Plant Physiol. (1999) 120: 1063-1074
Accumulation of Soybean Glycinin and
Its Assembly with the
Glutelins in Rice1
Tomoyuki Katsube,
Nobuyuki Kurisaka,
Masahiro Ogawa,
Nobuyuki Maruyama,
Reiko Ohtsuka,
Shigeru Utsumi2, *, and
Fumio Takaiwa2
Research Institute for Food Science, Kyoto University, Uji, Kyoto
611-0011, Japan (T.K., N.M., S.U.); Shimane Women's College, Matsue,
Shimane 690-0044, Japan (T.K.); Ehime Prefectural Agricultural
Experiment Station, Houjo, Ehime 799-2424, Japan (N.K.); Yamaguchi
Prefectural University, Department of Domestic Economy, Sakurabatake,
Yamaguchi 753-8502, Japan (M.O.); Faculty of Agriculture, Kyushu
University, Hakozaki, Higashiku, Fukuoka 812-8581, Japan (R.O.); and National Institute of Agrobiological Resources, Tsukuba, Ibaraki
305-0856, Japan (F.T.)
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ABSTRACT |
Saline-soluble
glycinins and insoluble glutelins are the major storage proteins in
soybean (Glycine max) and rice (Oryza
sativa), respectively. In spite of their differences in
solubility properties, both proteins are members of the 11S globulin
gene family based on their similarities in primary sequences and
processing of the coded protein. Wild-type and methionine-modified
glycinin coding sequences were expressed in transgenic rice plants
under the control of the rice glutelin GluB-1 promoter.
Glycinins were specifically synthesized in the endosperm tissue and
co-localized with glutelins in type II protein bodies. They assembled
into 7S and 11S species, similar to what was observed in developing
soybean seeds. This pattern was quite different from that displayed by
the rice glutelins in untransformed plants, in which processed subunits
sedimenting at 2S were apparent. In glycinin-expressing transgenic
plants, however, glutelins were observed sedimenting at 7S and 11S with lesser amounts in the 2S region. A portion of the glycinins was also
found associated in the insoluble glutelin fraction. Renaturation experiments suggested that the hybrid glycinin-glutelin oligomers were
formed through specific interactions. Overall, these results indicate
that despite significant differences in the assembly of soybean
glycinin and rice glutelin, both proteins can assemble with each other
to form soluble hexameric oligomers or insoluble aggregates.
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INTRODUCTION |
Seed storage proteins were initially classified into albumins
(water soluble), globulins (saline soluble), prolamins (alcohol soluble), and glutelins (residue) by Osborne (1924)
according to their
solubility properties. Based on more recent and extensive molecular and
biochemical analysis of the storage protein genes and their coded
products, the storage proteins fall into two major groups, the
globulins and the prolamins (Shewry and Tatham, 1990). The rice
(Oryza sativa) glutelins and soybean (Glycine
max) glycinins are excellent examples of this reclassification of
storage proteins. The rice glutelins, which comprise up to 70% to 80%
of the total seed protein, are insoluble in a neutral saline solution
but soluble in a diluted acid/alkaline solution. They exist as large
macromolecular complexes formed by disulfide and hydrophobic
interactions of acidic and basic polypeptides. The soybean glycinins,
which account for 40% of the total proteins (Utsumi, 1992; Utsumi et
al., 1997), are soluble in neutral saline solutions. These proteins
accumulate as 11S oligomers comprised of six pairs (subunits) of acidic
and basic polypeptides interlinked by a conserved disulfide bond. Although glutelin and glycinin have different properties (such as their
solubility), they nevertheless are related and are both members of the
11S globulin family of storage proteins. These proteins share 32% to
37% identity in their primary sequences. Moreover, both proteins are
synthesized as a larger precursor on the ER, are proteolytically
processed into acidic and basic polypeptides, and are accumulated and
stored in a vacuolar compartment.
The cellular events that lead from the synthesis of soybean glycinin to
their assembly into a protein storage vacuole are well understood. When
translocated in the ER lumen, the newly synthesized proglycinins (2S)
assemble into trimers that sediment at 7S to 8S in Suc density
gradients. These trimers are competent for transport to the protein
storage vacuole via the Golgi complex. At this storage site, each
proglycinin subunit of the trimer is proteolytically cleaved into
acidic and basic polypeptides (Staswick et al., 1984) whereupon
assembly into a hexamer (11S-12S) occurs. Soluble proteins destined for the vacuole must carry sorting
determinants that allow them to be identified and sorted from the
secretory pathway (Okita and Rogers, 1996). Horse bean 11S globulin,
legumin, was demonstrated to have an internal signal determinant
(Saalbach et al., 1991; Müntz, 1998) and a similar sorting signal
likely occurs for the soybean glycinin. Recently, physical aggregation was proposed as a sorting mechanism of pea legumin: the higher hydrophobicity of prolegumin than mature legumin would be a driving force of aggregate formation and binding to the membrane of cargo vesicles that are targeted to the protein storage vacuole (Hinz et al., 1997; Müntz, 1998; Neuhaus and Rogers, 1998; Robinson et
al., 1998).
The cellular processes that lead to the transport of rice glutelin to
the type II protein body (equivalent to the protein storage vacuole)
are not as well understood as the processes following the synthesis of
soybean glycinin. Pulse-chase labeling studies readily
demonstrate that the glutelin is initially synthesized as a larger
precursor, which is then proteolytically processed into acidic and
basic subunits. No evidence has been obtained showing that the rice
glutelins are transported to the vacuole by an ordered process of 7S
trimer formation, proteolysis, and 11S hexamer formation, as has been
demonstrated for the soybean glycinins. Instead, the precursor form
itself may be transported to the Golgi, where it is packaged into dense
vesicles associated with the Golgi cisternae (Krishnan et al., 1986).
