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Rice Science, 2020, 27(2): 152í161

Research Paper

Expression Profiles and Protein Complexes of Starch

Biosynthetic Enzymes from White-Core and Waxy Mutants
Induced from High Amylose Indica Rice

CHEN Yaling1, 2, PANG Yuehan1, BAO Jinsong1

(1Institute of Nuclear Agricultural Sciences, College of Agriculture and Biotechnology, Huajiachi Campus, Zhejiang University,
Hangzhou 310029, China; 2College of Life Sciences, Jiangxi Normal University, Nanchang 330022, China)

Abstract: Physicochemical properties of endosperm starches in milled rice determine its cooking and
eating quality. Amylose is synthesized by granule-bound starch synthase I (GBSSI), whilst amylopectin is
synthesized by the synergistic activities of starch synthases (SSs), branching enzymes (BEs) and
debranching enzymes (DBEs). However, the complexes formed by starch biosynthetic enzymes are not
well characterized. Gene expression profiles and protein complexes were determined in white-core
(GM645) and waxy (GM077) mutants derived from a high amylose indica rice Guangluai 4 (GLA4). In
GM645, genes including AGPS1, GBSSI, SSIIa, BEI, BEIIa, BEIIb, PUL, ISA1 and SP were significantly
downregulated during seed development. In GM077, the expression levels of AGPL2, AGPS1, AGPS2b,
SSIIIa, BEI, PUL and ISA1 were significantly upregulated. Co-immunoprecipitation assays revealed
interactions of SSs-BEs, SSs-PUL and BEs-PUL in developing seeds. However, weak SSI-SSIIa
interaction was detected in GM077, whilst SSI-PUL interaction was absent. Weak interaction signals for
SSI-SSIIa, SSIIa-BEI, SSIIa-BEIIb, BEI-BEIIb and SSI-BEI were also observed in GM645. These results
suggest that the protein-protein interactions for starch biosynthesis are modified in mutants, which
provides insight into the mechanisms of starch biosynthesis, particularly in indica rice.
Key words: amylose; endosperm mutant; indica rice; protein-protein interaction; starch biosynthetic
enzyme; waxy rice

The physicochemical properties of endosperm starch
in milled rice determine its cooking and eating quality
(Vandeputte and Delcour, 2004; Bao, 2012, 2019; Xu
et al, 2018). Starch is the major storage material in the
rice endosperm, accounting for 80%–90% of the seed
weight, and is composed of amylose and amylopectin
(Vandeputte and Delcour, 2004; Du et al, 2019). Starch
mutants are defective in either amylose or amylopectin
biosynthesis and have been used for the molecular
characterization of key enzymes and regulatory factors
during starch synthesis. Defects in granule-bound
starch synthase I (GBSSI) produce a waxy (wx)
endosperm composed of amylose-free starch grains,
confirming the essential role of GBSSI in amylose
synthesis (Jeng et al, 2009; Zhang et al, 2012). The
functions of other enzymes in amylopectin synthesis
have also been shown in starch mutants such as
ADP-glucose pyrophosphorylases (AGPases), soluble
starch synthases (SSs), branching enzymes (BEs), and
debranching enzymes (DBEs) (Nishi et al, 2001;
Fujita et al, 2006, 2007, 2009; Nakamura et al, 2010;
Toyosawa et al, 2016). Furthermore, mutations in these
genes exhibit abnormal features of starch storage in
the endosperm. For example, SSIIIa mutants produce
a chalky interior, and the amylose content and
physicochemical properties of the starch granules

