities between the allosteric regulatory
properties of the endosperm AGPases from these species.
In addition to AGPase, evidence has been obtained that SuSy
may also be a significant source of ADPglucose in both leaf and
heterotrophic organs (Muñoz et al. 2006, Baroja-Fernández
et al. 2009). This view has been extended to maize endosperm
where transgenic plants overexpressing a potato SuSy showed
an elevated grain starch content of 10–15% (Li et al. 2013). Our
results presented here show that loss of the major cytoplasmic
AGPase (shr2) results in a loss of about 75% of normal starch
content. If SuSy is a viable alternative pathway for ADPglucose,
this enzyme together with the plastidial AGPase is unable to
compensate for the loss of the major cytoplasmic AGPase,
reinforcing the concept that the major pathway leading to
ADPglucose is through the cytoplasmic AGPase (Tsai and
Nelson 1966, Johnson et al. 2003, Sakulsingharoj et al. 2004,
Lee et al. 2007) with only minor contributions by the plastidial
AGPase and SuSy. Hence, SuSy, like the plastidial AGPase, is
unlikely to be a major determinant of ADPglucose formation
and, in turn, of starch accumulation in developing rice endosperm. A likely basis for the stimulation of starch synthesis by
overexpression of the potato SuSy in maize endosperm was the
elevated increases in UDPglucose. As the pathway leading from
sucrose degradation to ADPglucose is at near equilibrium
(Gibson et al. 2011), net elevation of UDPglucose will result
in simultaneous increases in Glc 1-P and ADPglucose levels,
the latter affecting starch synthesis.
Collectively, our results show that the rice endosperm
AGPase is allosterically regulated by the effectors 3-PGA and
Pi, and the LS is essential for both the optimal regulation and
catalysis of the enzyme. More in-depth studies are underway to
obtain additional insights into this intriguing enzyme since the
regulatory mechanisms controlling its activity appear to be
more complex than for the AGPases from other cereal plants
studied so far.
Materials and Methods
Generation, screening and identification of
endosperm mutants
Fig. 9 Homology modeled structure of the rice AGPase L2. T139 and
A171 are both part of loop structures and are located close to the
putative substrate (ADP) and effector (3-PGA/Pi) binding sites SO4
mimics PO4. Carboxyl and amine groups of T139 form polar contacts
(indicated by dashed lines) with L170 and A172, respectively.
1178
Rice EM lines containing the shr1, shr1a or shr2 mutations were
generated by treatment of independent fertilized egg cells of
japonica rice cultivars, Oryza sativa cv. Kinmaze and
Taichung65 (TC65) with MNU as described by Satoh and
Omura (1979). The rice plants were grown under natural conditions at the experimental field plots affiliated with Kyushu
University, Japan. Missense mutations in the OsAGPL2 gene
were identified by TILLING as described by Suzuki et al. (2008)
Plant Cell Physiol. 55(6): 1169–1183 (2014) doi:10.1093/pcp/pcu057 ! The Author 2014.
Allosteric regulation of rice endosperm AGPase
using the primers listed in Supplementary Table S1. Protein
contents of seeds from >250 EM lines, which were selected
based on aberrant endosperm phenotype (e.g. shrunken,
chalky, waxy, etc.), were analyzed at developing or mature
stages by SDS–PAGE/immunoblotting. The amount of proteins
used was normalized by resuspending 1 mg of powdered rice
seeds in 20 ml of sample buffer (8 M urea, 4% SDS, 5%
2-mercapthoetanol, 0.125 M Tris–HCl, pH 6.8). The protein samples were analyzed on 10% SDS–polyacrylamide gels under denaturing conditions. Immunoblot analysis of the seed AGPases was
performed as described by Nishi et al. (2001). Antibodies raised
against the S2b and L2 subunits were used in the immunoblots.
Segregation analysis
The shr2 mutant has been previously demonstrated to have a
null mutation in the structural gene for AGPase SSs in leaf (S2a)
and endosperm cytosol (S2b) (Yano et al. 1984, Kawagoe et al.
