Analysis of the Rice ADP-Glucose Transporter (OsBT1)
Indicates the Presence of Regulatory Processes in the
Amyloplast Stroma That Control ADP-Glucose
Flux into Starch1[OPEN]
Bilal Cakir, Shota Shiraishi, Aytug Tuncel, Hiroaki Matsusaka, Ryosuke Satoh, Salvinder Singh,
Naoko Crofts, Yuko Hosaka, Naoko Fujita, Seon-Kap Hwang, Hikaru Satoh*, and Thomas W. Okita*
Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164 (B.C., A.T., S.-K.H.,
T.W.O.); Faculty of Agriculture, Kyushu University, Fukuoka 812–8581, Japan (S.Sh., H.M., R.S., H.S.);
Department of Agricultural Biotechnology, Assam Agricultural University, Jorhat–785013, Assam, India (S.Si.);
and Department of Biological Production, Akita Prefectural University, Akita City, Akita 010–01195, Japan
(N.C., Y.H., N.F.)
ORCID IDs: 0000-0003-4610-0317 (B.C.); 0000-0002-4666-5679 (A.T.); 0000-0003-1439-412X (R.S.); 0000-0002-6792-2232 (N.C.);
0000-0002-6792-2232 (N.F.); 0000-0001-9552-5341 (S.-K.H.); 0000-0002-2246-0599 (T.W.O.).
Previous studies showed that efforts to further elevate starch synthesis in rice (Oryza sativa) seeds overproducing ADP-glucose
(ADPglc) were prevented by processes downstream of ADPglc synthesis. Here, we identified the major ADPglc transporter by
studying the shrunken3 locus of the EM1093 rice line, which harbors a mutation in the BRITTLE1 (BT1) adenylate transporter
(OsBt1) gene. Despite containing elevated ADPglc levels (approximately 10-fold) compared with the wild-type, EM1093 grains
are small and shriveled due to the reduction in the amounts and size of starch granules. Increases in ADPglc levels in EM1093
were due to their poor uptake of ADP-[14C]glc by amyloplasts. To assess the potential role of BT1 as a rate-determining step in
starch biosynthesis, the maize ZmBt1 gene was overexpressed in the wild-type and the GlgC (CS8) transgenic line expressing a
bacterial glgC-TM gene. ADPglc transport assays indicated that transgenic lines expressing ZmBT1 alone or combined with GlgC
exhibited higher rates of transport (approximately 2-fold), with the GlgC (CS8) and GlgC/ZmBT1 (CS8/AT5) lines showing
elevated ADPglc levels in amyloplasts. These increases, however, did not lead to further enhancement in seed weights even when
these plant lines were grown under elevated CO2. Overall, our results indicate that rice lines with enhanced ADPglc synthesis and
import into amyloplasts reveal additional barriers within the stroma that restrict maximum carbon flow into starch.
Cereal grains contribute a significant portion of
worldwide starch production. Unlike other plant tissue, starch biosynthesis in the endosperm storage
organ of cereal grains is unique in its dependence on
1
This work was supported by grants from the Japan Society for
the Promotion of Science (to H.S.), the Division of Chemical Sciences,
Geosciences, and Biosciences, Office of Basic Energy Sciences, U.S.
Department of Energy (grant no. DE–FG02–12ER20216 to S.-K.H.
and T.W.O.), the U.S. Department of Agriculture National Institute
of Food and Agriculture (project no. WNP00590), and the Agricultural Research Center, College of Agricultural, Human, and Natural
Resource Sciences, Washington State University.
* Address correspondence to [email protected] and hikaru.satoh.682@
m.kyushu-u.ac.jp.
The authors responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the
Instructions for Authors (www.plantphysiol.org) is: Thomas W. Okita
([email protected]) and Hikaru Satoh ([email protected]).
B.C., S.Sh., A.T., H.M., R.S. S.Si., N.C., Y.H. and S.-K.H. designed
and performed the experiments and analyzed the data; N.F., S.-K.H.,
H.S., and T.W.O. supervised the research; B.C., H.S., and T.W.O.
wrote the article with critical inputs from the other authors.
[OPEN]
Articles can be viewed without a subscription.
www.plantphysiol.org/cgi/doi/10.1104/pp.15.01911
two ADP-Glc pyrophosphorylase (AGPase) isoforms
(Denyer et al., 1996; Thorbjørnsen et al., 1996; Sikka
et al., 2001), a major cytosolic enzyme and a minor
plastidial one, to generate ADP-glucose (ADPglc), the
sugar nucleotide utilized by starch synthases in the
amyloplast (Cakir et al., 2015). The majority of ADPglc
in cereal endosperm is generated in the cytosol from
AGPase (Tuncel and Okita, 2013) as well as by Suc
synthase (Tuncel and Okita, 2013; Bahaji et al., 2014)
and subsequently transported into amyloplasts by the
BRITTLE-1 (BT1) protein located at the plastid envelope
(Cao et al., 1995; Shannon et al., 1998).
The Bt1 gene, first identified in maize (Zea mays;
Mangelsdorf, 1926) and isolated by Sullivan et al.
(1991), encodes a major amyloplast membrane protein
ranging from 39 to 44 kD (Cao et al., 1995). The BT1
protein and its homologs belong to the mitochondrial
carrier family (Sullivan et al., 1991; Haferkamp, 2007),
which has a diverse range of substrates (Patron et al.,
2004; Leroch et al., 2005; Kirchberger et al., 2008). The
assignment of BT1 protein as the ADPglc transporter in
cereal endosperms was first proposed by Sullivan et al.
(1991), and then it was characterized based on the increased ADPglc levels and reduced ADPglc import rate
Plant PhysiologyÒ, March 2016, Vol. 170, pp. 1271–1283, www.plantphysiol.org Ó 2016 American Society of Plant Biologists. All Rights Reserved.
Downloaded from on June 14, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
1271
Cakir et al.
in endosperms of BT1-deficient maize and barley
(Hordeum vulgare) mutants (Tobias et al., 1992; Shannon
et al., 1996, 1998; Patron et al., 2004). Biochemical transport studies of the maize BT1 showed that it imported
ADPglc by counter exchanging with ADP (Kirchberger
et al., 2007). The wheat (Triticum aestivum) BT1 homolog
also transports ADPglc but has similar affinities for ADP
and AMP as the counter-exchange substrate (Bowsher
et al., 2007).
Evidence from previous studies by our laboratory
(Sakulsingharoj et al., 2004; Nagai et al., 2009) suggested the potential role of BT1 as well as other
downstream processes as a rate-limiting step in starch
biosynthesis in the transgenic rice (Oryza sativa) GlgC
(CS8) lines overexpressing an up-regulated AGPase
(Escherichia coli glgC-TM). In GlgC (CS8) rice lines, grain
weights (starch) are elevated up to 15% compared with
wild-type plants, indicating that the AGPase-catalyzed
reaction is a rate-limiting step in starch biosynthesis
under normal conditions. When transgenic GlgC (CS8)
plants were grown under elevated CO2 levels, no further increases in grain weight were evident compared
with those grown at ambient CO2. As Suc levels are
elevated in leaf blades, leaf sheaths, culms (RowlandBamford et al., 1990), and peduncle exudates (Chen
et al., 1994) in rice plants grown under elevated CO2,
developing GlgC (CS8) grains were unable to convert
the increased levels of sugars into starch. This lack of
increase indicated that the AGPase-catalyzed reaction
(ADPglc synthesis) was no longer rate limiting and that
one or more downstream processes regulated carbon
flux from source tissues in developing GlgC (CS8) endosperm (Sakulsingharoj et al., 2004). This view is also
supported by a subsequent metabolite study in which
several GlgC (CS8) lines were found to contain up to
46% higher ADPglc levels than wild-type plants (Nagai
et al., 2009). As this increase in ADPglc levels was
nearly 3-fold higher than the increase in grain weight,
starch biosynthesis is saturated with respect to ADPglc
levels and carbon flow into starch is restricted by one or
more downstream steps. Potential events that may limit
the utilization of ADPglc in starch in GlgC (CS8) lines
are the import of this sugar nucleotide via the BT1
transporter into amyloplasts and/or the utilization of
ADPglc by starch synthases. Mutant analysis of the two
major starch synthases indicated no significant impact
on grain weight when one of these starch synthases was
nonfunctional, suggesting that this enzyme activity,
contributed by multiple enzyme isoforms, is present at
excessive levels (Fujita et al., 2006, 2007). Therefore, we
suspected that BT1 is the likely candidate limiting carbon flow into starch in GlgC (CS8) endosperms.
Figure 1. A and B, Seed morphology (A) and starch content (B) of the
wild-type (WT; cv TC65) and EM1093. C, Scanning electron micrographs of isolated starch granules and the surface of wild-type and
EM1093 endosperms. D, Effects of the shr3 mutation on the chain length
distribution of amylopectin from EM1093 compared with the wild-type.