Received: 5 August 2019; Accepted: 13 November 2019

Corresponding author: BAO Jinsong (jsbao@zju.edu.cn)
Copyright © 2020, China National Rice Research Institute. Hosting by Elsevier B V
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)
Peer review under responsibility of China National Rice Research Institute
http://dx.doi.org/
http://dx.doi.org/10.1016/j.rsci.2020.01.006
CHEN Yaling, et al. Expression Profiles and Protein Complexes of Indica Rice Mutants
(SGs) are influenced (Fujita et al, 2007; Ryoo et al,
2007; Bao, 2019). Loss-of-function mutations in
BEIIb lead to a white-core endosperm, with altered
amylopectin structures and gelatinization properties of
the SGs (Nishi et al, 2001; Sawada et al, 2018).
Isoamylase1 (ISA1)-deficient mutants (isa1) are also
termed as sugary mutants in rice (sug-1) and maize
(su1) (James et al, 1995; Kubo et al, 2005). Residual
SGs in the isa1 mutants are of abnormal size, shape and
number (Kubo et al, 2010; Utsumi et al, 2011). Studies
in starch mutants have enhanced our understanding of
the mechanisms for starch biosynthesis.
Recent works also indicate that the protein complexes
of starch synthesis isozymes may exist during seed
development in wheat, maize, barley and rice
(Hennen-Bierwagen et al, 2008; Tetlow et al, 2008;
Ahmed et al, 2015; Crofts et al, 2015, 2017), but
complexes in the mutant endosperm may differ from
those in wild type (Liu et al, 2009, 2012a; Crofts et al,
2018). Tetlow et al (2004) first reported a multi-enzyme
complex of BEIIb, BEI and starch phosphorylase (SP)
in the amyloplasts of developing wheat endosperm.
Subsequently, other enzyme complexes, including SSI,
SSIIa, SSIIIa, BEIIa and/or BEIIb, in various
combinations have been identified (Hennen-Bierwagen
et al, 2008; Ahmed et al, 2015). In developing maize
seeds, Liu et al (2009) reported that a major protein
complex consists of SSI, SSIIa and BEIIb, but the null
amylose extender mutant of SBEIIb contains a novel
protein complex comprised of SSI, SSIIa, SBEI,
SBEIIa and SP. Liu et al (2012b) further reported that
a point mutation in SSIIa in a su2-mutant of maize
causes the loss of SSI and SBEIIb, in addition to SSIIa
from SGs. In the SSIIa mutants (sex6) of barely, no
protein complexes involving SBEIIa or SBEIIb are
detected in amyloplasts (Ahmed et al, 2015). The BEII
isoform in the formation of novel protein complexes is
substituted by BEI and SP (Ahmed et al, 2015). These
observations imply that alterations in the granule
proteome arise from genetic mutations or the down-
regulation of specific genes gives rise to variations in
protein complexes in the amyloplasts.
Rice has 10 SS isoforms, 3 BE isoforms, and 4 DBE
isoforms. These isozymes have been characterized in
studies of starch mutants, predominantly from japonica
varieties, including Nipponbare, Taichung 65 and
Kinmaze (Sawada et al, 2018). Co-immunoprecipitation
(Co-IP) assays of SSs, BEs and DBEs provide direct
evidence of the formation of multi-enzyme complexes
in soluble extracts of Nipponbare endosperm (Crofts
et al, 2015; Chen and Bao, 2016). However, compared
153
to japonica varieties, indica rice varieties have active
SSIIa isozymes (Nakamura et al, 2005) and high
expression level of GBSSI (Bligh et al, 1998; Cai et al,
1998; Dobo et al, 2010). Challenges remain regarding
the mechanisms for starch synthesis in indica rice,
including whether the elevated expression of GBSSI
affects the activities of other enzymes (Bao, 2012).
Whether novel protein complexes are present in indica
rice due to the active SSIIa isozyme is also unknown.
Whether the activities of enzymes and protein
complex change during starch synthesis in indica
mutants is also uncharacterized.
In previous studies, two stable starch mutants were
isolated from high amylose indica rice, Guangluai 4
(GLA4) (Kong et al, 2014). One is a waxy mutant
(GM077) with an opaque endosperm and very low
apparent amylose content (2.6%). The other is a white-
core mutant (GM645) with a chalky endosperm in the
center of the grain. The physicochemical properties of
the starch and chain length distributions of the
amylopectin extracted from the two mutants differ
(Kong et al, 2014). In this study, the expression levels
of genes and enzymes involved in starch synthesis,
including AGPases, GBSSI, SSs, BEs and DBEs, in
developing rice endosperms were measured by
qRT-PCR and western blotting. Co-IPs were performed
to characterize the protein complexes formed in the
mutants. Our results provided direct evidence for
changes in protein complexes in rice mutants and
enhanced our understanding of starch biosynthesis in
indica rice.
MATERIALS AND METHODS
Materials
High amylose indica rice GLA4 and two stable
endosperm mutants (GM077 and GM645) were grown
in the field at the Zhejiang University farm, Hangzhou,
China. Individual panicles were labeled during
flowering. Developing seeds at 5, 10 and 15 d after