The concentration of glutelins in dense vesicles and their
hydrophobicity may result in protein aggregation, which would follow an
aggregation mechanism for sorting (Okita and Rogers, 1996; Hinz
et al., 1997; Müntz, 1998; Neuhaus and Rogers, 1998; Robinson et
al., 1998).
Rice and soybean proteins are deficient in Lys and sulfur-containing
amino acids, respectively. When viewed nutritionally, these proteins
can compensate for one another and collectively provide a better
balance of essential amino acids. In addition to their desired
physicochemical properties, e.g. heat-induced gel forming and
emulsifying abilities, soybean proteins such as glycinin lower the
cholesterol levels in human serum (Kito et al., 1993), while rice
glutelins have no significant physicochemical and physiological
properties. Because of these functional properties, genetic engineering
efforts have been directed at improving the nutritional value and
physicochemical properties of soybean glycinins (Kim et al., 1990b;
Utsumi et al., 1993a; Katsube et al., 1994, 1998a), with the aim of
introducing these modified genes into rice (Katsube et al., 1998b).
We have previously demonstrated that glycinin can accumulate to
significant levels in the endosperm tissue of tobacco seeds using a
glutelin GluB-1 promoter (Takaiwa et al., 1995). In the present study, we expressed the soybean glycinin gene and a Met-rich glycinin gene in transgenic rice. We show that glycinin is assembled into trimeric and hexameric structures, is proteolytically processed into subunits, and is packaged into a protein storage vacuole by events
in rice much like those observed in developing soybean seeds. In
contrast, rice glutelins do not exhibit such trimeric and hexameric
structures. However, in transgenic plants, glutelins can assemble with
glycinin to form soluble hetero-trimers and -hexamers and insoluble
complexes. Our results indicate that the secondary and tertiary
structures of glycinin and glutelin are compatible to form higher order
protein structures. Hence, assembly and packaging of glycinin should
not be a limiting factor in expression in rice.
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MATERIALS AND METHODS |
Construction of Chimeric Genes and Transformation
The chimeric gene consisting of the GluB-1 promoter
(
1302 to +18) and the normal glycinin A1aB1b cDNA was recovered from pBGG1 (Takaiwa et al., 1995) after digestion with
HindIII and SacI, and then inserted into the
corresponding sites of pUC18 containing the 0.6-kb 3
noncoding region
of the GluB-1 gene to give pUGluB1Gly. A DNA fragment
containing the CaMV 35S promoter-bialaphos resistance gene
(Bar)-nopaline synthase gene terminator hybrid gene was then
inserted into the filled-in HindIII site of pUGluB1Gly to
give pGluB1Gly. A similar construct containing a modified glycinin cDNA
(encoding the modified glycinin IV+4Met where four contiguous Met
residues were inserted) was constructed from
pBGG1IV (Takaiwa et al., 1995) and inserted in
the binary plasmid pGPTV-bar/Fer (Goto et al., 1999) to give
pGluB1GlyIV.
The plasmids containing the chimeric genes for the normal and modified
glycinins were transferred into rice (Oryza sativa cv
Matsuyama-mii) protoplasts by electroporation (Tada et al., 1990) and Agrobacterium tumefaciens-mediated transformation
(Goto et al., 1999), respectively. Bialaphos-resistant calli were
selected and plants were regenerated and rooted in the presence of 10 mg/L bialaphos as described previously (Caplan et al., 1992). A total of 68 plants containing pGluB1Gly and 34 plants containing pGluB1GlyIV were obtained and analyzed for the inserted glycinin gene by PCR amplification.
Glutelin and glycinin cDNAs encoding the mature primary
sequences were amplified by PCR using the following pair of
oligonucleotides; 5-cagcagctattaggccagagcactag-3 and
5-gggaagctttatccgcaagccgacctaag-3 for GluA-1,
5-cagctatttaatcccagcacaaaccc-3 and
5-gggaagcttacattactctgaggtctcgc-3 for GluB-1,
5-ttcagttccagagagcagcc-3 and 5-cgcggatccatacaaaaagggctctaag-3 for
A1aB1b. The latter primer of each pair corresponds to the downstream
primer and contained either a HindIII site for GluA-1 and
GluB-1 or a BamHI site for A1aB1b. The ends of the PCR
products were polished using a blunting kit (TaKaRa-Shuzo, Shiga,
Japan) and then digested with HindIII or
BamHI. The resultant DNA fragments were inserted into the
filled-in NcoI and the HindIII sites or the
filled-in NcoI and the BamHI sites of the pET-21d
vector (Novagen) to yield the Escherichia coli expression
plasmids pEGluA-1, pEGluB-1, and pEA1aB1b.
For protein induction, E. coli BL21(DE3) cells
harboring individual expression plasmids were grown in Luria-Bertani
medium. At an A600 of 0.6, isopropyl-
-D-thiogalactopyranoside (final concentration 1 mM) was added and the cells were
allowed to grow for 24 h at 37°C for GluA-1 and GluB-1 and at
20°C for A1aB1b.
RNA Analysis
RNA was isolated from leaf and developing seeds as described by
Takaiwa et al. (1987). Fifteen micrograms of total RNA was electrophoresed in a 1.2% (w/v) formaldehyde-containing agarose gel and then blotted to a charged nylon membrane (Amersham). The resulting nylon membrane was then hybridized in 50% (v/v)
formamide, 5× Denhardts solution, 0.5% (w/v) SDS, and 250 µg/mL
denatured salmon-sperm DNA at 45°C for 24 h with
32P-labeled glycinin A1aB1b and a glutelin GluB-1
cDNA inserts of pUG1 (Utsumi et al., 1993b) and
pREEK1 (Takaiwa et al., 1989), respectively. The cDNA inserts were
labeled with 32P by the random-priming method
(Feinberg and Vogelstein, 1983). Membranes were washed under
high-stringency conditions to exclude cross-hybridization four times in
2× SSC and 0.1% (w/v) SDS at room temperature and then twice
in 0.1× SSC and 0.1% (w/v) SDS at 55°C.