The chain length distributions were characterized using 8-amino1,3,6-pyrenetrisulfonic capillary electrophoresis. The data are representative of three different experiments Average starch contents were
statistically different based on one-way ANOVA with Tukey’s multiple
comparison test (P , 0.05).
1272
Plant Physiol. Vol. 170, 2016
Downloaded from on June 14, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Role of ADPglc Transporter in Rice Endosperms
Table I. Segregation analysis of F2 seeds derived from crosses of EM1093 with EM540 and EM22 and of
EM1093 with wild-type cv TC65 lines
x 2 values are based on inheritance ratios of 9:7 for EM crosses and 3:1 for wild-type and EM crosses.
Cross Combination
EM540 (shr1) 3 EM1093
EM22 (shr2) 3 EM1093
Wild-type (cv TC65) 3 EM1093
F1 Seeds
Normal
Normal
Normal
The aim of this study was to investigate the role of
BT1 in mediating the transport of ADPglc into amyloplast and to determine whether this transport activity is
rate limiting in rice endosperm. In order to address
these questions, we show that BT1 is the major transporter of ADPglc by analysis of the EM1093 rice line,
which contains a mutation at the shrunken3 (shr3) locus
and, specifically, in the OsBt1-1 gene. Second, we
assessed the impact of the expression of the maize
ZmBt1 gene in wild-type and GlgC (CS8) seeds to determine the potential limiting role of BT1 transport activity on starch biosynthesis. Our results indicate that
BT1 is essential for starch synthesis but is not rate limiting and that one or more stroma-localized processes
limit maximum carbon flow into starch.
RESULTS
Genetic and Biochemical Characterization of the shr3
Mutant (EM1093)
EM1093 plants (shr3) produce seeds that have a
shriveled phenotype due to the severe reduction in starch
content (Fig. 1A). This phenotype is likely caused by a
defect in one of the major enzymes involved in starch
biosynthesis (Kawagoe et al., 2005; Tuncel et al., 2014).
Starch levels were significantly decreased around 80% in
shr3 seeds compared with the wild-type (Fig. 1B). The
morphology of starch granules and the chain length
distribution of amylopectin in wild-type and shr3 mutant
endosperms were investigated by scanning electron microscopy (SEM) and 8-amino-1,3,6-pyrenetrisulfonic
capillary electrophoresis, respectively. SEM imaging
showed that the starch granules in endosperms of shr3
were smaller and more spherical compared with those of
the wild-type (Fig. 1C). Moreover, the shr3 mutation
strongly affected the chain length distribution of amylopectin, as the proportion of chains with degree of polymerization less than 13 were elevated, whereas those of
chains with degree of polymerization greater than 14
were decreased (Fig. 1D). These results clearly indicate
that mutation in the shr3 locus results in depressing
starch content as well as altering starch granule morphology and amylopectin structure.
The shriveled grain phenotype of shr3 is similar to
that seen for the mutant lines EM541 (shr1) and EM22
(shr2), which also produce shrunken seeds due to the
lack of large (L2) and small (S2b) subunits, respectively, of the endosperm cytosolic AGPase (Kawagoe
et al., 2005; Tuncel et al., 2014). To determine whether
Segregation in F2
Wild-Type
shr
199
168
105
148
142
31
Total
x2
347
310
136
0.17
0.54
0.35
EM1093 contained a mutation in one of the structural
genes for the major cytoplasmic AGPase, the EM1093
line was crossed with EM541 (Tuncel et al., 2014) or
EM22 (Kawagoe et al., 2005), marker lines for the shr1
and shr2 loci, respectively. F1 seeds derived from both
the EM1093 3 EM541 and EM1093 3 EM22 crosses
were normal in appearance, whereas F2 seeds demonstrated a ratio of wild-type to shrunken phenotype that
fitted well to the expected 9:7 ratio of independent gene
inheritance (Table I). This F2 segregation pattern verifies that the shr3 gene locus of EM1093 is independent of both shr1 and shr2 loci. In addition, EM1093 was
also crossed with the wild-type parental cv Taichung
65 (TC65). F1 seeds exhibited a wild-type phenotype,
while the segregation mode of normal to shrunken
seeds fitted well to the expected ratio of a single inheritance, 3:1 (105:31), in the F2 progeny (Table I). These
results indicated that the shr3 mutation in EM1093 is
controlled by a single gene locus and is unrelated to the
shr1 and shr2 loci.
To further verify that the shr3 locus was not an AGPase
structural gene, immunoblot studies were conducted.
Total proteins, extracted from developing seeds of
EM1093, were subjected to immunoblot analysis using
anti-S2b (AGPase small subunit) and anti-L2 (AGPase
large subunit) antibodies. The results demonstrated that
the EM1093 line contains both S2b and L2 proteins (Fig.
2A), suggesting that the shriveled phenotype is likely due
to one of the other major starch biosynthetic enzymes.
In order to identify the proximate chromosome location of shr3, linkage analysis was performed by
crossing EM1093 (japonica) with the indica rice cv
Kasalath. Mapping studies showed that the shr3 gene
cosegregated with three markers, RM1347, C10005, and
C63223, on chromosome 2 (Fig. 2B; Supplemental Table
S1). The F2 population derived from the cross between
rice cv Kasalath and EM1093 indicated that the shr3
mutation cosegregated with the RM1347 marker
(Supplemental Table S1). This result clearly demonstrated that the shr3 locus is located on the short arm of
chromosome 2 (Fig. 2B).
Mutation in the Bt1 Gene Is Responsible for shr3
Analysis of the rice genomic map for genes located
near the RM1347 marker implicated the OsBt1-1 as a
potential candidate gene. A mutation in the OsBt1-1 gene,
which was initially determined by targeting induced local lesions in genomes (TILLING; Suzuki et al., 2008),
Plant Physiol. Vol. 170, 2016
1273
Downloaded from on June 14, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Cakir et al.
To verify the loss of the BT1 polypeptide, amyloplast membrane fractions were prepared from developing wild-type and EM1093 seeds, and extracted
proteins were resolved by SDS-PAGE. Polyacrylamide
gel slices containing polypeptide in the vicinity of 41
kD, the size of OsBT1, were treated with trypsin, and
eluted peptides were analyzed by high-resolution liquid
chromatography-tandem mass spectrometry (LC-MS/MS).
This analysis showed the presence of unique peptide
sequences spanning 45% coverage of the intact BT1
protein in wild-type membranes. By contrast, no BT1
peptides were detected from EM1093 (Table II), indicating that amyloplasts of EM1093 lack intact BT1 protein
(Table II). Overall, the results from crossing experiments,
proteomic analysis, and DNA sequencing verify that the
mutation at the shr3 locus is due to a disruption in the
OsBt1-1 gene and a loss of BT1 protein in EM1093.
Plastids Isolated from shr3 Endosperm Fail to Efficiently
Transport ADPglc
Figure 2. Characterization of the shr3 mutant (EM1093). A, Immunoblot analysis of proteins extracted from developing (14-DAF) seeds of
the wild-type (cv TC65) and endosperm mutant (EM) lines. EM1093
contains both AGPase subunits, as assessed by immunoblotting using
antibodies raised against AGPase L2 and S2b. B, Linkage map of chromosome 2. shr3 maps near the marker RM1347, where OsBt1-1 closely
resides. C, Location of the nonsense mutation in the OsBT1-1 gene.
A G-to-A mutation at the first exon results in a premature termination
codon in EM1093. WT, Wild-type.
was verified by DNA sequencing of OsBt1-1 genomic
DNA (GenBank accession no. NM_001052765). In
EM1093, a G-to-A replacement at position 513 in the
first exon was detected, resulting in a Trp codon being
converted to a nonsense codon (Trp-171Stop; Fig. 2C).
Such an error generates a truncated polypeptide (170
amino acids) that would be subject to rapid degradation
in the cell (Laughlin et al., 1998) or cause nonsensemediated mRNA decay (Isshiki et al., 2001). In either
instance, loss of intact BT1 polypeptide was expected.
Purified amyloplasts from the wild-type and EM1093
were tested for their transport of ADP-[14C]glc. An
approximate ADPglc import rate was determined
by measuring the uptake of ADP-[14C]glc and incorporation into starch (Table III; Fig. 3A). Significantly higher levels of ADP-[14C]Glc (approximately
245 nmol g21 fresh weight) were transported and incorporated into wild-type (cv Kitaake and cv TC65)
amyloplasts compared with EM1093 (shr3) endosperms (49 nmol g21 fresh weight) in the absence of
any counter-exchange adenylate nucleotides (Table
III; Fig. 3A). ADPglc import rates nearly doubled
when wild-type amyloplasts were preincubated with
either ADP or AMP, whereas EM1093 amyloplasts
failed to exhibit such an increase in ADPglc import
(Table III; Fig. 3A). ATP had no influence on ADPglc
transport (Fig. 3A), a result similar to those from an
earlier study of the wheat BT1 (Bowsher et al., 2007).