flowering (DAF) were collected, and immediately
frozen on ice and stored at -80 ºC. Antisera against
rice GBSSI, SSI, SSIIa, BEI, BEIIb and PUL were a
kind gift of Dr. Naoko FUJITA at Akita Prefectural
University, Akita, Japan.
RNA extraction and quantitative real-time PCR
(qRT-PCR)
Total RNAs from the rice endosperm at different
developmental time points were isolated according to
154
the manufacturer’s protocols using the SV Total RNA
Isolation System (Promega, Beijing, China). cDNA
was synthesized using the First-Strand Synthesis cDNA
kit (Promega, Beijing, China). Gene-specific primers
of starch synthesis related genes were used in qRT-PCRs,
referring to Ohdan et al (2005). Additionally, the
housekeeping gene Actin was used as an internal
control to normalize cDNA levels in each sample.
qRT-PCRs were performed using StepOneTM &
StepOne PlusTM Real-Time PCR Systems (ABI, USA)
accompanying with SYBR® Premix Ex TaqTM (TaKaRa,
Dalian, China). Relative gene expression was calculated
using the 2-ǻǻCT method (Jain et al, 2006). Measurements
at each time point were performed in triplicate.
Extraction of soluble proteins and starch granule-
bound proteins
The isolation of soluble proteins was performed as
previously described (Chen and Bao, 2016). Proteins
bound with starch granules were extracted according to
Fujita et al (2006), with minor modifications. Following
protein extraction, the starch was washed twice with
three volumes of cold sodium dodecyl sulfate (SDS)
solution containing 55 mmol/L of Tris-HCl (pH = 6.8),
2.3% of SDS, 5% of 2-mercaptoethanol and 10% of
glycerol, to remove residual proteins attached to the
surface (Fujita et al, 2006). Residual pellets (starch
granules) were washed with 1 mL of distilled water
and 1 mL of acetone twice. Extracts were dried using
a freeze drier. Equivalent amounts of starch (50 mg)
were boiled in 10 volumes of SDS solution for 10 min.
After cooling, 20 volumes of SDS solution were added
with stLrUing, and samples were centrifuged at 12
000 × g for 10 min at 4 ºC. Supernatants were
assessed for starch granule-bound proteins.
Protein concentrations were measured on a
NanoDrop 2000 spectrophotometer (Thermo, Canada).
Co-immunoprecipitation (Co-IP) assay
Co-IP assays were performed as described by Crofts et al
(2015) and Chen and Bao (2016) with some
modifications. Soluble proteins (200 ȝL, 10 mg/mL)
were mixed with different volumes of primary
antibodies (15 ȝL anti-SSI, 20 ȝL anti-SSIIa, 10 ȝL
anti-BEI, 15 ȝL anti-BEIIb, or 15 ȝL of anti-PUL) for
2 h at 4 ºC. A 200 ȝL aliquot of reconstituted 50%
protein A-sepharose resin (TransGen, China) was
added and incubated on a rotator for 3 h at 4 ºC.
Protein A-sepharose-antibody-protein complexes were
centrifuged at 6 000 × g for 3 min at 4 ºC and
Rice Science, Vol. 27, No. 2, 2020
supernatants were discarded. The resins were washed
eight times with PBS (137 mmol/L NaCl, 10 mmol/L
Na2HPO4, 2.7 mmol/L KCl, 1.8 mmol/L KH2PO4, pH
7.4). Bound proteins were released by boiling for 10
min in 1× SDS buffer. After centrifugation at 12 000
r/min for 3 min, 10 ȝL of the lysates were used for
western blotting.
Western blotting
Proteins were resolved by 10% SDS-PAGE (SDS-
polyacrylamide gel electrophoresis), and transferred
onto polyvinylidene fluoride (PVDF) membranes
using a transblotter. Western blotting procedure was
performed as described by Crofts et al (2015). Blots
were repeated at least three times, while blots for the
Co-IPs were performed at least two times.
Data analysis
Western blots were quantitated using the Image J
software. t-tests of the differences in expression levels
between the mutant and wild type were performed
using the SPSS 20.0 software (SPSS, Inc., Chicago IL,
USA).
RESULTS
Expression profiles of starch synthesis genes
Previous studies showed that major starch genes
including AGPL1, AGPL2, AGPS1, AGPS2b, GBSSI,
SSI, SSIIa, SSIIIa, BEI, BEIIa, BEIIb, ISA1, PUL and
SP are expressed in rice endosperms (Duan and Sun,
2005). The expression levels of these genes in two
endosperm mutants (GM077 and GM645) during the
developing of rice grains at 5, 10 and 15 DAF were
examined by qRT-PCR.
Compared to the wild type GLA4, the 14 genes
displayed four different expression profiles in GM077.
The expression of GBSSI was downregulated, whereas
the expression levels of AGPL2, AGPS1, AGPS2b, SSIIIa,
BEI, PUL and ISA1 were significantly upregulated
(Fig. 1). The expression levels of SSI and BEIIb were
downregulated at 5 DAF but subsequently upregulated.
The expression levels of AGPL1, SSIIa, BEIIa and SP
were upregulated from 5 to 10 DAF but subsequently
downregulated. These data revealed that the expression
levels of most starch synthesis genes were upregulated
in GM077 seeds during the grain-filling stage.
In the white-core endosperm mutant GM645, the
expression levels of AGPS1, GBSSI, SSIIa, BEI, BEIIa,
CHEN Yaling, et al. Expression Profiles and Protein Complexes of Indica Rice Mutants 155