Preparation of Anti-Proglutelin GluA-1 and GluB-1 Sera
E. coli cells were resuspended in 35 mM Na-Pi buffer (pH 8.0) containing 0.1 M NaCl, 1 mM EDTA, and 1.5 mM PMSF and disrupted by sonication. After
centrifugation, the pellet was extracted with a 1% (v/v) lactic acid,
1 mM EDTA solution to solubilize the recombinant
expressed proglutelins GluA-1 and GluB-1. The extracted proglutelins
were dialyzed against buffer A composed of 50 mM
Tris-HCl buffer (pH 8.0) containing 6 M urea, 0.1 M ME, 1 mM EDTA, and 1 mM PMSF, and then subjected to fast-flow
chromatography (2.6- × 10-cm columns; SP Sepharose FF, Pharmacia). The
proglutelins were eluted with a linear gradient of 0 to 0.2 M NaCl in buffer A. GluA-1 and GluB-1 were
further purified by SDS-PAGE (11%, w/v) (Laemmli, 1970), yielding a
single purified band. The purified proglutelins were electroeluted from
the polyacrylamide gel and then thoroughly dialyzed against 10 mM K-Pi buffer (pH 7.6) containing 6 M urea to remove excess SDS. Rabbit antibodies
were raised against the purified GluA-1 and GluB-1 as described
previously (Utsumi et al., 1993b).
Screening of Transgenic Rice Plants Accumulating Glycinin at a High
Level and Its in Situ Localization by Tissue Printing
Six seeds from independent transgenic plant were soaked overnight
at 4°C in 10 mM phosphate buffer (pH 7.4) containing
0.8% (w/v) NaCl, 0.2% (w/v) KCl, 10 mM
NaN3, and 0.4 M Suc, and then horizontally dissected into two pieces using a razor blade as described
by Manteuffel and Panitz (1993). The pieces were soaked in
acetone for 20 s to remove lipids and then pressed onto a
nitrocellulose membrane for 5 to 10 s. After the prints had been
made the membrane was baked for 30 min at 60°C. Glycinins were
detected with anti-glycinin serum followed by goat anti-rabbit
IgG-alkaline phosphatase conjugate (Promega).
For in situ localization of glycinin, a grain from a rice plant
expressing glycinin at the highest level was vertically dissected into
two pieces and treated as described above.
Electron Microscope Immunocytochemical Localization of
Glycinin
Analysis of immunocytochemical localization was carried out
essentially as described by Takeuchi and Nishimura (1993). Developing rice seeds were cut into 1.5- to 2.0-mm sections and fixed overnight at
4°C in 3% (v/v) glutaraldehyde solution. Tissues were washed three
times (10 min for each wash) with phosphate buffer and then dehydrated
through a 30%, 50%, 60%, 70%, 80%, 90% (v/v) series of ethanol
washes (15 min for each wash). The specimens were then further
dehydrated by three wash steps in 95% ethanol (15 min for each wash)
followed by three wash steps in 100% ethanol (30 min per wash). The
dehydrated tissues were infiltrated for 2 h in a 1:1 (v/v) London
white resin:ethanol mixture, in 2:1 (v/v) overnight, followed by 100%
London white resin over the next 24 h. Polymerization was in
gelatin capsules for 2 to 7 d at 52°C under nitrogen gas.
Ultrathin sections (60-80 nm) were obtained with a glass knife and
placed onto formal/carbon-coated grids. The sections were blocked with
5% (w/v) BSA-PBS and then incubated for 1 h at room temperature on a drop of anti-glycinin serum diluted 1:20 in PBS (10 mM Na-Pi buffer, pH 7.4, containing 150 mM
NaCl) supplemented with 0.8% (w/v) BSA (Sigma), 0.1% (w/v) gelatin,
and 2 mM NaN3 (BSA-PBS). The sections
were washed four times for 5 min each on a drop of BSA-PBS and then
incubated for 1 h at room temperature on a drop of goat antirabbit
IgG (H+L) (Auro Probe EM, Amersham) diluted 1:40 in BSA-PBS. After
washing twice with BSA-PBS, the sections were treated with 2.5%
(w/v) glutaraldehyde solution for 10 min and washed twice with
PBS and then once with distilled water (5 min per step). The sections
were stained for 30 min with 2% (w/v) uranyl acetate followed by a
1-min incubation in 80 mM lead nitrate, 120 mM
trisodium citrate dihydrate, and 0.16 N NaOH for 1 min. The
grids were examined and photographed using an electron microscope
(model H-700H, Hitachi, Tokyo).
Protein Extraction and Immunological Detection
For segregation analysis, 50 mature grains from each primary
transgenic rice plant (T0), which accumulated
significant amounts of normal or modified glycinins, were ground
separately with a mortar and pestle in 62.5 mM Tris-HCl
buffer (pH 6.8) containing 2% (w/v) SDS, 30% (v/v) glycerol, 0.1 M ME, 1.5 mM PMSF, and 1 mM EDTA at
4°C. Aliquots were then spotted on a nitrocellulose membrane and
screened immunologically with anti-glycinin serum. The glycinin
accumulation levels were estimated by comparing the densitometry
signals obtained from extracts prepared from the transgenic plants
against those of known amounts of purified glycinin.
Analysis of Self-Assembly by Suc Density Gradient
Centrifugation
Soluble protein fractions were obtained by extracting 100 mg of
mature rice seeds in 35 mM K-Pi buffer (pH 7.6) containing 0.4 M NaCl, 1.5 mM PMSF, and 1 mM
EDTA (buffer B). A sample was then resolved by Suc density gradient
centrifugation and the fractions analyzed by SDS-PAGE followed by
immunoblotting as described previously (Utsumi et al., 1993b). The 2S,
7S, and 11S fractions purified from soybean (Glycine max)
seeds according to the method of Thanh and Shibasaki (1976) were run in
parallel as size markers.