Overall, these results showed that the loss of BT1
protein in EM1093 results in poor ADPglc import and
incorporation into starch by intact amyloplasts and, in
turn, lower starch content (Figs. 1 and 3A; Table III).
The shr3 Mutant Accumulates Higher Levels of ADPglc
The loss of ADPglc transport activity into amyloplast
would likely elevate cytoplasmic ADPglc levels due to
their active synthesis by AGPase (Tuncel and Okita,
2013) or by Suc synthase (Bahaji et al., 2014). Estimation
of ADPglc levels from developing seed extracts showed
that EM1093 contained dramatically increased (approximately 10-fold) ADPglc levels compared with the
wild-type (Fig. 3B; Table IV). This result is consistent
with an earlier study in maize (Shannon et al., 1996),
where bt1 kernels contained 13-fold more ADPglc than
normal kernels. Additionally, compartmentalization of
ADPglc levels demonstrated that wild-type amyloplasts contained around 52% of the total ADPglc,
whereas most of the ADPglc in EM1093 existed in the
1274
Plant Physiol. Vol. 170, 2016
Downloaded from on June 14, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Role of ADPglc Transporter in Rice Endosperms
Table II. Identification of proteins from the wild-type and EM1093
Seed extracts from the wild-type and EM1093 were resolved by SDS-PAGE and the polyacrylamide gel
area containing 41-kD polypeptides treated with trypsin and resulting peptides analyzed by LC-MS/MS.
BT1 peptides were detected only in the wild-type and not in EM1093. Matched peptide values indicate the
number of unique peptides matched. Values in parentheses signify the percentage of sequence coverage
by peptide mass fingerprinting (PMF) using LC-MS/MS. ND, Not detected.
Identifier
Matched Peptides
Protein Name
Wild-Type
LOC_Os02g10800
LOC_Os10g26060
LOC_Os01g64700
LOC_Os05g33570
LOC_Os07g46460
ADPglc transporter (BT1)
Glutelin
Tubby-like F-box protein3
Pyruvate, phosphate dikinase1
Ferredoxin-dependent Glu synthase
7
2
1
1
1
(45)
(16)
(9)
(11)
(10)
EM1093
ND
3 (21)
1 (12)
1 (13)
1 (11)
et al., 1998) was transformed via Agrobacterium tumefaciens
into the various rice lines.
The generated transgenic plants were screened for the
expression of both GlgC-TM (bacterial AGPase) and
ZmBT1 proteins by immunoblot analysis using antiGlgC and anti-ZmBT1 antibodies. Transgenic plants
expressing the ZmBt1 cDNA (AT5 lines) alone or coexpressed with glgC-TM (CS8) during seed development
were identified by immunoblotting (Fig. 4). All ZmBt1expressing transgenic plants showed two polypeptide
bands corresponding to ZmBT1 at approximately 41 kD
(Shannon et al., 1998), whereas transgenic plants containing glgC-TM expressed a single band at 48 kD (Fig. 4).
These transgenic lines were further propagated until
homozygous T3 lines were identified in order to characterize the role of BT1 on starch biosynthesis.
cytosol (Fig. 3B; Table IV). Thus, the loss of BT1 protein
resulted in dramatically decreased ADPglc transport
into amyloplasts and, in turn, higher ADPglc levels in
cytosol of EM1093 seeds.
Introduction of ZmBt1 into Wild-Type and GlgC (CS8)
Transgenic Rice Lines
Results from previous studies suggested the possibility
that the OsBt1 activity limited carbon flux into starch,
especially in the starch-overaccumulating GlgC (CS8) rice
lines expressing an up-regulated bacterial AGPase
(Sakulsingharoj et al., 2004; Nagai et al., 2009). This potential rate-limiting role of the BT1 transporter could be
tested directly by overexpressing the native BT1 transporter in wild-type and GlgC (CS8) transgenic rice lines.
To verify the expression of native BT1 at the protein level,
an antibody was required. Despite repeated attempts,
however, we were unable to generate an antibody to the
OsBT1 polypeptide or selected peptide fragments. A BT1
antibody was available to the maize ortholog, ZmBT1.
Unfortunately, the antibody to the maize transporter did
not cross act with the rice OsBT1 protein. In view of these
circumstances, the maize ZmBt1 complementary DNA
(cDNA) under the control of the Globulin promoter (Wu
Higher ADPglc Uptake Is Readily Detected in Amyloplasts
Isolated from ZmBT1 (AT5) Transgenic Plants
Transgenic Lines Harboring ZmBt1 Exhibit Higher ADPglc
Transport Capacity
Intact amyloplasts were isolated from developing rice
seeds (12–15 d after pollination [DAP]) to investigate the
Table III. Uptake and incorporation of ADP-[14C]glc into starch by purified intact amyloplasts in the
presence or absence of the counter-exchange ADP
Total starch synthase activity was estimated by lysing the amyloplasts and measuring the amount of
incorporation of ADP-[14C]glc into starch. Each value represents the mean 6 SE of three independent
experiments.
Intact
Lines
Buffer
Wild-type (cv Kitaake)
ZmBT1 (AT5-1)
ZmBT1 (AT5-19)
GlgC (CS8)
GlgC/ZmBT1 (CS8/AT5-18)
GlgC/ZmBT1 (CS8/AT5-27)
GlgC/ZmBT1 (CS8/AT5-37)
Wild-type (cv TC65)
EM1093 (cv TC65)
235
445
450
265
466
440
454
245
49
6
6
6
6
6
6
6
6
6
33
43
34
23
41
44
50
33
7
Starch Synthase Activity
+ADP
nmol h21 g21
437 6 45
850 6 47
860 6 39
440 6 63
881 6 70
897 6 71
985 6 72
480 6 49
51 6 6
Buffer
fresh wt
145 6 13
153 6 11
150 6 9
125 6 7
147 6 12
144 6 11
149 6 13
125 6 18
43 6 5
Plant Physiol. Vol. 170, 2016
+ADP
140
152
154
120
141
154
139
135
41
6
6
6
6
6
6
6
6
6
11
10
11
8
16
19
17
15
3
1275
Downloaded from on June 14, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Cakir et al.
higher ADP-[14C]glc transport-incorporation rates
than the wild-type and GlgC (CS8; Fig. 5; Table III).
Irrespective of the rice line, preincubation of the intact amyloplasts with ADP stimulated ADP-[ 14 C]glc
transport-incorporation rates by nearly 2-fold. By contrast, ADP-[14C]glc incorporation into starch by ruptured
amyloplasts from the various rice lines was considerably
lower than the rates measured for intact amyloplasts.
Moreover, the rates of ADP-[14C]glc incorporation by
ruptured amyloplasts were not stimulated by ADP, unlike the rates observed for intact amyloplasts (Table III),
and did not vary from one rice line to another. Thus, the
observed increases in ADP-[14C]glc transport and incorporation into starch by intact amyloplasts from the various ZmBT1-expressing rice lines are due to enhanced
transport into the amyloplast stromal compartment and
not to differences in starch synthase activities. These results are consistent with the view that overexpression of
ZmBT1 leads to an increase in ADPglc uptake across the
amyloplast membrane.
ADPglc Levels in Transgenic Seeds
The contribution of elevated ADPglc transport capacity to ADPglc levels in transgenic rice seeds was
investigated. Transgenic ZmBT1 (AT5) plants expressing the maize BT1 alone contained ADPglc levels similar to those observed for the wild-type. Consistent with
our earlier results (Nagai et al., 2009), total ADPglc
levels in GlgC (CS8) plants were elevated around
40% compared with the wild-type (Table IV). ADPglc
levels were also increased in the double transgenic lines
(GlgC/ZmBT1) to the same extent (approximately 40%)
observed in GlgC (CS8) compared with the wild-type
(Table IV). Even though ZmBT1 (AT5) lines exhibited
higher in vitro transport rates of ADPglc (Fig. 5),
these transgenic plants failed to accumulate increased
ADPglc levels in amyloplasts compared with the
wild-type (Table IV). In both rice lines, about 50%
of the total ADPglc was evident in isolated amyloplasts. A much higher proportion (65%–69%) of total
ADPglc was associated with the amyloplasts of GlgC
(CS8) and GlgC/ZmBT1 (CS8/AT5) endosperms
(Table IV).
Figure 3. ADPglc incorporation and levels in wild-type and EM1093 rice
seeds. A, Uptake and incorporation of ADPglc into starch of purified amyloplasts
from the wild-type (WT) and EM1093 in the presence or absence of adenylates.