Fig. 1. Expression profiles of rice starch synthesis genes during seed development in GLA4 and two endosperm mutants.
GLA4, Guangluai 4. ADP-glucose pyrophosphorylase (ADPase) genes: AGPL1, AGPL2, AGPS1 and AGPS2b; Granule-bound starch synthase
(GBSS) gene: GBSSI; Soluble starch synthase (SS) genes: SSI, SSIIa and SSIIIa; Branching enzyme (BE) genes: BEI, BEIIa and BEIIb; Debranching
enzyme (DBE) genes: ISAI and PUL; Starch phosphorylase (SP) gene: SP.
Total RNAs were extracted from developing seeds at 5, 10 and 15 d after flowering (DAF). Data are Mean ± SD from three replicates. The
asterisks indicate statistical significance between GLA4 and the mutants, as determined by the Student’s t-test (*, P < 0.05; **, P < 0.01).

BEIIb, PUL, ISA1 and SP were significantly down-
regulated during endosperm development. The expression
levels of AGPS2b, SSI and SSIIIa were downregulated
from 5 to 10 DAF but subsequently upregulated (Fig. 1).
Accumulation of starch synthesis related proteins
To assess the levels of protein accumulation in the two
endosperm mutants (GM077 and GM645), we performed
western blotting of the wild type and the two
endosperm mutants during rice endosperm development
with various antibodies. As shown in Fig. 2-A, GBSSI
protein was undetectable in rice endosperm of GM077
during grain filling. SSIIa protein at 5 DAF increased
in GM077, which was comparable to GLA4 at 10 and
15 DAF. There were no significant changes for SSI,
BEI and BEIIb from 5 to 15 DAF (Fig. 2-B).
In the white-core endosperm mutant GM645, a
significant reduction in SSIIa, BEI, BEIIb and PUL
expression was observed. SSI at 10 and 15 DAF in
GM645 was similar to that in GLA4 (Fig. 2-B). These
results suggested that the white-core mutation reduced
the levels of soluble proteins in relation to starch
biosynthesis in the endosperms.
Analysis of protein-protein interactions
To investigate possible interacting partners amongst
starch biosynthetic isozymes in the wild type (GLA4)
and mutants of indica rice, Co-IPs were performed
using soluble endosperm extracts at 10 DAF (Table 1
and Fig. 3).
Strong pairwise associations through Co-IPs were
observed for SSI-SSIIa, SSI-BEIIb, SSI-PUL, SSIIa-
BEI, SSIIa-BEIIb, BEI-BEIIb, BEI-PUL and BEIIb-
PUL in the GLA4 endosperm. Interactions were observed
156 Rice Science, Vol. 27, No. 2, 2020