Solubility Analysis of Glycinin Accumulated in Rice Seeds
Rice seeds (18 mg) were finely ground and proteins were
sequentially extracted six times for 1 h each with 1.8 mL of
saline solution (buffer B) containing 25 mM NEM with
vigorous shaking at room temperature. The residual proteins were then
extracted three times with 1.8 mL of 1% (v/v) lactic acid containing 1 mM EDTA and 25 mM NEM.
Renaturation Analysis of Reduced-Denatured Glutelin, Glycinin, and
Proglycinin
Finely ground rice seeds (1.5 g) were sequentially extracted with
10 mL of buffer B containing 25 mM NEM followed by 10 mL of
55% (v/v) n-propyl alcohol to solubilize albumins,
globulins, and prolamins. The residue was then extracted with 10 mL of
1% (v/v) lactic acid containing 1 mM EDTA and 25 mM NEM from the insoluble fraction to yield an
enriched glutelin fraction. Glycinins were purified from soybean as
described by Nagano et al. (1992). Recombinant proglycinin A1aB1b was
purified as described previously (Kim et al., 1990a).
Glutelin, glycinin, and recombinant proglycinin A1aB1b (all at 100 µg/mL), and mixtures (100 µg each/mL) of glutelin with glycinin,
recombinant proglycinin A1aB1b, ovalbumin (Sigma) or BSA (Sigma), and
zein (Nacalai, Kyoto) with glycinin or proglycinin A1aB1b were
denatured and reduced in 35 mM K-Pi buffer (pH7.6) containing 1 mM EDTA, 1 mM PMSF, 6 M urea, and 0.1 M ME. The denatured and reduced
proteins were renaturated using a two-step dialysis method (Katsube et
al., 1998a) based on the procedures of Utsumi et al. (1980) and Hirose
et al. (1989). The denatured and reduced samples were dialyzed against
35 mM K-Pi buffer (pH 7.6) containing 0.1 M
NaCl, 40% (w/v) glycerol, and 0.1 M ME for 12 h at 4°C, followed by dialysis for 24 h at 20°C, and then
against the same buffer without ME for 24 h at 20°C. The
renatured samples were dialyzed against 35 mM K-Pi buffer
(pH 7.6) containing 0.1 M NaCl. The supernatant and the
precipitate fractions of the dialysate were subjected to SDS-PAGE.
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RESULTS |
Transformation of Rice with Chimeric Normal and Modified Glycinin
Genes
Normal and modified preproglycinin A1aB1b cDNAs composed of 27-bp
5 noncoding, 1,488- or 1,506-bp coding, and 189-bp 3 noncoding sequences were fused to the 5-flanking region (1,302 and +18 nucleotides from the transcriptional start site) of the rice storage protein glutelin GluB-1 gene (Fig. 1).
These chimeric constructs were introduced into rice protoplasts by
electroporation or into rice callus cells by A. tumefaciens-mediated transformation, and regenerated plants were
obtained.
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| Figure 1.
Schematic representation of the normal (A) and
Met-modified (B) glycinin chimeric genes. White and shaded boxes
indicate the coding region and the 5 and 3 noncoding regions of
glycinin cDNA, respectively. Thin and light-shaded lines depict vector
DNA. Arrows indicate direction of transcription. 35S Pro, CaMV 35S
promoter; Bar, the bialaphos resistance gene; Nos Ter, nopaline
synthase gene terminator; g7, gene 7 terminator; Glutelin Pro,
GluB-1 promoter; Glycinin cDNA, preproglycinin A1aB1b
cDNA; Glutelin Ter, GluB-1 terminator; R, right T-DNA
border; L, left T-DNA border.
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To investigate the temporal- and spatial-specific expression of the
introduced chimeric genes, total RNA was prepared from developing seeds
and leaves of T2 transgenic homozygous plants. As
shown in Figure 2A, glycinin mRNA levels
increased from 6 to 16 DAF, reached a plateau at 16 DAF, and then
gradually decreased at 22 DAF. These profiles were identical to those
of the glutelin GluB mRNA detected by GluB-1 cDNA (Fig. 2B), indicating
that the glycinin gene was expressed specifically in the maturing seeds under the control of the GluB-1 promoter. Glycinin mRNAs
were not detected in leaves or any nonseed tissues. There was no
apparent difference in temporal and spatial patterns between the normal and modified glycinin constructs (data not shown).
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| Figure 2.
Northern analysis of developing seed and leaf RNAs
isolated from transgenic rice plants. Total RNAs were isolated and
fractionated by electrophoresis on a 1.2% agarose gel containing
formaldehyde and then blotted onto nylon membranes. The RNAs were
hybridized with the coding regions of preproglycinin A1aB1b cDNA (A) or
proglutelin GluB-1 cDNA (B). L, Leaf.
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Screening of Transgenic Rice Plants Accumulating Soybean Glycinin
and Its Localization
The insertion of normal and modified glycinin genes into the rice
genome of bialaphos-resistant plants was verified by PCR analysis. The
accumulation levels of glycinin in individual plants (total of 70 plants) were roughly estimated immunologically by tissue printing. Most
seeds were positively stained with anti-glycinin sera, although the
antigen levels varied considerably among independent plants. Transgenic
lines 11[pGluB1Gly] and 417[pGluB1GlyIV] exhibited the highest
accumulation level among independent transgenic plants for each
construct.
Immunological analysis of tissue prints prepared from longitudinally
sectioned rice seeds revealed that glycinin was confined to the
endosperm (Fig. 3). The same results were
observed for seeds containing the modified glycinin. This accumulation
pattern is consistent with the GUS staining pattern directed by the
GluB-1 promoter (Wu et al., 1998a, 1998b), indicating that
the accumulation pattern driven by the GluB-1 promoter was
retained in the transgenic rice.
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| Figure 3.