Amyloplasts were preloaded with buffer (black vertical lines), 20 mM ATP (blue
horizontal lines), 20 mM ADP (red checkered lines), or 20 mM AMP (green
crosswise lines) for 10 min. The reaction containing 100 mL of isolated amyloplasts was started by adding 4 mM ADP-[14C]glc. Uptake experiments were
performed at 30˚C for 30 min. FW, Fresh weight. B, ADPglc levels in wild-type
and EM1093 seeds were determined by a reverse direction assay. Data represent
means 6 SE of three independent experiments. Black vertical lines represent total
ADPglc levels, and blue horizontal lines represent amyloplast ADPglc levels.
role of the ZmBT1 transporter on ADPglc uptake capacity. Amyloplasts isolated from the transgenic lines
containing ZmBt1 (AT5-1, CS8/AT5-18, CS8/AT5-27,
and CS8/AT5-37) exhibited approximately 2-fold
Table IV. ADPglc levels in total and amyloplast fractions of wild-type and transgenic lines
Each value represents the mean 6
Lines
Wild-type (cv Kitaake)
ZmBT1 (AT5-1)
ZmBT1 (AT5-19)
GlgC (CS8-18)
GlgC/ZmBT1 (CS8/AT5-18)
GlgC/ZmBT1 (CS8/AT5-27)
GlgC/ZmBT1 (CS8/AT5-37)
Wild-type (cv TC65)
EM1093 (cv TC65)
SE
of three independent experiments.
Total
(nmol/seed)
Amyloplast
Percentage
of Amyloplast
6
6
6
6
6
6
6
6
6
nmol seed21
0.46 6 0.02
0.45 6 0.04
0.44 6 0.02
0.76 6 0.02
0.80 6 0.02
0.83 6 0.04
0.78 6 0.03
0.47 6 0.05
0.16 6 0.03
52.9
49.5
49.5
65.0
69.6
69.7
67.2
51.1
1.6
0.87
0.91
0.89
1.17
1.15
1.19
1.16
0.92
10.3
0.07
0.06
0.04
0.05
0.08
0.09
0.01
0.09
0.19
1276
Plant Physiol. Vol. 170, 2016
Downloaded from on June 14, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Role of ADPglc Transporter in Rice Endosperms
Figure 4. Immunoblot analysis of proteins extracted from developing
(14-DAF) seeds of the wild-type (WT) and GlgC (CS8) and ZmBT1 (AT5)
transgenic lines. CS8 lines, harboring the E. coli glgC-TM gene and AT5
lines, expressing the ZmBt1 gene, were analyzed using antibodies
raised against GlgC-TM and ZmBT1.
Analysis of Amylose and Amylopectin Content from
Wild-Type and Transgenic Endosperms
To test whether elevated ADPglc levels lead to a
modified starch form, the amylose and amylopectin
contents were investigated by gel filtration chromatography. GlgC (CS8) and GlgC/ZmBT1 (CS8/AT5)
lines contained higher amylose contents (fraction 1)
compared with wild-type endosperms (Table V). This is
most likely due to elevated ADPglc levels in these
transgenic lines (Table IV), since granule-bound starch
synthase I (GBSSI), which is responsible for amylose
formation, possesses lower affinity toward ADPglc
than the other starch synthase isozymes (Clarke et al.,
1999). By contrast, the amylose content from ZmBT1
(AT5) lines was not significantly different from that
seen for wild-type endosperms (Table V). There were
also no significant changes in the fraction III-fraction II
ratio between the transgenic lines and the wild-type
indicating that the chain length distribution of amylopectin was not affected (Table V).
Comparison of Seed Weight and Seed Morphology
between Wild-Type and Transgenic Plants
The effect of ZmBt1 introduction on starch biosynthesis was examined by measuring the average weight
of mature seeds from transgenic lines grown under
ambient and elevated CO2 conditions. Under ambient
conditions, ZmBT1 (AT5-1 and AT5-19) transgenic lines
did not demonstrate any significant changes in grain
weight compared with the wild-type (Table VI). By
contrast, as shown earlier (Nagai et al., 2009), the GlgC
(CS8) transgenic lines exhibited about an 11% increase
in grain weight, an increase that was also reflected in
grain size (Table VI). GlgC/ZmBT1 (CS8/AT5) lines
produced heavier seeds but were nearly identical in
weight to those from the CS8 line (Table VI).
As sugars provided by photosynthetic source leaves
may be limiting under ambient conditions, transgenic
plants were grown under elevated CO2 levels (1000 ml L21)
during the reproductive stage to investigate whether
these plants could utilize the extra Suc and incorporate it
into starch. Similar to the results obtained under ambient
CO2 levels, ZmBT1 (AT5) transgenic lines exhibited the
same grain weight as those from the wild-type (Table VI).
Although GlgC (CS8) lines produced heavier seeds, the
increases in seed weights were similar to those produced
under ambient CO2 conditions (Table VI). Likewise,
GlgC/ZmBT1 (CS8/AT5) lines also showed a similar
increase in grain weight, varying 11% to 13% from the
wild-type, but this increase was nearly identical to those
obtained under ambient conditions. Overall, ZmBT1
(AT5) and GlgC/ZmBT1 (CS8/AT5) lines failed to incorporate the excess assimilate generated by higher photosynthesis under elevated CO2 into starch compared with
the wild-type and CS8 lines, respectively.
Starch Turnover Does Not Account for the Lack of
Increased Starch Synthesis When ADPglc Is in Excess
The expression of a plastidial targeted bacterial
AGPase gene in Russet Burbank potato (Solanum
tuberosum) tubers resulted in a dramatic elevation of
starch content (Stark et al., 1992). A comparable study
(Sweetlove et al., 1996) in the potato cv Desiree, however, failed to observe enhanced starch content. Metabolic labeling analysis indicated that the failure of cv
Desiree tubers to accumulate more starch was likely
due to increased starch turnover (Sweetlove et al.,
1996). To directly assess whether starch turnover is a
major factor limiting carbon flux into starch in rice,
Figure 5. Uptake and incorporation of ADP-[14C]glc
into starch by purified amyloplasts from wild-type
(WT) and transgenic rice seeds. ADPglc import into
amyloplasts is shown for the wild-type, GlgC (CS8),
ZmBT1 (AT5), and GlgC/ZmBT1 (CS8/AT5) lines in
the presence or absence of adenylates. Amyloplasts
were preloaded with buffer (black vertical lines),
20 mM ATP (blue horizontal lines), 20 mM ADP (red
checkered lines), or 20 mM AMP (green crosswise
lines) for 10 min. Uptake experiments were performed at 30˚C for 30 min. Data represent means 6 SE
of three independent experiments. FW, Fresh weight.
Plant Physiol. Vol. 170, 2016
1277
Downloaded from on June 14, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Cakir et al.
Table V. Composition of carbohydrate in endosperm fractions of transgenic lines
Three fractions (I, II, and III) were detected by refractive index detectors. Fraction I is enriched with
amylose, whereas fractions II and III are enriched with amylopectin chains. Total carbohydrate content is
100%. Each value represents the mean 6 SE of three independent experiments.
Lines
Wild-type (cv Kitaake)
GlgC (CS8-3)
ZmBT1 (AT5)
GlgC/ZmBT1(CS8/AT5)
a
Fraction I
Fraction II
6
6
6
6
weight %
22.6 6 0.37
22.0 6 0.90
22.2 6 0.27
21.9 6 0.08
15.1
16.9
15.7
16.1
0.19
0.70a
0.33
0.16a
Fraction III
62.3
61.1
62.1
62.0
6
6
6
6
Fraction III:II Ratio
0.42
0.30
0.34
0.15
2.76
2.79
2.79
2.84
6
6
6
6
0.06
0.13
0.04
0.01
Significant differences between transgenic lines and the wild-type by Student’s t test at P , 0.05.
ADP-[14C]glc pulse-chase labeling studies were conducted (Fig. 6). Purified amyloplasts were incubated
with ADP-[14C]glc for 20 min and then chased with
excess nonradioactive ADPglc. No obvious loss of radioactivity from starch was detected in both wild-type
and CS8 amyloplasts up to 60 min of incubation. Hence,
starch turnover rates are very low and not responsible
for the inability of CS8 rice lines to increase starch
production when ADPglc is in excess.
DISCUSSION
In cereals, the majority of the endosperm ADPglc is
synthesized in the cytosol. Therefore, cereals are dependent on the BT1 transporter to import cytosolic
ADPglc into amyloplast, which is subsequently incorporated into starch (Denyer et al., 1996; Thorbjørnsen
et al., 1996; Sikka et al., 2001). This view is supported by
BT1-deficient mutants of maize kernels (Shannon et al.,
1998) and barley seeds (Patron et al., 2004), which had
drastically reduced starch content. Until now, however,
a detailed analysis of BT1 function in starch biosynthesis in rice endosperms was lacking.
In this study, EM1093 seeds contained dramatically
decreased starch content due to mutation in the shr3
locus. OsBt1-1 was initially identified as a potential
candidate gene underlying the shr3 locus by TILLING.