A
GLA4 GM077 GM645 whilst the reciprocal Co-IPs were relatively weaker.
5 10 15 5 10 15 5 10 15 DAF In the GM077 endosperm, strong pairwise associations
were obtained by reciprocal Co-IPs for SSI-BEI,
GBSSI
SSI-BEIIb, SSIIa-BEI, SSIIa-BEIIb, SSIIa-PUL, BEI-
1 0.95 1.11 0.90 0.87 0.93 BEIIb, BEI-PUL and BEIIb-PUL. Compared to GLA4,
0.02 0.04 0.05 0.01 0.05
the pairwise interaction signals of SSI-SSIIa were
B 5 DAF 10 DAF 15 DAF weaker. Of note, SSI-PUL interactions were not detected
in the GM077 endosperm.
GLA4 GM077 GM645 GLA4 GM077 GM645 GLA4 GM077 GM645
In the GM645 endosperm, the interactions of SSI-
SSI BEIIb, SSI-PUL, BEI-PUL, BEIIb-PUL and SSIIa-
PUL were similar to the GLA4 endosperm. Pairwise
1 0.94 0.54 1.16 1.19 1.17 1.28 1.31 1.25
interactions for SSI-SSIIa, SSIIa-BEI, SSIIa-BEIIb
0.01 0.09 0.05 0.01 0.05 0.06 0.20 0.05
and BEI-BEIIb in the GM645 endosperm were weaker
SSIIa than those observed in the wild type. Furthermore, a
clear, but less intense signal for SSI-BEI was obtained
1 1.14 0.71 0.94 0.83 0.51 0.63 0.48 0.12
0.01 0.01 0.01 0.02 0.01 0.01 0.01 0.00 from only one side of Co-IP in the GM645 endosperm.

BEI DISCUSSION
1 1.07 0.91 1.39 1.32 0.79 1.19 1.18 0.77
0.03 0.06 0.02 0.08 0.10 0.08 0.14 0.15
Protein complexes of starch biosynthetic enzymes
BEIIb from high amylose indica rice GLA4
1 1.15 0.84 1.44 1.59 1.22 1.64 1.65 1.22 Interactions between starch biosynthetic isozymes
0.01 0.09 0.02 0.02 0.16 0.09 0.10 0.14
have been shown in wheat, maize, barley and japonica
rice developing endosperms (Hennen-Bierwagen et al,
PUL
2008; Tetlow et al, 2008; Ahmed et al, 2015; Crofts et al,
1 0.95 0.82 1.19 0.98 0.76 0.96 0.95 0.93 2015). Co-IP assays revealed associations of SSs-BEs,
0.04 0.02 0.01 0.01 0.01 0.01 0.02 0.02 BEIIa-Pho1, and pullulanase-type DBE-BEI present in
Fig. 2. Western blotting of starch synthetic enzymes extracted from japonica rice Nipponbare (Crofts et al, 2015; Hayashi
developing endosperms of GLA4, GM077 and GM645 at 5, 10, et al, 2018; Miura et al, 2018), which possesses inactive
15 d after flowering (DAF) was performed using antisera against SSIIa and lower GBSSI expression levels compared to
GBSSI, SSI, SSIIa, BEI, BEIIb and PUL from soluble proteins.
A, Starch granule-bound protein for GBSSI blot. B, Soluble
indica rice. In high amylose indica rice GLA4 that
proteins for SSI, SSIIa, BEI, BEIIb and PUL enzymes blots. Values in possesses active SSIIa and higher GBSSI expression,
the first line under the gel are mean relative levels to the GLA4 at 5 SSI-BEs, SSI-PUL and PUL-BEI complexes were
DAF, while those in the second lines are the standard deviation. GLA4,
similar to japonica rice Nipponbare (Crofts et al, 2015;
Guangluai 4.
Hayashi et al, 2018), but SSI-SSIIa, SSIIa-BEs and
for SSI-BEI and SSIIa-PUL (the first acronym: PUL-SSIIa were found only in GLA4 (Fig. 3 and
antibodies used for immunoprecipitation; the second Table 1). These discoveries provide a basis for the
acronym: antibodies detected by western blotting), comprehensive analysis of protein complexes of starch