2005, Ohdan et al. 2005). EM22 was used as the shr2 marker line
for segregation analysis. Rice plants were crossed by hand pollination and grown under natural conditions to obtain mature
seeds. F1 seeds were sown in plastic pods filled with sterilized
organic soil, grown in the greenhouse for about 1 month and
then transplanted and cultivated in the experimental field
plots. Self-pollinated F2 seeds were harvested after maturation.
Determination of seed weight and starch content
Seed weight and starch content of the seeds were determined
as described previously (Satoh et al. 2008).
Isolation and cloning of rice endosperm cytosolic
AGPase genes
Total RNA was extracted from immature rice seeds at 5–10 d
after pollination. Seeds were pulverized in liquid nitrogen using
a mortar and pestle and mixed with Trizol reagent (Invitrogen).
The RNeasy Minelute Cleanup (Qiagen) and the M-MLV reverse transcriptase (Promega) kits were used to produce cDNA
according to the manufacturers’ instructions. The OsAGPS2b
(GenBank accession No. AK103906) and OsAGPL2 (GenBank
accession No. AK071497) genes, including 50 - and 30 -untranslated regions (UTRs), were PCR amplified using gene-specific
primers (Supplementary Table S2) and cloned into pBS II SK(Stratagene). PCR conditions were 25 cycles at 94 C for 30 s,
55 C for 30 s and 68 C for 2 min. Subsequent cloning of the
protein-coding regions of the genes into the bacterial expression vectors, pSH558 and pSH476, was done by PCR amplification under the same conditions using gene-specific primers
(Supplementary Table S2). The S2b-coding sequence was
cloned into pSH558, a codon-optimized form of pSH208
(Hwang et al. 2007), which carries rare tRNA genes derived
from the pRARE plasmid (Novagen). The L2-coding sequence
was cloned into pSH476, a 6 His-tag-carrying plasmid which
was derived from pQE30 (Qiagen) (Hwang et al. 2007)
(Supplementary Fig. S1). The S2b-coding sequence was also
cloned into pSH476 for expression and affinity purification of
the S2b homotetramer. The L2 mutants carrying T139I (EM540
and EM817) or A171V (EM715) were generated using the
QuickChange site-directed mutagenesis kit (Stratagene) with
the primers listed in Supplementary Table S2. Mutagenesis
PCRs were performed with the L2-coding sequence in the
pUC58 vector (GenBank accession No. AF253496) to avoid
changes in the expression vector. PCR conditions were 18
cycles at 95 C for 30 s, 55 C for 30 s and 68 C for 14 min. The
resulting plasmid DNAs were sequenced for verification of the
mutations and then subcloned into pSH476.
Expression and purification of AGPases
Expression and purification of the rice AGPases were performed
according to previously described protocols (Hwang et al. 2006,
Hwang et al. 2007). The plasmid DNAs containing the AGPase
S2b- and L2-coding sequences were sequentially transformed
(first the S2b and then the L2 gene) into E. coli EA345 cells
which lack endogenous AGPase activity due to the null mutation in its structural gene, glgC (Hwang et al. 2007). Three
colonies were inoculated into 25 ml of Luria Broth medium
supplemented with 0.4% (w/v) glucose, 50 mg ml–1 kanamycin
and 200 mg ml–1 penicillin G. The starter culture grown overnight at 37 C was transferred to 1 liter of liquid NZCYM
medium (10 g of NZ-amine, 5 g of yeast extract, 5 g of NaCl,
2 g of MgSO47H2O and 1 g of casamino acid, pH 7.0) containing
the same antibiotic concentrations. Isopropyl-b-D-thiogalactopyranoside (IPTG) was added to a final concentration of
0.1 mM when the OD600 of the culture reached 1.0–1.2 and
the protein expression was induced for 18 h at room temperature. The S2b homotetramer was expressed under the same
conditions except that EA3457 cells harboring the pRARE plasmid were used and the cells were grown in the presence of
