Title of Article:
Accumulation of a Sesame 2S Albumin Enhances Methionine and
Cysteine Levels of Transgenic Rice Seeds
Tiger T. T. Lee*, and Jason T. C. Tzen*
*Graduate
Institute of Biotechnology, National Chung-Hsing University, Taichung,
Taiwan 40227 ROC
1
Abstract
With the aim of improving rice nutritive value, a chimeric gene encoding a
precursor polypeptide of sesame 2S albumin, a sulfur-rich seed storage protein, was
expressed in transgenic rice plants under the control of glutelin promoter.
Rice grains
harvested from the first generation of ten different transformed lines inherited the
transgene, and the accumulated sesame 2S albumin was presumably processed correctly
as its mature form in sesame seed. This transgene was found to express specifically in
maturing rice seeds with its encoded sesame 2S albumin exclusively accumulated in
seeds.
Crude protein contents in rice grains of five putative homozygous lines were
increased by 0.64-3.54%, and the methionine and cysteine contents of these transgenic
rice grains were separately elevated by 29-76% and 31-75% compared with those of
wild-type rice grains.
Key words: methionine, sulfur amino acid, sesame 2S albumin, transgenic rice
Abbreviation: anti-2SL, antibody against the large subunit of sesame 2S albumin;
CaMV, cauliflower mosaic virus; S2SA, sesame 2S albumin; S-amino acid,
sulfur-containing amino acid
2
Introduction
Rice is a staple food supplying principal energy for more than one third of world’s
population.
Moreover, rice polishing provides an important agricultural by-product
used as animal feed.
With a protein content ca. 6-8% (on dry weight basis), rice is a
primary protein source for numerous people, especially where vegetable-based diets are
the major food sources.
Nevertheless, the low contents of sulfur-containing amino
acids (S-amino acids), 0.18% methionine and 0.11% cysteine, considerably limit the
nutritive value of rice. 1)
Animals are unable to synthesize methionine, and have to acquire this essential
amino acid from their food. 2) Since animals are capable of converting methionine to
cysteine, but not the reverse, methionine is the only essential S-amino acid in terms of
food nutrition.
Dietary deficiency of methionine results in an imbalanced uptake of
amino acids and may retard the growth and development of human and animals.3)
In
addition to nutritive value, methionine plays an essential role in cellular metabolism; it is
the precursor for the biosynthesis of S-adenosylmethionine, a substrate in numerous
transmethylation reactions that occur in the biosynthesis of many secondary metabolites.4)
To sustain a minimal amount of S-amino acids for optimal growth and development of
human and livestock, methionine-rich additive or even pure methionine has been
constantly supplemented to diet or animal feed.
Sesame seed (Sesamum indicum L.), due to its high methionine content, is
considered as a source for nutritionally important proteins.
Thirty years ago, Protein
Advisory Group of the United Nations declared that sesame flour is an adequate
supplement for methionine enrichment in human diets.5) Based on the deduced amino
acid sequence of its corresponding gene, 2S albumin, the major soluble protein in sesame
3
seed has been demonstrated to be a sulfur-rich protein, which apparently accounts for the
high quality of nutritive content in sesame.6) Similar to other known seed 2S albumins,
the sesame 2S albumin (S2SA) is first synthesized as a precursor protein (17 kDa) with a
cleavable N-terminal signal sequence for ER penetration. After cleavage of the signal
sequence and further post-translational processing and folding, the resulting mature
S2SA comprises two subunits of 9 kDa and 4 kDa linked by disulfide bonds, and is
accumulated in seed protein bodies.
In light of the limited nutritive value of rice caused by its low S-amino acid
content, we attempted to increase S-amino acid content of rice by introducing the edible
sulfur-rich protein, S2SA, via transgenic technique.
Transgenic rice plants with
detectable S2SA in their seeds were obtained under the control of a rice seed-specific
glutelin promoter.
Five putative homozygous lines were screened from these transgenic
plants; significant increases in both methionine and cysteine contents were detected in
their rice grains.
Materials and Methods
Plasmid construction. A chimeric gene, pGlu2S, constructed with a rice
seed-specific glutelin promoter (1200 bp) 7) and a cDNA fragment encoding the S2SA
precursor polypeptide (450 bp), 6) was ligated into pCAMBIA 1300 (Accession No.