Table 1. Comparison of protein-protein interactions among starch synthetic related enzymes in wild type and mutant endosperms determined
by co-immunoprecipitation assay.
Reciprocal One sided a
Material No signal
Strong signal Weak signal Strong signal Weak signal
GlA4 SSI-SSIIa, SSI-BEIIb, SSI-PUL, SSIIa-BEI, SSI-BEI, PUL-SSIIa
SSIIa-BEIIb, BEI-BEIIb, BEI-PUL, BEIIb-PUL
GM077 SSI-BEI*, SSI-BEIIb, SSIIa-BEI, SSIIa-BEIIb, SSI-SSIIa* SSI-PUL*
SSIIa-PUL*, BEI-BEIIb, BEI-PUL, BEIIb-PUL
GM645 SSI-BEIIb, SSI-PUL, BEI-PUL, BEIIb-PUL SSI-SSIIa*, SSIIa-BEI*, SSIIa-PUL SSI-BEI*
SSIIa-BEIIb*, BEI-BEIIb*
a
The antibody used for immunorecipitation on the left and the coprecipitated enzymes detected by western blotting on the right. * indicates the
different interactions between the wild type and mutants.
CHEN Yaling, et al. Expression Profiles and Protein Complexes of Indica Rice Mutants
Antibody used for IP
kDa M SSI SSIIa
90
75
90
75
90
75
90
75
90
75
a b c a b c
Fig. 3. Protein-protein interactions between starch synthetic related enzymes in wild type and mutant endosperms by co-immunoprecipitation
(Co-IP).
Black arrows represent the western blotting signals. M, Protein marker; a, Guangluai 4 (GLA4); b, GM077; c, GM645.
biosynthetic enzymes in rice, although the possibility
remains that the differences arise not only from each
plant-specific function, but from differences in the
experimental approaches, such as the choice of
endosperm development and whether amyloplasts or
whole-cell extracts are used as the starting material.
Protein complexes of starch biosynthetic enzymes
from waxy mutant GM077
The waxy mutants of cereals and the responsible gene
(GBSSI) were discovered many years ago (Bligh et al,
1998; Cai et al, 1998; Dobo et al, 2010). Due to the
loss of amylose accumulation, reallocation of the
carbon source to amylopectin synthesis in GM077 has
been reported by Zhang et al (2012), who found that
the elevated expression levels of AGPS and ISA1 in
the rice waxy mutants drive carbon flux from amylose
to amylopectin synthesis. In this study, qRT-PCR analysis
indicated that GM077 was a waxy mutant with high
expression levels of 11 amylopectin synthetic related
genes, and that higher AGPS1 and ISA1 expression
levels were detected during the whole endosperm
developmental stage compared to the wild type GLA4,
which is consistent with Zhang et al (2012). The
mechanism behind the elevated expression of AGPS
and ISA1 in the rice mutants remains unclear. The
Į-1,4-glucosidic link chains of both amylose and
amylopectin are elongated through the addition of the
157
BEI BEIIb PUL
SSI
SSIIa
Antibody used for western blotting
BEI
BEIIb
PUL
a b c a b c a b c
glucose moiety from ADP-glucose. One possibility is
that the extra AGPase is required in GM077 to convert
Glc1-P to ADP-glucose in the cytosol. ISA1 is
particularly important for amylopectin formation, and
therefore, it is unsurprising that ISA1 expression in
GM077 significantly increased when excess amylopectin
was produced in the endosperm (Utsumi et al, 2011;
Sun et al, 2015).
Novel complexes of starch biosynthetic enzymes
have been reported in rice mutants lacking SSI and
BEIIb (Crofts et al, 2018), but reports on starch
biosynthetic protein complexes in rice waxy mutants
are sparse. Co-IP analysis indicated the pairwise
interaction signals of SSI-SSIIa in the waxy mutant
GM077 were weaker compared to GLA4. The weak
interaction may be due to the low levels of SSIIa in
GM077 compared to GLA4, although the expression
level and protein content of SSI were higher in
GM077 than in GLA4 (Fig. 2). Moreover, it was
interesting to note that no SSI-PUL interaction was
detected in reciprocal Co-IPs in GM077 endosperm.
Western blotting also detected a low amount of PUL
protein (Fig. 2-B). However, the expression of ISA1 in
the GM077 endosperm was significantly higher than
that of GLA4 (Fig. 1). Fujita et al (2009) provided
evidence that the role of PUL is to supplement ISA
when it is absent. Utsumi et al (2011) reported that the
ISA1-ISA1 homomer is the only functional form in
158
rice endosperm. Although interaction of SSI and ISA
has not been reported, it is worth noting that a close
correlation between ISA and SS activities during
starch accumulation occurs in Arabidopsis leaves,
suggesting functional or physical interaction exists
between these two enzyme classes (Pfister et al, 2014).
This may explain the loss of SSI-PUL in GM077 due
to increased function of the ISA1-ISA1 homomer or
the interaction between SSI and ISA1.
Protein complexes of starch biosynthetic enzymes