Histochemical localization of glycinin in mature
rice seeds. Seed section imprints from nontransgenic (A) and transgenic
rice 11 [pGluB1Gly] (B) plants on nitrocellulose were visualized
using anti-glycinin serum and a goat anti-rabbit IgG-alkaline
phosphatase conjugate. Em, Embryo; En, endosperm.
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Glycinin is deposited into the protein storage vacuole in developing
soybean seeds. Similarly, the rice glutelins are packaged in a vacuolar
compartment called protein body II (Yamagata et al., 1982; Yamagata and
Tanaka, 1986). Rice also contains a second storage site called protein
body I, which contains the prolamins. Immunocytochemical studies at the
electron microscope level were conducted to determine the intracellular
location of glycinin in transgenic rice plants. Figure
4, A and B, shows the separate accumulation and packaging of prolamins and glutelins into protein bodies I and II, respectively. A similar immunocytochemical analysis for the soybean glycinin showed that the gold particles that denote reactivity with anti-glycinin were restricted to the matrix of the
glutelin-containing protein body II. In addition to its absence in
protein body I (Fig. 4D), immunogold particles were not evident on the
ER or the Golgi apparatus, presumably due to the low concentration of
glycinin in these organelles. These results indicate that glycinins are
correctly targeted to the matrix of protein body II. Moreover, similar
results were obtained for the modified glycinin, indicating that the
modification did not cause mistargeting.
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| Figure 4.
Immunolocalization of accumulated glycinin in
transgenic rice endosperm. Sections of embedded tissue were
labeled with anti-prolamin (A), anti-glutelin (B), or anti-glycinin (C
and D) sera, followed by gold-conjugated anti-rabbit IgG (A and B, 30 nm gold particles; C and D, 10 nm). A, B, and D, Transgenic rice
11[pGluB1Gly]; C, nontransgenic rice.
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Progeny Segregation Analysis of the Expression Level
Total proteins extracted from 50 T1 seeds of
the transgenic plants 11[pGluB1Gly] and 417[pGluB1GlyIV] were
spotted onto nitrocellulose membranes and glycinin levels were
estimated immunologically (Fig. 5, A and
C). The highest levels of both normal and modified glycinins were
around 5% of total proteins. Comparison of the relative accumulation levels of 50 grains within each plant indicated that the ratio of the
grains with high (1%-5%), medium (0.3%-1.0%), low (0.1%-0.3%), and little (<0.1%) expression was approximately 1:1:1:1, as shown in
Figure 5, B and D. The bialaphos resistance trait segregated into a 3:1
ratio of resistant to sensitive seeds. These segregation patterns are
consistent with the view that the transgene was inserted in the rice
genome at a single locus or closely linked loci. This was confirmed by
Southern analysis of genomic DNA isolated from selected transgenic
plants, which showed that one to a few copies of the intact fragment
were integrated into the rice genome (data not shown).
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| Figure 5.
Comparison of the accumulation levels of normal
and Met-modified glycinins in individual rice seeds by dot
immunoblotting. Extracts (10 µg of proteins) of matured seeds from
transgenic rice plants 11[pGluB1Gly] (A) and 417[pGluB1GlyIV] (C)
were analyzed by immunoblotting. Between 0.001 and 1 µg of purified
glycinin mixed with 10 µg of proteins of seed extract from
nontransgenic rice plant was used as standard. The number below each
dot indicates an individual seed. Frequencies of the accumulation
levels of the expressed proteins in the individual seeds of
11[pGluB1Gly] (B) and 417[pGluB1GlyIV] (D) were calculated from dot
blots.
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Ten T2 generation seeds, each derived from
T1 plants that gave the highest accumulation of
normal and modified glycinins by half-seed analysis, were analyzed for
the glycinin accumulation level, and the degree of glycinin
accumulation was stably inherited in the third generation (data not
shown).
Assembly of Glycinin in Rice Seeds
To investigate the ability of glycinin to self-assemble into
hexamers in rice seeds, the salt-soluble proteins from the
nontransgenic rice seeds and the T2 seeds of
11[pGluB1Gly] and 417[pGluB1GlyIV] were subjected to Suc density
gradient centrifugation. After fractionation, proteins in each fraction
were analyzed by SDS-PAGE in the absence of ME, followed by
immunoblotting using anti-glycinin serum (Fig. 6, A and C). No immunoreactive bands were
detected in the extract from nontransgenic rice seeds (Fig. 6A). The
band with a molecular size similar to that of the mature
disulfide-bonded subunit of glycinin was mainly observed in fractions
5, 9, and 15 of the extract from the transgenic rice seeds,
corresponding to a size of 2S (monomer), 7S to 8S (trimer), and 11S to
12S (hexamer), respectively (Fig. 6C). In addition, the band with a
molecular size similar to that of the proglycinin subunit expressed in
E. coli (which is larger than that of mature glycinin) was
detected in fractions 5 and 9 to 11. Soluble protein extracts from
417[pGluB1Gly IV] gave similar sedimentation patterns of 2S, 7S to
8S, and 11S to 12S, as shown in Figure 6C. These results indicate that
newly synthesized glycinins assemble into trimeric and hexameric
oligomers in a manner similar to that observed in developing soybean
seeds.
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| Figure 6.
Analysis of the assembly of the normal glycinins
accumulated in the seeds of the transgenic rice plant. Seed extracts
were subjected to centrifugation on a 12-mL, 10% to 30% (w/v) linear
Suc density gradient and analyzed by SDS-PAGE in the absence (A
and C) or presence (B and D) of ME followed by immunoblotting.
Sedimentation is from left to right. Sedimentation standards in
Svedberg units are given. Arrowheads indicate the positions of
recombinant proglycinin (Pro) synthesized in E. coli
(lane P) and native mature glycinin (A-B; lane M). A and B,
Nontransgenic rice; C and D, 11[pGluB1Gly].