AF234296) between the restriction enzyme sites of HindIII and EcoRI.
The resulting
plasmid, harboring a selectable marker hygromycin phosphotransferase gene, was
transferred into Agrobacterium tumefaciens strain EHA105 by electroporation.
4
Plant transformation and selection. Oryza sativa L., japonica cv. TNG67, a major
rice variety cultured in Taiwan, was selected as the target plant for transformation.
Rice
calli derived from immature embryos were used as the target material for
Agrobacterium-mediated transformation according to the report of Hiei et al. 8) with the
following modifications.
Rice calli were incubated in a suspension of Agrobacteria
tumefaciens containing pCAMBIA 1300 for 15-30 min.
Transformants were removed
from the suspension and transferred to solid 2N6-AS medium (2-fold N6 salts,
2,4-dichlorophenoxyacetic acid 2 ppm, casamino acid 1 g/L, sucrose 30 g/L, pH 5.2),
and cocultivated in a plant incubator at 28C for 72 h in the dark. After cocultivation,
transformants were washed in a liquid medium with cefotaxime (250 mg/L) and plated
on solid 2N6-CH medium (2-fold N6 salts, 2,4-dichlorophenoxyacetic acid 2 ppm,
sucrose 30 g/L, pH 5.7) for 3 weeks.
Subcultured transformants were then transferred
to RS-selection medium (MS salts, casamino acid 1 g/L, naphthaleneacetic acid 0.02
ppm, kinetin 2 ppm, sorbitol 30 g/L, sucrose 30 g/L, pH 5.7) containing hygromycin 50
ppm every ten days until shoot regenerated.
Regenerated plantlets (T0 plants) were
transplanted to soil in pots in a restricted greenhouse in the Taiwan Agricultural
Research Institute, Taichung.
Transgenic lines were detected by screening with
hygromycin resistance and PCR amplification.
In PCR detection, genomic DNA
extracted from leaves of different transformed lines was used as template with two
primers designed according to the S2SA gene.
Southern and Northern hybridization. Total genomic DNA for Southern blot
analysis was isolated from 3-week-old seedlings of ten independently transgenic rice
lines using the method of Dellaporta et al.. 9) Aliquots of DNA (15 µg) were digested
5
with HindIII/EcoRI or BglI, fractionated on a 1% agarose gel, transferred onto the
Zeta-probe GT membrane (Bio-Rad, USA), and hybridized with a 32P-labeled probe
containing the S2SA full-length coding region (450 bp). Hybridization and detection
were carried out according to the manufacturer’s instructions.
Total RNA was extracted from seeds of wild-type or trangenic plants and other
tissues such as root, stem, and leaf, according to the method described in Sambrook et al..
10)
Extracted RNA of 10 µg was fractionated on a 1.2% agarose/formaldehyde gel,
blotted onto the Zeta-probe blotting membrane (Bio-Rad, USA), and hybridized as
described above.
Antibody preparation and Western blotting. Large subunit (9 kDa) of S2SA was
purified from the soluble fraction of sesame seed extract in the presence of
-mercaptoethanol as described before. 11) Antibody against large subunit of S2SA was
raised in chickens, and immunoglobulin, anti-2SL, was purified from egg yolks for the
immunoassays. 12)
In the immunoassays, proteins were extracted from rice seeds,
fractionated on a 15% SDS-PAGE gel, and transferred onto nitrocellulose membrane in a
Bio-Rad Trans-Blot system according to the manufacturer’s instructions.
The
membrane was first probed with primary anti-2SL and then treated with secondary
anti-chicken antibody conjugated with horse radish peroxidase (Sigma, St. Louis, MO, U.
S. A.). After antibody recognition, the membrane was incubated with
4-chloro-1-naphthol containing H2O2 for color development as described by Chen et al..
13)
Immunological tissue printing. In situ localization of the S2SA in transgenic rice
seeds was detected using the immunological tissue printing method. 14) Rice grains
6
from wild-type and transgenic plants were cut in half at the vertical section and subjected
to immunodetection as described in Western blotting.
Selection of homozygous lines. T1 seeds from four transgenic lines were selected for
germination in the medium containing hygromycin as described previously, and ten
seedlings from each transgenic line were grown in the restricted greenhouse.
To screen
homozygous transgenic rice plants containing S2SA, forty T2 seeds harvested from each
transgenic plant were tested for their germination rate in the presence of hygromycin.