from white-core mutant GM645
In the white-core mutant GM645, reductions in
expression level of starch synthesis genes, including
AGPS1, GBSSI, SSIIa, BEI, BEIIa, BEIIb, PUL, ISA1
and SP, were observed (Fig. 1). Western blot analysis
revealed an extreme reduction of BEI, BEIIb and PUL
(Fig. 2). Co-IPs indicated that pairwise interactions
between BEI-SSIIa and BEI-BEIIb were weaker than
those of wild type. Furthermore, weaker signal of
SSI-BEI was obtained from only one of the Co-IPs in
the GM645 endosperm.
Gene mutations have not been characterized in
GM645. Previous studies isolated and functionally
characterized four white-core endosperm mutants,
including flo4, flo5, rsr1 and flo12 (Kang et al, 2005;
Ryoo et al, 2007; Fu and Xue, 2010; Zhong et al,
2019). Amongst them, flo12 shows the reduced
expression of starch synthesis genes, including SSI,
SSIIb, SSIII, SSIV, AGPL, ISA1 and BEI (Zhong et al,
2019). The expression levels of SSI and SSIIIa in
GM645 remained unchanged, so GM645 is not mutated
at the flo12 locus. The floury-white endosperm flo2
exhibits reduced levels of BEI in the developing rice
endosperm, along with decreased levels of other
starch-synthetic enzymes, including AGPase, GBSS,
SS and BEIIb (Kawasaki et al, 1996). She et al (2010)
reported that the flo2 mutant shows lower expression
of genes involved in the production of storage starch
in the endosperm. Wu et al (2015) reported that three
flo2 mutants perturb the expression of starch synthesis-
related genes including OsAGPL2, OsAGPS2b, OsGBSSI,
OsBEI, OsBEIIb, OsISA1 and OsPUL. Consistent with
these findings, both gene expression and western blot
analysis indicated that starch synthesis genes are
down-regulated in GM645 (Figs. 1 and 2). According
to the mutant phenotypes, GM645 had higher numbers
of short and long chains consisting of 6௅9 degrees of
polymerization (DP) and • 44 DP, respectively (Kong
et al, 2014), but flo2 has lower numbers of short and
Rice Science, Vol. 27, No. 2, 2020
long chains (She et al, 2010). Mutations in GM645
await further map-based cloning, but the mechanisms
by which these protein complexes influence starch
biosynthetic enzymes now require further investigation.
Proposed models for starch synthesis in high amylose
indica rice, waxy mutants and white-core mutants
Functional redundancy has been observed for mutants
lacking specific rice starch biosynthetic enzymes
(Nakamura, 2002; Fujita, 2014). GBSSI is primarily
involved in amylose biosynthesis. SSI elongates short
chains (8–12 DP) of amylopectin generated by BEIIb,
and the 8 DP chains can be elongated to form
intermediate intra-cluster chains (12–24 DP) by SSIIa.
SSIIIa generates long chains (> 30 DP) connecting
multiple clusters of amylopectin. ISA and PUL
directly debranch improper branches. In our previous
studies, waxy mutant GM077 had more chains with
6–10, 18–33 and > 44 DP in addition to higher
gelatinization temperature, whilst white-core mutants
GM645 had more chains with 6–9, 22–35 and > 44 DP
as well as with lower gelatinization temperature than
GLA4 (Kong et al, 2014). Through the analysis of the
chain length distribution of amylopectin, expression
profiles and protein complexes from GLA4, GM077
and GM645, mechanisms for the formation of enzyme
complexes involved in starch biosynthesis in the
endosperms of high amylose indica rice, waxy mutant
and white-core mutant were proposed (Fig. 4).
In the GLA4 endosperm that possesses higher
levels of SSIIa expression, amylose and amylopectin
are elongated by the addition of the glucose moiety
from ADP-glucose (Fig. 4-A). The trimeric complex
of BEI, SSI and SSIIa or BEIIb, SSI and SSIIa binds
to the glucan, branches and elongates the polymer,
generating intermediate intra-cluster chains (12–24
DP) and acting as a ‘glucan-chaperone’ (Tetlow et al,
2015). SSI-SSIIa contributes to the initial short chain
elongation of 6–7 DP produced by BEIIb to form
12–24 DP chains. SSIIIa activity, which is abundant at
the starch filling stage, elongates the intermediate
chains to 30 DP. The complex may be unable to
proceed as a result of the formation of disorganized
branches that lead to steric hindrance. ISA1-ISA1
homomers or the complexes of BEs-SSs-PUL remove
the disorganized branches (Fig. 4).
In the GM077 endosperm, higher SSIIIa expression
may increase the synthesis of intermediate chains that
lead to BEI-SSI-SSIIa from 18–33 DP chains (Fig. 4).
BEIIb-SSI or SSI branches and elongates the polymer,
CHEN Yaling, et al. Expression Profiles and Protein Complexes of Indica Rice Mutants 159