200 mg ml–1 penicillin G and 30 mg ml–1 chloramphenicol. The
cells were harvested and resuspended in 25 ml of buffer A
(25 mM HEPES-NaOH, pH 8.0, 5% glycerol) containing
0.5 mg ml–1 lysozyme and 1 mg ml–1 each of leupeptin and pepstatin A. Following incubation of the cell suspension for 30 min
on ice, the cells were disrupted by sonication after addition of
1 mM phenylmethylsulfonyl fluoride. To avoid potential proteolytic degradation of the S2b homotetramer during purification, 1 EDTA-free protease inhibitor cocktail (Roche) was
additionally supplied to the cell suspension before cell disruption. After centrifugation at 20,000 g for 20 min at 4 C, the
crude extract was loaded on a DEAE-Sepharose Fast Flow
(Amersham) column (bed volume: 25 ml) pre-equilibrated
with buffer A and then the column was washed with the
same buffer until the absorbance at 280 nm reached background levels. AGPase activities were eluted with a 0–0.5 M
NaCl gradient in buffer A. Fractions containing AGPase activity
were pooled and passed through a 5 ml bed volume of immobilized metal affinity resin (IMAC-TALON Superflow, Clontech)
pre-equilibrated with buffer B (25 mM HEPES-NaOH, pH 8.0, 5%
glycerol, 0.3 M NaCl) using a BioLogic DuoFlow chromatography system (BioRad). After extensively washing the column
Plant Cell Physiol. 55(6): 1169–1183 (2014) doi:10.1093/pcp/pcu057 ! The Author 2014.
1179
A. Tuncel et al.
with 5 mM imidazole in buffer B, proteins were eluted with
100 mM imidazole in buffer B and then precipitated in 67%
ammonium sulfate for 1 h. The protein sample was centrifuged
at 20,000 g for 15 min, and the precipitate was resuspended in
4 ml of buffer A and then centrifuged again at 15,000 g for
10 min to remove denatured proteins. The supernatant was
carefully collected and diluted 1 : 10 in 40 ml of buffer A to
load on a 2 ml bed volume of POROS 20 HQ resin (Applied
Biosystems) pre-equilibrated with buffer A. After extensive
washing with buffer A, the column was subjected to a 0–
0.5 M NaCl gradient in buffer A. Fractions containing AGPase
activity were combined and concentrated to approximately
1 mg ml–1 using a 30 kDa cut-off ultrafiltration membrane
(Millipore). The concentrated enzyme solution was divided
into small aliquots and stored at –80 C until use.
Protein, SDS–PAGE and immunoblot analyses of
the recombinant proteins
Protein concentrations were measured using the Advanced
Protein Assay Reagent (Cytoskeleton, Inc.) and bovine serum
albumin (BSA) as the standard. SDS–PAGE and Western blot
analyses of the recombinant proteins were performed as
described previously (Hwang et al. 2004). Protein bands specific
to the rice recombinant AGPase small and large subunits were
detected using either the potato AGPase SS or maize LS
antibodies.
Enzyme assays
Kinetic characterizations of the recombinant WT and mutant
enzymes were performed as described previously (Hwang et al.
2004). AGPase activities were measured in the forward synthesis reaction by measuring the amount of [14C]Glc 1-P incorporated into ADPglucose. One unit of enzyme is defined as the
amount that produces 1 mmol of ADPglucose per minute. The
reaction mixture in a total volume of 100 ml contained 100 mM
HEPES-NaOH, pH 7.0, 8 mM dithiothreitol (DTT), 10 mM
MgCl2, 0.4 mg ml–1 BSA, 0.15 U of inorganic pyrophosphatase
(Sigma), 10 mM 3-PGA, 2 mM ATP, 2 mM Glc 1-P and 1,000–
1,200 d.p.m. nmol–1 [14C]Glc 1-P (Moravek), and an appropriate
amount of enzyme. The reaction mixtures were pre-warmed
prior to the addition of enzyme. The assays were performed at
37 C for 10 min and terminated by placing the tubes in boiling
water for 2 min. AGPase activities were linear with respect to
time and enzyme amount.