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To investigate whether the expressed glycinin was processed into mature
form, protein fractions from the Suc density gradients were analyzed by
SDS-PAGE in the presence of ME. Although immunoreactive bands were not
detected in the extract from nontransgenic rice seeds (Fig. 6B), the
band with a molecular size corresponding to the acidic polypeptide of
mature glycinin was observed in fractions 5, 9, and 15 of the extract
from the transgenic rice seeds, with the concomitant disappearance of
the mature disulfide-bonded subunit (Fig. 6D). The band with a
molecular size corresponding to proglycinin was also detected in
fractions 5 and 9 to 11. These results indicate that processing from
proglycinin to the mature form occurred in transgenic rice seeds in a
manner similar to that in soybean seed, although a part of glycinin
remained as proglycinin in the state of a monomer or a trimer. The
anti-proglycinin antibody reacted very weakly with the basic
polypeptide.
Glutelins Display a Distinct Sedimentation Pattern
The processing and assembly of rice glutelins are poorly
understood. To determine whether the rice glutelins are assembled into
higher order oligomeric structures (as observed for the soybean glycinins), the same Suc density gradient fractions depicted in Figure
6, A and B, were analyzed by immunoblotting using anti-proglutelin GluA-1 serum. Trace amounts of the proglutelin were evident between the
2S and 7S regions of the Suc density gradient (Fig.
7A). The bulk of the soluble glutelin
polypeptides was observed in the 2S region, with a molecular size
comparable to the acidic subunit of 32 kD (Fig. 7A). Reactive glutelin
polypeptides were essentially absent or at very low levels in the 7S
and 11S regions of the Suc density gradients. When subjected to
reducing conditions, bands corresponding to the acidic and basic
polypeptides as well as a smaller reactive polypeptide band were
evident (Fig. 7B). These results show that even though glutelin shares
considerable homology with glycinin, the rice protein is processed and
assembled in higher order structures by a different mechanism.
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| Figure 7.
Analysis of the formation of hybrid-11S storage
protein complexes between glycinin and glutelin in seeds of transgenic
rice plants. The same Suc density gradient fractions depicted in Figure
6 were analyzed by SDS-PAGE in the absence (A and C) and presence (B
and D) of ME followed by immunoblotting using anti-proglutelin GluA-1
serum. Sedimentation is from left to right. Sedimentation standards are
given in Svedberg units. Arrowheads indicate the positions of
recombinant proglutelin (Pro) (lane B) and the native acidic
polypeptide (A) of glutelin. One microgram each of recombinant
proglycinin (lane P) and native mature glycinin (lane M) was
electrophoresed as negative controls. A and B, Nontransgenic rice; C
and D, 11[pGluB1Gly].
|
|
Formation of Hybrid Proteins between Glycinin and Glutelin
The sedimentation pattern of glutelin polypeptides was also
investigated in the glycinin-expressing transgenic plants and yielded a
much different pattern than that evident in normal plants. In addition
to the polypeptide pattern at the 2S region observed earlier in
nontransgenic plants, proglutelin polypeptides were evident at the 11S
region with significantly smaller amounts at the 7S region of the Suc
density gradient (Fig. 7C). In the presence of ME, the proglutelin
polypeptides in 11S region dissociated into constituent polypeptides
with a molecular size corresponding to the acidic polypeptides (Fig.
7D). Likewise, the bulk of the proglutelin in the 7S region dissociated
into constituent polypeptides but a proportion remained undissociated,
indicating intact unprocessed proglutelin. These results indicate that
glutelin polypeptides assembled with glycinin proteins to form
hetero-trimeric (7S) and hetero-hexameric (11S) structures. The amount
of glutelin forming hybrid globulins with glycinin was estimated by
densitometric measurements to be around 5% of the total glycinin
extracted with a neutral saline solution.
Rice glutelins are composed of two classes, class A and class B. To
examine whether polypeptides of both glutelin classes or only a single
class can assemble with glycinin, proteins from the 11S fraction of the
Suc density gradient (from 11[pGluB1Gly]) were examined by immunoblot
analysis using anti-proglutelin GluA-1 and GluB-1 sera (Fig.
8). Anti-proglutelin GluA-1 serum reacts strongly with proglutelin GluA-1 and weakly with GluB-1 (Fig. 8A, lanes
1 and 2), whereas the reactivity of anti-proglutelin GluB-1 serum is
just the opposite (Fig. 8B, lanes 1 and 2). Immunoblot analysis
indicated that both antibodies reacted to glutelin polypeptides to
about the same level irrespective of the absence or presence of ME
(Fig. 8, lanes 4 and 6). These results indicate that glutelins A and B
contributed equally to the formation of hybrid globulins with glycinin
subunits.
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| Figure 8.
Analysis of the glutelin type involved in the
hybrid globulin in seeds of transgenic rice plants. Proteins in
fraction 15 in Figure 7 were analyzed immunologically using antibodies
specific for GluA-1 (A, lanes 3-6) and GluB-1 (B, lanes 3-6) glutelin
polypeptides. GluA-1 (100 ng, lane 1) and GluB-1 (100 ng, lane 2) were
electrophoresed as positive controls. Lanes 1 to 4 were electrophoresed
in the absence of ME; lanes 5 and 6 in the presence of ME. Lanes 3 and
5, Nontransgenic rice; lanes 4 and 6, 11[pGluB1Gly].
|
|
Glycinin was extracted repeatedly from the seeds of transgenic rice to
examine whether glycinin is incorporated into the glutelin fraction.
Glycinin was effectively solubilized by neutral saline solution after
five extractions (Fig. 9, lane 6). When
the residual protein fraction was extracted with dilute lactic acid
solution, significant levels of glycinins were released (Fig. 9, lane
7), suggesting that glycinin also assembled with glutelins to form an
insoluble complex. The amount of glycinin in the insoluble fraction
was estimated to be around 30% of the total accumulated glycinin.
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| Figure 9.