GLA4 12–24 DP

12–24 DP
12–24 DP

8–12 DP
GM077 8–12 DP
18–33 DP

GM645
6–9 DP 6–9 DP
22–35 DP

Fig. 4. Hypothesis models for starch synthesis in high amylose indica rice Guangluai 4 (GLA4) and two endosperm mutants.
The predicted protein-protein complexes in developing seed of wild type indica rice GLA4 were demonstrated referring to Nakamura (2002);
Nakamura et al (2005) and Fujita (2014). Only the difference in the protein-protein interactions in GM077 and GM645 from GLA4 was shown.
Alterations of chain length distribution in GM077 and GM645 referred to Kong et al (2014). The small rounds of different color represent glucosyl
residues from different starch synthases. DP, Degree of polymerization.

generating intermediate chains (8–12 DP) due to the
weaker interaction of SSI-SSIIa (Table 1). Disorganized
branches were removed by ISA1-ISA1 homomers due
to the lack of SSI-PUL complex in GM077 (Table 1).
In the GM645 endosperm, complexes of BEI-SSIIa,
BEI-BEIIb and SSI-BEI were weaker, but SSI-BEIIb
interactions were comparable to GLA4. The complex
of SSI-BEIIb forms more chains with 6–9 DP (Fig. 4).
Similar SSIIIa expression may elongate the short
chains to 22–35 DP. Disorganized branches were
removed by the same complexes as wild type.
It is noteworthy that Pang et al (2018) identified
phosphorylated proteins (SSIIa, SSIIIa, BEI, BEIIb,
PUL and SP) related to starch synthesis in indica rice.
We hypothesized that the formation of multiple
enzyme complexes are related to the phosphorylation
of rice starch biosynthesis related enzymes. As in
wheat and maize, complexes are catalyzed as a result
of phosphorylation (Tetlow et al, 2004, 2008; Liu et al,
2009). Future studies focusing on the cloning and
functional characterization of genes for the white-core
mutant, validation of the mechanisms for the differences
in protein-protein interactions, and assessments of the
involvement of protein phosphorylation during protein
complex formation in amylopectin synthesis between
the wild type and mutant endosperms are required.
ACKNOWLEDGEMENTS
This work was financially supported by the National
Key Research and Development Program of China
(Grant No. 2016YFD0400104), and the Natural Science
Foundation of China (Grant Nos. 31800640 and
31871531).
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