Subsequent DNA sequencing studies showed the
presence of a nonsense mutation in the OsBt1-1 gene
from EM1093 plants, resulting in a premature stop codon and, in turn, a loss of protein (Fig. 2C). Proteomic
analysis confirmed the absence of tryptic fragments
corresponding to intact OsBT1 from amyloplast membranes from EM1093 (Table II). Moreover, amyloplasts
from EM1093 (shr3) exhibited very low rates of ADPglc
transport into amyloplast (Fig. 3A; Table III), which is
consistent with the decreased seed weight. The severe
reduction in grain weight also indicates that the hexose
sugar (e.g. Glc-1-P and Glc-6-P) transporters, located in
the plastidial membrane, as well as the amyloplastlocalized AGPase are unable to compensate for the
loss of a functional BT1 transporter in shr3 endosperm.
Although EM1093 plants accumulated dramatically
increased ADPglc levels in the endosperm compared
with wild-type plants (Fig. 3B), their seeds only contained around 20% of normal starch content due to the
loss of BT1 protein and its transport activity. Overall,
BT1 is required for a normal rate of starch synthesis
during rice seed development.
ZmBT1 is targeted to the amyloplast via the presence
of a plastid-targeting leader sequence. Expression of
ZmBT1 produced a polypeptide doublet at approximately 41 kD in developing rice seeds as viewed by
immunoblotting (Fig. 4). An identical polypeptide
doublet was also observed in developing maize kernels by Shannon et al. (1998), who suggested that the
Table VI. Average weights of individual seeds from various rice lines
Mean weight values (mg) were measured from five plants per line. Asterisks indicate that average seed
weights were statistically different at the 95% confidence level compared with the wild-type. Percentage
values indicate the percentage of seed weight compared with wild-type cv Kitaake, with the exception of
EM1093, which was compared with wild-type cv TC65. ND, Not determined.
Wild-type (cv Kitaake)
ZmBT1 (AT5-1)
ZmBT1 (AT5-19)
GlgC (CS8-18)
GlgC/ZmBT1 (CS8/AT5-18)
GlgC/ZmBT1 (CS8/AT5-27)
GlgC/ZmBT1 (CS8/AT5-37)
Wild-type (cv TC65)
EM1093 (cv TC65)
High CO2
Ambient CO2
Lines
Seed Weight
Percentage
Seed Weight
Percentage
6
6
6
6
6
6
6
6
6
100
101
101
111
112
113
110
100
25
24.4 6 0.8
25.3 6 0.9
24.9 6 0.7
27.1 6 0.9*
27.4 6 1.0*
27.6 6 1.4*
27.1 6 0.8*
ND
ND
100
103
102
111
112
113
111
ND
ND
23.3
23.5
23.6
25.8
26.1
26.3
25.7
22.9
5.7
0.6
0.7
0.6
0.4*
0.7*
0.5*
0.5*
0.7
0.3
1278
Plant Physiol. Vol. 170, 2016
Downloaded from on June 14, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Role of ADPglc Transporter in Rice Endosperms
Figure 6. Pulse-chase studies using ADP-[14C]glc to
assess starch turnover in intact amyloplast. Wild-type
(WT; A) and GlgC (CS8; B) amyloplasts, preloaded
with ADP, were incubated in the presence of 0.1 mM
ADP-[14C]glc. After 20 min of incubation, a 100-fold
excess (10 mM) of unlabeled ADPglc was added to the
amyloplast mixture, and the extent of radioactivity in
starch was assessed. Data represent means 6 SE of
three independent experiments. FW, Fresh weight.
ZmBT1 precursor contained two peptide leader processing sites.
In vitro transport and starch incorporation studies
demonstrated that ADPglc transport was stimulated by
the presence of ADP or AMP (Figs. 3A and 5). This result is consistent with an earlier study from wheat
(Bowsher et al., 2007), in which ADPglc transport
was enhanced by ADP and AMP as counter-exchange
substrates. Interestingly, ATP had no impact on the
ADPglc uptake rate. As shown earlier (Bowsher et al.,
2007), ATP is the primary substrate for ADP/ATP
carriers on the plastid membranes but not for BT1.
Under physiological conditions, ADPglc is most likely
exchanged with ADP, since this adenylate nucleotide is
generated by the action of starch synthases during the
polymerization of Glc residues.
The transgenic lines, ZmBT1 (AT5) and GlgC/
ZmBT1 (CS8/AT5), had significantly higher ADP[14C]glc transport and starch incorporation rates by intact amyloplasts than those isolated from the wild-type
and GlgC rice lines lacking the maize BT1 transporter
(Fig. 5; Table III). Despite the significantly elevated
ADP-[14C]glc transport and starch incorporation rates
seen for amyloplasts from the ZmBT1 (AT5) transgenic
lines, these did not translate into higher amyloplast
content of ADPglc and higher seed weights, which
were no different from the wild-type values (Table VI).
Hence, ADP-[14C]glc transport and starch incorporation rates by purified amyloplasts are poor indicators of
the potential for seed weight increases. Better indicators
of increased carbon flux into starch and, in turn, higher
seed weights are total ADPglc levels per seed and
amounts in amyloplasts. While ADPglc levels in the
ZmBT1 rice line were no different from the quantities
measured in the wild-type, significantly elevated levels
of total ADPglc and amounts in amyloplasts were
readily evident in GlgC and GlgC/ZmBT1, rice lines
that bore seeds of 11% to 13% higher weight. Overall,
these results indicate that OsBT1 is not a limiting factor
in controlling carbon flux into starch.
The measured levels of ADPglc in developing rice
seeds were 0.87 nmol seed21 (38.8 nmol g21 fresh
weight) for the wild-type and up to 1.19 nmol seed21 for
the GlgC transgenic lines (Table IV). These estimated
Plant Physiol. Vol. 170, 2016
1279
Downloaded from on June 14, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Cakir et al.
amounts of ADPglc in developing rice seed (approximately 40 nmol g21 fresh weight) are about 3- to 4-fold
lower than the amounts (146 nmol g21 fresh weight)
enzymatically estimated in developing barley grains
(Tiessen et al., 2012). Moreover, the majority of ADPglc
(approximately 80%) was located in the cytosol of
barley endosperms, whereas approximately 50% of total ADPglc was found in the nonamyloplast fraction in
developing rice seeds (Table IV). It should be pointed
out that measured ADPglc levels in this study were
similar to the amounts estimated by LC-MS/MS analysis in an earlier study from this laboratory (Nagai
et al., 2009). For example, the wild-type was estimated
to contain about 0.72 nmol seed21 by LC-MS/MS, an
amount similar to the 0.87 nmol seed21 measured in this
study. Hence, the large 3- to 4-fold differences in ADPglc
levels between developing rice and barley grains likely
represent the innate properties of each plant species
and are not due to differences in the way the metabolite samples were prepared or measured between the
two studies.
The lack of further enhancement in seed weights by
CS8 or ZmBT1/CS8 transgenic rice lines grown under
elevated CO2 conditions compared with those grown
under ambient conditions (Table VI) clearly indicate
that BT1 transport or AGPase activities do not limit
carbon flux into starch. The expression of maize sh2
AGPase genes in maize (Hannah et al., 2012), rice
(Smidansky et al., 2003), and wheat (Smidansky et al.,
2002) increased crop yields by increasing the number of
grains produced per plant. Analyses of the various
GlgC and/or ZmBT1 transgenic plants cultured in
growth chambers indicate no significant increases in
total seed number or total number of panicles per plant
(data not shown). We should emphasize, however, that
such yield studies require field-grown plants to eliminate limitations in photosynthesis and root growth
from attaining maximum seed yields per plant.
Since the increases in ADPglc amounts in CS8 transgenic lines (Table IV) did not result in corresponding
increases in grain weight (Table VI), these results point
out that one or more processes located in the stroma
control starch synthesis when ADPglc is in excess (Fig.
7). If ADPglc transport activity and levels are not barriers, what is responsible for limiting carbon flow into
starch in the GlgC (CS8) rice lines? One possible factor
limiting the carbon flux into starch could be simultaneously occurring degradation of starch coupled with
synthesis, as observed in GlgC-overexpressing cv Desiree
tubers (Sweetlove et al., 1996). Similarly, down-regulation
of glucan, water-dikinase, an enzyme that facilitates
starch degradation, resulted in enhanced wheat grain
yields and increases in biomass (Ral et al., 2012). Hence,
increases in starch turnover, which may counterbalance
increases in starch synthesis, may be responsible for the
absence of any further increases in starch synthesis and
accumulation, especially when rice plants are grown
under elevated CO2 conditions. Results from the ADP[14C]glc pulse-chase study using purified amyloplasts
indicate, however, that very little, if any, starch turnover
occurs in the CS8 line (Fig. 6). Hence, starch turnover is
unlikely to be the causal basis for the inability of CS8 lines
to increase starch production.