Solubility analysis of glycinins accumulated in
the seeds of transgenic rice plants. Seed proteins were extracted six
successive times with saline solution followed by three successive
times with lactic acid solution. Each saline extract (lanes 1-6) and a
mixture of the lactic acid solution extract (lane 7) were
electrophoresed in the absence of ME and then analyzed by
immunoblotting using anti-glycinin serum. A-B denotes the position of
the native mature glycinin (lane M).
|
|
The results obtained here strongly suggest that glycinin and glutelin
subunits form hybrid-oligomeric complexes in addition to their
homologous assembly in the transgenic rice seeds. Alternatively, there
is also the possibility that the formation of hybrid proteins is caused
by nonspecific disulfide bonding and/or hydrophobic interaction. We
analyzed the renaturation behavior of glutelin, glycinin, and
proglycinin and mixtures of glutelin with either glycinin, proglycinin,
ovalbumin or BSA, and glycinin or proglycinin with zein by SDS-PAGE in
the absence (Fig. 10A) and presence
(Fig. 10B) of ME. Ovalbumin contains four free Cys residues and one
disulfide bond, and BSA contains one free Cys residue and 17 disulfide
bonds. Both of these proteins are soluble in a neutral saline solution. The latter protein binds fatty acids and bile acids, indicating that
the molecular surface has hydrophobic patches.
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| Figure 10.
Renaturation of glutelin, glycinin, and
proglycinin either alone or in combination with other proteins.
Purified glutelin, glycinin, or proglycinin was denatured and reduced
in urea solution containing ME and then slowly renatured by dialysis
either alone or in combination with other proteins. The dialysates were
then centrifuged to obtain supernatant (S) and pellet (P) fractions,
which were analyzed by SDS-PAGE in the absence (A) or presence (B) of
ME. Lanes 1 and 2, Proglycinin; lanes 3 and 4, glutelin; lanes 5 and 6, mixture of glutelin and proglycinin; lanes 7 and 8, mixture of glutelin
and ovalbumin (OVA); lanes 9 and 10, mixture of glutelin and BSA; lanes
11 and 12, glycinin; lanes 13 and 14, mixture of glutelin and glycinin;
lanes 15 and 16, mixture of glycinin and zein; lanes 17 and
18, mixtures of proglycinin and zein. Protein molecular size
markers were loaded onto lane M (200, 116, 97.4, 66, 45, 31, 21.5, and
14.5 kD).
|
|
Zein is not soluble in a neutral saline solution and contains a
variable number of Cys residues (1-15) depending on the zein species
(Shewry and Tatham, 1990). These proteins were denatured and reduced in
urea containing ME and slowly renatured by dialysis as outlined in
``Materials and Methods''. Upon renaturation, the bulk of the
glutelin formed a precipitate with a small amount of acidic and basic
polypeptides present in the supernatant (Fig. 10, lanes 3 and 4). This
pattern was more readily apparent when the proteins were resolved by
SDS-PAGE in the presence (Fig. 10B) of ME than in its absence (Fig.
10A). All of the glycinin (Fig. 10, lanes 11 and 12) and
proglycinin A1aB1b (Fig. 10, lanes 1 and 2) renatured in a soluble
form. However, when glutelin was renatured along with glycinin (Fig.
10, lanes 13 and 14) or proglycinin A1aB1b (Fig. 10, lanes 5 and 6),
about one third of the total glycinin and proglycinin A1aB1b was
recovered in the insoluble fraction.
These results suggest that glycinin and proglycinin A1aB1b were
incorporated into the insoluble glutelin network through disulfide bonding. In contrast, much of the ovalbumin and BSA was recovered in
soluble fraction when renatured in the presence of glutelin (Fig. 10,
lanes 7-10), indicating no significant interaction among these
proteins. Likewise, most of zein was recovered in the insoluble fraction and did not interact with glycinin (Fig. 10, lanes 15 and 16)
or proglycinin (Fig. 10, lanes 17 and 18). These results suggest that
the formation of hybrid proteins between glutelin and glycinin is
through specific interactions, especially in the case of insoluble
hybrid proteins through specific disulfide bonding of Cys residues.
| |
DISCUSSION |
In this study, we expressed the soybean glycinin genes in rice to
determine whether they would be synthesized, processed, and assembled
into higher order oligomeric structures, as was observed in developing
soybean seeds. Our results clearly demonstrate that the majority (70%)
of the newly synthesized glycinin molecules accumulate as salt-soluble
proteins (globulins) and are assembled into trimeric (7S) and hexameric
(11S) structures. Moreover, the glycinin polypeptides are properly
processed into acidic and basic polypeptides interlinked by disulfide
bonding in a manner similar to that in soybean embryonic tissue and
cotyledons. In contrast, rice glutelins do not appear to be assembled
into such oligomeric structures, as the only soluble form was
proteolytically processed and sedimented at the 2S region of the Suc
density gradient. Since proteolytic processing has been suggested to
occur in the protein storage vacuole, these results indicate that
glutelins are transported either as a monomer or as aggregates much
larger than 11S. Alternatively, glutelins are assembled into trimers
and hexamers similar to glycinin, but the transport from the ER to the
protein storage vacuole and the processing step occurs at a much faster
rate than for glycinins, resulting in low levels of these higher order
oligomeric forms.
An important finding of this study was that a portion of the trimers
and hexamers resolved by Suc density gradient centrifugation were
hybrid globulins containing both glutelin and glycinin subunits. About
30% of newly synthesized glycinin molecules formed hybrid glutelins
through specific interactions with glutelin subunits. Thus, glycinin
and glutelin have the capacity to assemble with one another to form
higher order protein structures. It is known that the formation of
glycinin hexamers requires the processing of proglycinin to the mature
form (Dickinson et al., 1989). Both proglycinin and proglutelin were
observed in the trimer fraction but not in the hexamer fraction,
indicating that glycinin and glutelin assemble into a trimeric and
hexameric structural conformation very similar to the homoglycinin
oligomers. These common structural features together with the
similarities in their processing and sorting mechanisms further
supports the hypothesis that these proteins are derived from a common
ancestral gene (Takaiwa et al., 1987; Utsumi, 1992). Moreover, it may
be concluded that transport of the rice glutelin occurs by an ordered
process of protein folding at the secondary, tertiary, and quaternary
levels, much like what occurs with soybean glycinin.