Protein samples for native enzyme assays were prepared
from developing endosperm of the seeds at the milky stage
(10–15 d after pollination) according to the protocol described
by (Kawagoe et al. 2005). AGPases in the seed extracts were
then precipitated in 67% ammonium sulfate and resuspended
in buffer A following centrifugation at 20,000 g for 15 min. A
100 ml aliquot of the sample was desalted using a desalting
column (EdgeBio) and kept at –80 C after freezing in liquid
nitrogen. The activities of the native AGPases were measured
in the synthesis direction and under the same conditions as the
1180
recombinant enzymes except that concentrations of DTT, ATP
and Glc 1-P were 3, 1.5 and 1.0 mM, respectively.
Determination of kinetic parameters
Kinetic parameters were calculated by fitting the experimental
data to the Hill equation, V = v0 + Vmax [S]n/Ksn + [S]n, where V
represents the reaction rate, v0 the initial reaction rate in the
absence of substrate (or effector), Vmax the maximum reaction
rate, [S] the substrate (or effector) concentration, Ks the reaction constant (S0.5, A0.5 or I0.5) and n the Hill coefficient (Hwang
et al. 2007). IGOR 6.22 (WaveMetrics) and VisualEnzymics 2010
(Softzymics) were used to calculate the kinetic parameters. The
S0.5 values of ATP, Glc 1-P and Mg2+ are the amounts of substrates (or cofactor) required to obtain 50% of the maximum
reaction rates and were determined in the presence of saturating 3-PGA (10 mM) and non-variable substrate concentrations
(up to 4 mM). The A0.5 value of 3-PGA is the amount of activator required for half the maximum reaction rate and was
determined at saturating concentrations of ATP (2 mM), Glc
1-P (2 mM) and Mg2+ (10 mM). The I0.5 value of Pi is the
amount of inhibitor required for 50% inhibition of enzyme activity at 0.6 and 6 mM 3-PGA. The I0.5 values were calculated
using the same Hill equation by plotting the Pi concentration vs.
the percentage inhibition.
Iodine staining
Iodine staining was performed by exposing cells to iodine vapor
after the cells were grown overnight on Kornberg’s medium
(1.1% K2HPO4, 0.85% KH2PO4, 0.6% yeast extract, 1.5% agar,
pH 7.0) supplemented with 0.25% (w/v) glucose, 50 mg ml–1
kanamycin and 200 mg ml–1 penicillin G.
Sequence alignment and structural modeling
Sequence alignment of the selected AGPase LSs was obtained
using the CLUSTALW2 (Larkin et al. 2007) server (http://www.
ebi.ac.uk/Tools/msa/clustalw2/) with default parameters.
Homology modeling of the L2 protein was performed using
the SWISS-MODEL (Arnold et al. 2006) (http://swissmodel.
expasy.org/) automated mode. The crystal structure of the
potato tuber AGPase SS (Jin et al. 2005) (protein data bank
id: 1yp4) was used as the template for homology modeling.
The homology modeled L2 protein was then superimposed
on to the C-chain of potato tuber AGPase SS using the
DeepView-Swiss-PdbViewer software (Guex and Peitsch 1997)
keeping the sulfates and ADP fixed. The resulting structure was
energy minimized using the energy minimization module to
eliminate side chain clashes and rendered in PyMol (http://
pymol.org/). Molecular weights of the L2 and S2b proteins
were predicted using the Expasy–compute pI/Mw tool server
(http://web.expasy.org/compute_pi/) (Walker et al. 2005).
Supplementary data
Supplementary data are available at PCP online.
Plant Cell Physiol. 55(6): 1169–1183 (2014) doi:10.1093/pcp/pcu057 ! The Author 2014.
Allosteric regulation of rice endosperm AGPase
Funding
This work was supported by the Japan Society for the
Promotion of Science [a Grant-in-Aid for Scientific Research
to H.S.]; the Division of Chemical Sciences, Geosciences, and
Biosciences, Office of Basic Energy Sciences of the US
Department of Energy [Grant DE-FG02-12ER20216 to T.W.O
and
S.-K.H.];
the
National
Science
Foundation
Intergovernmental Personnel Act Funds [T.W.O]; Project
0590, Agricultural Research Center, College of Agricultural,
Human, and Natural Resource Sciences, Washington State
University.
Disclosures
The authors have no conflicts of interest to declare.
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