A second process that may limit carbon flow into
starch centers on the starch synthases. We had earlier
discounted this possibility and focused on the ADPglc
transporter, as genetic mutant studies showed that loss
of action of the major starch synthase isoforms, SSI and
Figure 7. Summary of ADPglc compartmentalization in the wild-type and transgenic lines. A, ADPglc levels are present at nearly
equal amounts in the cytosol and amyloplasts in wild-type (WT) and ZmBT1 (AT5) lines. B, ADPglc levels in GlgC (CS8) and GlgC/
ZmBT1 (CS8/AT5) lines are increased significantly (approximately 80%) in amyloplasts while decreased slightly (approximately
9%) in the cytosol compared with the wild-type and ZmBT1 (AT5) lines. This nearly doubling of ADPglc levels, however, only
translated into an approximately 11% increase (on average based on three independent transgenic lines) in starch content,
indicating that the maximum flux of carbon into starch is tightly regulated. This slowing in net starch synthesis, mediated by
elevated ADPglc levels, is probably not caused by starch turnover but more likely by a reduction in one or more starch synthase
(SS) isoform enzyme activities.
1280
Plant Physiol. Vol. 170, 2016
Downloaded from on June 14, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Role of ADPglc Transporter in Rice Endosperms
SSIII, did not have a significant impact on starch synthesis and accumulation. Mutant lines that lack one of
these major starch synthase isoforms had starch levels
similar to that seen for the wild-type (Fujita et al., 2006,
2007). The loss or reduction in catalytic activity of one of
these enzymes is apparently compensated by one or
more of the other starch synthase isoforms.
Although genetic studies (Fujita et al., 2006, 2007) imply
that the major starch synthases (SSI and SSIIIa) do not limit
starch synthesis in endosperm under normal conditions,
their catalytic activities may be rate determining in the
transgenic rice GlgC (CS8) lines, where ADPglc is in excess
(Fig. 7). Indeed, this view is supported by the intracellular
distribution of ADPglc in the GlgC (CS8) transgenic lines
(Table IV). Irrespective of the presence or absence of maize
BT1, the transgenic GlgC (CS8) lines showed elevated
amounts of ADPglc in the amyloplast compared with the
wild-type or ZmBT1 (AT5) lines (Table IV). This result
supports the view that the catalytic activity of one or more
of the starch synthase isoforms is insufficient to accommodate the excess ADPglc present in the amyloplast.
A significant reduction in starch synthase activity in
the GlgC (CS8) lines may be the result of the plant’s
responses to excess ADPglc. Earlier metabolite analysis showed that CS8 lines not only contain elevated
levels of ADPglc but also proportional increases in
upstream metabolites, UDPglc, Fru, Glc-6-P, and Glc-1-P,
and possibly Glc and Suc (Nagai et al., 2009). The
hexose sugars and Suc are well-documented signaling
molecules that control gene and protein expression
(Tognetti et al., 2013; Sheen, 2014), and the elevation of
these sugars will likely lead to the reprogramming of
gene expression. Indeed, preliminary RNA sequencing
studies of the wild-type and CS8 lines show significant
alterations in the steady-state RNA levels for several
starch biosynthetic enzymes, including significant depression of SSIII expression and an increase in invertase
RNA sequences. Moreover, elevated expression of a
starch-binding domain protein (SBDP) was observed.
This SBDP gene has only been identified by computational analysis of the rice genome; hence, nothing is
known about whether it has a role in starch synthesis.
Other studies have demonstrated that nonenzymatic
carbohydrate-binding proteins play major roles in starch
biosynthesis. A novel gene, FLOURY ENDOSPERM6
(FLO6), encodes for a CBM48-containing protein, which
interacts with ISOAMYLASE1 and presumably facilitates its debranching activity in rice (Peng et al., 2014).
Transgenic plants lacking the FLO6 protein have lower
starch content and modified starch granules. Additionally, a novel protein containing a CBM48 domain localizes the GBSS to starch granules (Seung et al., 2015). The
absence of this starch-targeting protein resulted in amylosefree starch in Arabidopsis (Arabidopsis thaliana) leaves.
Therefore, other carbohydrate-binding module-containing
proteins may take part in starch biosynthesis, either as
accessory proteins to known starch biosynthetic enzymes, which normally lack carbohydrate-binding domains, or potentially as regulators controlling the flux of
carbon into starch.
Overall, our results demonstrated that ADPglc
transport by BT1 is essential for the normal rate of
starch synthesis in rice endosperm. On the other hand,
enhanced ADPglc import did not further increase the
sink strength of developing rice seeds, indicating that
one or more biochemical processes in the amyloplast
stroma control carbon flux into starch. Ongoing efforts
by this laboratory are directed at identifying these
limiting factors and elucidating their roles in regulating
starch biosynthesis as a means to increase the yields of
this cereal grain that feeds a large proportion of the
world’s population.
MATERIALS AND METHODS
Generation and Screening of the Endosperm Mutant
The sh3 mutation in the rice (Oryza sativa) EM1093 line was generated by
N-methyl-N-nitrosourea treatment of independent fertilized egg cells of the
japonica cv TC65, as described previously (Satoh and Omura, 1979). Segregation
analysis was performed by crossing shr3 with cv TC65 (wild type), EM541
(shr1), and EM22 (shr2). RFLP analysis was performed by crossing the indica cv
Kasalath with EM1093. The rice plants were grown under natural conditions at
the Kyushu University experimental field plots. A mutation in the OsBt1-1 gene
of EM1093 was initially identified by TILLING (Suzuki et al., 2008) and subsequently confirmed by DNA sequencing of the OsBt1-1 gene.
Plasmid Construction and Rice Transformation
The plant expression vector (pAT5) was generated by cloning the ZmBt1
cDNA under the control of the rice endosperm-specific Globulin promoter
(Zheng et al., 1993) into pCAMBIA 1303. pAT5 was then transformed into wildtype cv Kitaake and the transgenic CS8 line via Agrobacterium tumefaciens AGL1
(Toki, 1997). Identification of the transgenic plants expressing both the glgC-TM
(Sakulsingharoj et al., 2004) and ZmBT1 genes was carried out by histochemical
GUS staining. These plants were further screened for the expression of both
GlgC-TM and ZmBT1 proteins by immunoblot analysis using anti-GlgC and
anti-ZmBT1 antibodies as described previously (Cao et al., 1995; Sakulsingharoj
et al., 2004). T3 lines homozygous for both transgenes were used in this study.
Plant Growth under the Elevated CO2 Condition
Wild-type, single transgenic (ZmBT1 overexpressing), and double transgenic
(ZmBT1 and GlgC-TM overexpressing) rice plants were grown in a controlled
chamber under a 12-h photoperiod at 26°C during the day and 22°C at night.
The relative humidity and photosynthetic flux density was set to 70% and
2,000 mmol m22 s21, respectively. The plants were grown at ambient CO2 levels
(400 ml L21) until the reproductive stage and then were subjected to elevated
CO2 levels (1000 ml L21) to maturity.
Seed Weight Measurements
At maturity, all seeds from individual plants were harvested, dried, and
measured for total yield and average grain weight as described previously
(Nagai et al., 2009). Briefly, panicles from transgenic plants were grown in a
growth chamber for two consecutive periods and then harvested. The average
grain weight of each transgenic plant was measured by weighing 100 seeds
from five panicles.
Amyloplast Isolation from Developing Rice Endosperm
Amyloplasts were isolated from developing rice seeds (12–15 DAP) as described previously (Sikka et al., 2001). After removal of the seed hull, the ends of
developing seeds (approximately 2 g) were carefully removed with forceps and
transferred into 5 mL of extraction buffer (0.8 M sorbitol, 50 mM HEPES-KOH,
pH 7.5, 1 mM EDTA, 2 mM MgCl2, 10 mM KCl, 5 mM dithiothreitol, and
0.1% (w/v) bovine serum albumin) for plasmolysis. After incubation in the
extraction buffer on ice for 30 min, the seeds were gently chopped with a razor
Plant Physiol. Vol. 170, 2016
1281
Downloaded from on June 14, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Cakir et al.
blade. The homogenate was filtered through four layers of Miracloth (Calbiochem),
and the filtrate was kept on ice for 3 h to sediment amyloplasts via gravity. The
pellet, which consists of enriched amyloplasts, was resuspended in extraction
buffer. To check the intactness of the plastids, one-half of the isolated amyloplasts
were disrupted by the addition of 0.5% Triton X-100 (v/v), agitated for 1 min, and
centrifuged at 20,000g for 10 min. The yield of intact amyloplasts was estimated at
about 25%, based on the relative amounts of the plastid enzyme marker, inorganic
pyrophosphatase, recovered in the amyloplast fraction, as described previously
(Sikka et al., 2001).
Analysis of Starch Granules of cv TC65 and
EM1093 Endosperms
Starch granule morphology was investigated by SEM as described previously (Wong et al., 2003). The chain length distribution of amylopectin isolated
from wild-type and EM1093 endosperms was performed by a modified method
(O’Shea et al., 1998) as described previously (Nakamura et al., 2002).