Assembly and processing of glycinins synthesized in rice were not
complete, as shown in Figure 6. Glycinin monomers, trimers, and
hexamers were observed in the proportion around 1:1:1, and unprocessed
proglycinins were observed in the monomer and trimer fractions. A
similar pattern of insufficient assembly and processing was observed
for glycinins expressed under the control of the GluB-1
promoter in the endosperm tissue of tobacco seeds (Takaiwa et al.,
1995). In contrast, the assembly and processing of glycinin into 11S
species was much more efficient when the transgene was expressed under
the control of CaMV 35S promoter in transgenic tobacco seeds. The CaMV
35S promoter drives maximum levels of glycinin expression of only
approximately 0.1% of the total proteins (Utsumi et al., 1993b)
compared with the stronger GluB-1 promoter, which yields up
to 4% to 5% of the total proteins in rice or tobacco (Takaiwa et al.,
1995). As the formation of the 11S hexamer is ultimately dependent on
the transport of the 7S trimer to the protein storage vacuole, these
observations suggest that the endomembrane system of rice and tobacco
endosperm cells has a limiting capacity for protein transport to the
vacuole.
Glutelin subunits are classified into two types, GluA and GluB, on the
basis of their amino acid sequences (Takaiwa et al., 1991). GluA and
GluB contain eight and five Cys residues, respectively. Four of these
Cys residues are conserved in the glutelins and in all of the 11S
globulins found in legume and nonlegume seeds (Katsube et al., 1998b).
These four conserved Cys residues form two disulfide bonds; one is an
interchain bond between the acidic (Cys residue III of both types) and
a basic polypeptide (VII of GluA and IV of GluB), while the other is an
intrachain disulfide bond in the acidic polypeptide (I and II of both
types) (Fig. 11) (Utsumi, 1992; Katsube
et al., 1998b). Cys residues VIII of GluA and V of GluB are probably
free, since most of glycinin-type proteins have a free Cys residue at
the homologous site; e.g. cysteine residue VIII of glycinin A1aB1b
(Katsube et al., 1998b).
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| Figure 11.
Schematic representation of the proposed
positions of free Cys residues and disulfide bonds in glutelins A and B
and glycinin A1aB1b. White and hatched areas are the acidic and the
basic polypeptides, respectively. N and C represent the NH2
and COOH termini, respectively. The Cys residues are numbered by Roman
numerals from the NH2 terminus. Disulfide bonds are
represented by lines connecting two Cys residues.
|
|
We had speculated that some or all of the Cys residues IV, V, and VI of
GluA contribute to the formation of multiple interchain disulfide
bonds, resulting in the formation of insoluble glutelin aggregates, and
that GluB is able to assemble into hexamers together with glycinin
subunits (Katsube et al., 1998b). However, we observed both types of
glutelins can assemble with glycinin to form hexamer structures (Fig.
8). This observation does not eliminate the possibility that Cys
residues are responsible for the formation of glutelin aggregate, but
indicates that the interaction of glutelin with glycinin is independent
of the number of Cys residues in the glutelin primary sequences.
This glutelin-glycinin interaction not only results in soluble protein
complexes but insoluble ones as well, since glycinin is observed in the
glutelin fraction. In the highest glycinin-expressing transgenic rice
seeds, glutelin is present at around 15-fold excess over glycinin.
Therefore, we can speculate that: Interaction between glycinin subunits
is stronger than that between glycinin and glutelin subunits, since
homoglycinin is dominant (around 70% of the expressed glycinin), and
once a glycinin subunit interacts with a glutelin subunit, the
formation of hybrid globulin or glutelin proceeds depending on the
population of glycinin and glutelin. Since glutelin levels are much
larger than glycinin levels, the proportion of glycinin in the
hybrid-glutelin complexes is much larger than in the hybrid globulin.
It has been demonstrated that soybean globulins exhibit a significant
hypocholesterolemic effect if the dietary intake of these proteins is
more than 6 g/d (Imura et al., 1996). Glycinin is likely to be
primarily responsible for this effect on cholesterol levels (Makino et
al., 1988; Sugano, 1996). Since glycinin accounts for around 40% of
the soybean globulins, a dietary intake of 2.4 g/d of glycinin would be
required. Seeds of 11[pGluB1Gly] contain 8% protein (Momma et al.,
1999), including 400 mg of glycinin/100 g seeds. Since the average
daily consumption of rice in Japan is 200 to 250 g, a transgenic
rice plant containing 3 times higher glycinin than our present highest
level would be necessary to fulfill this dietary requirement. By
exploiting the processing and assembly of glycinin proteins into homo-
and hetero-hexameric structures and into insoluble aggregates with
glutelin, such heterologous expression levels in rice may be achieved
in the near future.
| |
FOOTNOTES |
1
This work was supported in part by grants from
the Program for Promotion of Basic Research Activities for Innovative
Biosciences (to S.U. and F.T.), the Ministry of Agriculture, Forestry,
and Fisheries of Japan (to T.K., S.U., and F.T.), and Takano Life Science Research Foundation (to S.U.).
2
These authors contributed equally to the
paper.
*
Corresponding author; e-mail utsumi{at}soya.food.kyoto-u.ac.jp;
fax 81-774-38-3761.
Received March 12, 1999;
accepted May 6, 1999.
| |
ABBREVIATIONS |
Abbreviations:
DAF, days after flowering.
ME, 2-mercaptoethanol.
NEM, N-ethylmaleimide.
| |
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Top
Abstract
Introduction