Size Fractionation of Debranched Starch and Amylose
Content Analysis from Wild-Type and Transgenic Lines
Powdered mature rice endosperm samples were gelatinized and debranched
by Pseudomonas spp. isoamylase (Hayashibar) as described previously (Crofts
et al., 2012). The samples were then analyzed using a Toyopearl HW55S gel
filtration column (300 3 20 mm) connected to three Toyopearl HW50S columns
(300 3 20 mm; Fujita et al., 2007). The eluted glucans were detected with a
refractive index detector (Tosoh RI-8020).
reverse AGPase reaction coupled with phosphoglucomutase and Glc-6-P dehydrogenase reactions as described previously (Seferoglu et al., 2014). The reaction was initiated by the addition of excess amounts of near homogenously
pure potato AGPase and performed at 37°C for 20 min in reaction buffer containing 100 mM HEPES, pH 7, 5 mM dithiothreitol, 5 mM 3-phosphoglyceric acid,
10 mM MgCl2, 0.6 mM NAD+, 0.4 mg mL21 bovine serum albumin, 0.5 units of
Glc-6-P dehydrogenase, and 0.5 units of phosphoglucomutase. The reaction
was terminated at 100°C and clarified by centrifugation, and the amount of
NADH was determined by measuring the A340. A standard curve for determining the amounts of ADPglc was generated by incubating known amounts of
ADPglc in the coupled enzyme reaction mixtures. The total amount of ADPglc
in amyloplasts was estimated by factoring in the percentage recovery of intact
amyloplasts using inorganic pyrophosphatase as a plastid marker enzyme as
described earlier.
Supplemental Data
The following supplemental materials are available.
Supplemental Table S1. Linkage analysis of shr3 with three marker genes
on chromosome 2.
ACKNOWLEDGMENTS
We thank Thomas D. Sullivan (University of Wisconsin School of Medicine)
for providing the maize ZmBt1 cDNA and BT1 antibody.
Received December 17, 2015; accepted January 7, 2016; published January 11,
2016.
Synthesis of ADP-[14C]glc
ADP-[14C]glc was prepared from [14C]Glc-1-P as described previously
(Preiss and Greenberg, 1972), except that the product was separated by thinlayer chromatography (TLC) instead of paper chromatography. Briefly, 25 mCi
of [14C]Glc-1-P was converted to ADP-[14C]glc using purified potato (Solanum
tuberosum) AGPase as described (Hwang et al., 2004). The final reaction mixture
was resolved by TLC for 7 h in solvent containing 95% ethanol:1 M ammonium
acetate, pH 7.5 (5:2). Hexose sugars were detected by spraying the TLC plate
with 10% (v/v) H2SO4 in methanol and then drying at 120°C for 10 min to visualize
the sugars. The top of the chromatogram containing ADPglc was collected and
then washed with absolute ethanol for 3 h to remove residual ammonium acetate.
After drying, ADP-[14C]glc was eluted with water. Based on the initial starting
amount of [14C]Glc-1-P, the yield of ADP-[14C]glc was about 70%, as determined by
absorption at 260 nm and liquid scintillation spectrometry.
ADP-[14C]glc Transport Assay
The transport of ADPglc by intact amyloplasts was performed as described
previously (Shannon et al., 1998). Isolated amyloplasts (100 mL) were transferred into a reaction mixture containing 100 mM Bicine, 0.5 M sorbitol, 12.5 mM
EDTA, 50 mM potassium acetate, 10 mM glutathione, and 4 mM ADP-[14C]glc
(specific activity of 150 cpm nmol21). When evaluating the counter-exchange
substrates, amyloplasts were preloaded with 20 mM AMP, ADP, or ATP before
carrying out uptake-starch incorporation assays. The reaction was carried out at
30°C for 30 min and terminated by the addition of 2 mL of 75% methanol
containing 1% (w/v) KCl. The insoluble starch was collected by centrifugation
at 2,000g for 10 min and washed twice with methanol/KCl to remove excess
substrate. The pellet was then resuspended in water, and the amount of ADP[14C]glc converted into starch was quantified by liquid scintillation spectrometry.
Starch turnover was investigated by performing pulse-chase studies. Briefly,
amyloplasts (100 mL), preincubated with 20 mM ADP, were used for the incorporation assay in the presence of 0.1 mM ADP-[14C]glc (specific activity of
900 cpm nmol21). After 20 min of uptake and incorporation, a 100-fold excess of
unlabeled ADPglc was added, and the reaction was incubated for up to an
additional 60 min.
Measurement of ADPglc Levels
Purified amyloplasts were isolated from 50 developing rice seeds (10–13
DAP), lysed by the addition of 0.5% [v/v] Triton X-100, and then boiled for
2 min to inactivate native AGPase activity. ADPglc content was then estimated
for the purified amyloplast fraction and total seed extract by performing the
LITERATURE CITED
Bahaji A, Li J, Sánchez-López ÁM, Baroja-Fernández E, Muñoz FJ,
Ovecka M, Almagro G, Montero M, Ezquer I, Etxeberria E, et al (2014)
Starch biosynthesis, its regulation and biotechnological approaches to
improve crop yields. Biotechnol Adv 32: 87–106
Bowsher CG, Scrase-Field EF, Esposito S, Emes MJ, Tetlow IJ (2007)
Characterization of ADP-glucose transport across the cereal endosperm
amyloplast envelope. J Exp Bot 58: 1321–1332
Cakir B, Tuncel A, Hwang SK, Okita T (2015) Increase of grain yields by
manipulating starch biosynthesis. In Y Nakamura, ed, Starch. Springer,
Tokyo Japan, pp 371–395
Cao H, Sullivan TD, Boyer CD, Shannon JC (1995) Btl, a structural gene
for the major 39-44 kDa amyloplast membrane polypeptides. Physiol
Plant 95: 176–186
Chen C, Li C, Sung J (1994) Carbohydrate metabolism enzymes in CO2‐
enriched developing rice grains of cultivars varying in grain size.
Physiol Plant 90: 79–85
Clarke BR, Denyer K, Jenner CF, Smith AM (1999) The relationship between the rate of starch synthesis, the adenosine 59-diphosphoglucose
concentration and the amylose content of starch in developing pea
embryos. Planta 209: 324–329
Crofts N, Abe K, Aihara S, Itoh R, Nakamura Y, Itoh K, Fujita N (2012)
Lack of starch synthase IIIa and high expression of granule-bound starch
synthase I synergistically increase the apparent amylose content in rice
endosperm. Plant Sci 193-194: 62–69
Denyer K, Dunlap F, Thorbjørnsen T, Keeling P, Smith AM (1996) The
major form of ADP-glucose pyrophosphorylase in maize endosperm is
extra-plastidial. Plant Physiol 112: 779–785
Fujita N, Yoshida M, Asakura N, Ohdan T, Miyao A, Hirochika H, Nakamura Y
(2006) Function and characterization of starch synthase I using mutants in
rice. Plant Physiol 140: 1070–1084
Fujita N, Yoshida M, Kondo T, Saito K, Utsumi Y, Tokunaga T, Nishi A,
Satoh H, Park JH, Jane JL, et al (2007) Characterization of SSIIIadeficient mutants of rice: the function of SSIIIa and pleiotropic effects
by SSIIIa deficiency in the rice endosperm. Plant Physiol 144: 2009–2023
Haferkamp I (2007) The diverse members of the mitochondrial carrier
family in plants. FEBS Lett 581: 2375–2379
Hannah LC, Futch B, Bing J, Shaw JR, Boehlein S, Stewart JD, Beiriger R,
Georgelis N, Greene T (2012) A shrunken-2 transgene increases maize
yield by acting in maternal tissues to increase the frequency of seed
development. Plant Cell 24: 2352–2363
1282
Plant Physiol. Vol. 170, 2016
Downloaded from on June 14, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Role of ADPglc Transporter in Rice Endosperms
Hwang SK, Salamone PR, Kavakli H, Slattery CJ, Okita TW (2004) Rapid
purification of the potato ADP-glucose pyrophosphorylase by
polyhistidine-mediated chromatography. Protein Expr Purif 38: 99–107
Isshiki M, Yamamoto Y, Satoh H, Shimamoto K (2001) Nonsensemediated decay of mutant waxy mRNA in rice. Plant Physiol 125:
1388–1395
Kawagoe Y, Kubo A, Satoh H, Takaiwa F, Nakamura Y (2005) Roles of
isoamylase and ADP-glucose pyrophosphorylase in starch granule
synthesis in rice endosperm. Plant J 42: 164–174
Kirchberger S, Leroch M, Huynen MA, Wahl M, Neuhaus HE, Tjaden J
(2007) Molecular and biochemical analysis of the plastidic ADP-glucose
transporter (ZmBT1) from Zea mays. J Biol Chem 282: 22481–22491
Kirchberger S, Tjaden J, Neuhaus HE (2008) Characterization of the
Arabidopsis Brittle1 transport protein and impact of reduced activity on
plant metabolism. Plant J 56: 51–63
Laughlin MJ, Chantler SE, Okita TW (1998) N- and C-terminal peptide
sequences are essential for enzyme assembly, allosteric, and/or catalytic
properties of ADP-glucose pyrophosphorylase. Plant J 14: 159–168
Leroch M, Kirchberger S, Haferkamp I, Wahl M, Neuhaus HE, Tjaden J
(2005) Identification and characterization of a novel plastidic adenine nucleotide uniporter from Solanum tuberosum. J Biol Chem 280: 17992–18000
Mangelsdorf PC (1926) Genetics and Morphology of Some Endosperm Characters in Maize. Connecticut Agricultural Experiment Station, New Haven
Nagai YS, Sakulsingharoj C, Edwards GE, Satoh H, Greene TW, Blakeslee
B, Okita TW (2009) Control of starch synthesis in cereals: metabolite
analysis of transgenic rice expressing an up-regulated cytoplasmic ADPglucose pyrophosphorylase in developing seeds. Plant Cell Physiol 50:
635–643
Nakamura Y, Sakurai A, Inaba Y, Kimura K, Iwasawa N, Nagamine T
(2002) The fine structure of amylopectin in endosperm from Asian cultivated rice can be largely classified into two classes. Starke 54: 117–131
O’Shea MG, Samuel MS, Konik CM, Morell MK (1998) Fluorophoreassisted carbohydrate electrophoresis (FACE) of oligosaccharides: efficiency of labelling and high-resolution separation. Carbohydr Res 307:
1–12
Patron NJ, Greber B, Fahy BF, Laurie DA, Parker ML, Denyer K (2004)
The lys5 mutations of barley reveal the nature and importance of plastidial ADP-Glc transporters for starch synthesis in cereal endosperm.
Plant Physiol 135: 2088–2097
Peng C, Wang Y, Liu F, Ren Y, Zhou K, Lv J, Zheng M, Zhao S, Zhang L,
Wang C, et al (2014) FLOURY ENDOSPERM6 encodes a CBM48
domain-containing protein involved in compound granule formation
and starch synthesis in rice endosperm. Plant J 77: 917–930
Preiss J, Greenberg E (1972) ADP-[14C]glucose. Methods Enzymol 28: 279–281
Ral JP, Bowerman AF, Li Z, Sirault X, Furbank R, Pritchard JR, Bloemsma
M, Cavanagh CR, Howitt CA, Morell MK (2012) Down-regulation of
glucan, water-dikinase activity in wheat endosperm increases vegetative
biomass and yield. Plant Biotechnol J 10: 871–882
Rowland-Bamford AJ, Allen LH Jr, Baker JT, Boote KJ (1990) Carbon
dioxide effects on carbohydrate status and partitioning in rice. J Exp Bot
41: 1601–1608
Sakulsingharoj C, Choi SB, Hwang SK, Edwards GE, Bork J, Meyer CR,
Preiss J, Okita TW (2004) Engineering starch biosynthesis for increasing
rice seed weight: the role of the cytoplasmic ADP-glucose pyrophosphorylase. Plant Sci 167: 1323–1333
Satoh H, Omura T (1979) Induction of mutation by the treatment of fertilized egg cell with N-methyl-N-nitrosourea in rice. Journal of the
Faculty of Agriculture-Kyushu University (Japan) 24: 165–174
Seferoglu AB, Koper K, Can FB, Cevahir G, Kavakli IH (2014) Enhanced
heterotetrameric assembly of potato ADP-glucose pyrophosphorylase
using reverse genetics. Plant Cell Physiol 55: 1473–1483
Seung D, Soyk S, Coiro M, Maier BA, Eicke S, Zeeman SC (2015) PROTEIN
TARGETING TO STARCH is required for localising GRANULE-BOUND
STARCH SYNTHASE to starch granules and for normal amylose synthesis
in Arabidopsis. PLoS Biol 13: e1002080
Shannon JC, Pien FM, Cao H, Liu KC (1998) Brittle-1, an adenylate
translocator, facilitates transfer of extraplastidial synthesized ADPglucose into amyloplasts of maize endosperms. Plant Physiol 117:
1235–1252
Shannon JC, Pien FM, Liu KC (1996) Nucleotides and nucleotide sugars in
developing maize endosperms (synthesis of ADP-glucose in brittle-1).
Plant Physiol 110: 835–843
Sheen J (2014) Master regulators in plant glucose signaling networks. J
Plant Biol 57: 67–79
Sikka VK, Choi SB, Kavakli IH, Sakulsingharoj C, Gupta S, Ito H, Okita
TW (2001) Subcellular compartmentation and allosteric regulation of the
rice endosperm ADPglucose pyrophosphorylase. Plant Sci 161: 461–468
Smidansky ED, Clancy M, Meyer FD, Lanning SP, Blake NK, Talbert LE,
Giroux MJ (2002) Enhanced ADP-glucose pyrophosphorylase activity in
wheat endosperm increases seed yield. Proc Natl Acad Sci USA 99:
1724–1729
Smidansky ED, Martin JM, Hannah LC, Fischer AM, Giroux MJ (2003)
Seed yield and plant biomass increases in rice are conferred by deregulation of endosperm ADP-glucose pyrophosphorylase. Planta 216: 656–
664
Stark DM, Timmerman KP, Barry GF, Preiss J, Kishore GM (1992) Regulation of the amount of starch in plant tissues by ADP glucose pyrophosphorylase. Science 258: 287–292
Sullivan TD, Strelow LI, Illingworth CA, Phillips RL, Nelson OE Jr (1991)
Analysis of maize brittle-1 alleles and a defective Suppressor-mutatorinduced mutable allele. Plant Cell 3: 1337–1348
Suzuki T, Eiguchi M, Kumamaru T, Satoh H, Matsusaka H, Moriguchi K,
Nagato Y, Kurata N (2008) MNU-induced mutant pools and high performance TILLING enable finding of any gene mutation in rice. Mol
Genet Genomics 279: 213–223
Sweetlove LJ, Burrell MM, ap Rees T (1996) Starch metabolism in tubers of
transgenic potato (Solanum tuberosum) with increased ADPglucose
pyrophosphorylase. Biochem J 320: 493–498
Thorbjørnsen T, Villand P, Denyer K, Olsen OA, Smith AM (1996) Distinct isoforms of ADPglucose pyrophosphorylase occur inside and
outside the amyloplasts in barley endosperm. Plant J 10: 243–250
Tiessen A, Nerlich A, Faix B, Hümmer C, Fox S, Trafford K, Weber H,
Weschke W, Geigenberger P (2012) Subcellular analysis of starch metabolism in developing barley seeds using a non-aqueous fractionation
method. J Exp Bot 63: 2071–2087
Tobias RB, Boyer CD, Shannon JC (1992) Alterations in carbohydrate intermediates in the endosperm of starch-deficient maize (Zea mays L.)
genotypes. Plant Physiol 99: 146–152
Tognetti JA, Pontis HG, Martínez-Noël GM (2013) Sucrose signaling in
plants: a world yet to be explored. Plant Signal Behav 8: e23316
Toki S (1997) Rapid and efficient Agrobacterium-mediated transformation
in rice. Plant Mol Biol Rep 15: 16–21
Tuncel A, Kawaguchi J, Ihara Y, Matsusaka H, Nishi A, Nakamura T,
Kuhara S, Hirakawa H, Nakamura Y, Cakir B, et al (2014) The rice
endosperm ADP-glucose pyrophosphorylase large subunit is essential
for optimal catalysis and allosteric regulation of the heterotetrameric
enzyme. Plant Cell Physiol 55: 1169–1183
Tuncel A, Okita TW (2013) Improving starch yield in cereals by overexpression of ADPglucose pyrophosphorylase: expectations and unanticipated outcomes. Plant Sci 211: 52–60
Wong KS, Kubo A, Jane JL, Harada K, Satoh H, Nakamura Y (2003)
Structures and properties of amylopectin and phytoglycogen in the
endosperm of sugary-1 mutants of rice. J Cereal Sci 37: 139–149
Wu CY, Adach T, Hatano T, Washida H, Suzuki A, Takaiwa F (1998)
Promoters of rice seed storage protein genes direct endosperm-specific
gene expression in transgenic rice. Plant Cell Physiol 39: 885–889
Zheng Z, Kawagoe Y, Xiao S, Li Z, Okita T, Hau TL, Lin A, Murai N
(1993) 59 distal and proximal cis-acting regulator elements are required
for developmental control of a rice seed storage protein glutelin gene.
Plant J 4: 357–366
Plant Physiol. Vol. 170, 2016
1283
Downloaded from on June 14, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.