Transgenic plants whose T2 seeds could 100% germinate in the selective antibiotic were
picked up as putative homozygous transgenic lines. The inheritance of S2SA gene in
the germinated seedlings was also confirmed by PCR detection as described previously.
Analysis of crude protein content and amino acid composition. Crude protein
content was estimated according to the method described by the Association of Official
Agricultural Chemists. 15) To analyze amino acid composition, rice seed proteins
(approximately 100 mg, sampled from the extract of 50 seeds) from one wild-type and 5
homozygous transgenic lines were individually subjected to performic acid oxidation
prior to acidic hydrolysis in 15 ml 6 N HCl solution at 105C for 22 h according to the
procedure of Sibbald.16) Amino acid composition and S-amino acid content were
analyzed in the Department of Production Management, Animal Technology Institute,
Taiwan, by a Beckman Model 126 amino acid analyzer (Paloo Alto, CA).
7
Results
Detection of the S2SA gene and its expression in transgenic rice plants
Ten transgenic rice lines (G1-G10) were obtained by Agrobacterium-mediated
transformation, and the insertion of pGlu2S in their genomes was confirmed by PCR
detection (data not shown).
The result indicated that S2SA gene directed by the rice
glutelin promoter was transferred reliably to these ten transformed rice plants.
To
further confirm the success of transformation, four transformant T1 seeds (G1, G3, G5,
and G6) were selected from the ten lines and subjected to restriction enzyme digestion
and Southern hybridization. As expected, a HindIII/EcoRI fragment of approximately 2
kb was hybridized with the 32P-labelled S2SA clone in the genomes of these transgenic
plants (Fig. 1A).
Based on Southern hybridization of BglI digested fragments, these
four transgenic lines appeared to possess one or low copy number of the transgene in
their genomes (Fig. 1B).
Expression of S2SA gene was also detected in maturing rice
seeds of the four representative transgenic plants as shown in Fig. 1C.
Accumulation of S2SA in rice seed
To examine the accumulation of S2SA in transgenic rice seeds, proteins extracted
from T1 seeds of the ten transformants were subjected to analyses of SDS-PAGE and
Western blotting (Fig. 2).
An extra minor polypeptide of 14 kDa (estimated in the
SDS-PAGE gel without -mercaptoethanol), equivalent to the mature S2SA in sesame
seed, was found in the ten transformants compared with the wild-type plant (Fig. 2A).
The extra polypeptide was converted into a 9 kDa polypeptide, equivalent to the large
subunit of S2SA, in the presence of -mercaptoethanol (Fig. 2B). The presence of the 9
kDa large subunit of S2SA in the transgenic rice seeds was further confirmed by
8
immunodetection with anti-2SL (Fig. 2C). The results revealed that S2SA was
successfully accumulated in seeds of the ten transgenic plants, and the expressed 17 kDa
S2SA precursor was presumably processed correctly into two subunits linked by
disulfide bonds though the small subunit (4 kDa) of S2SA in transgenic seeds could not
be clearly resolved in the SDS-PAGE gel (Fig. 2B) of this study.
Putative homozygous transgenic rice plants
Five putative homozygous transgenic plants, G1-5, G3-4, G3-5, G5-2, and G6-5,
were screened from the four representative T1 lines.
T2 seeds harvested from these
putative homozygous transgenic plants showed 100% germination (using 40 seeds in
each line) in the presence of the selective antibiotic, hygromycin; genomic DNA
extracted from their seedlings inherited the S2SA gene as detected by PCR amplification
(data not shown). Accumulated amount of S2SA in T2 seeds was substantially higher
than that in T1 seeds as shown in Fig. 3 for the G6 and G6-5 transgenic plants.
According to Northern hybridization (Fig. 4A) and Western blot analysis (Fig. 4B), the
S2SA gene was specifically expressed in maturing seeds with its encoded S2SA correctly
processed and exclusively accumulated in seeds of the G6-5 plant.
Distribution of the expressed S2SA in transgenic rice seeds
Milled rice and bran polished from T2 seeds of the G6-5 putative homozygous line
were subjected to analyses of SDS-PAGE and Western blotting (Fig. 5).
The results
showed that S2SA was accumulated in both portions; milled rice (mainly endosperm)
and bran (mainly embryo and aleurone layer) possessed approximately 90% and 10% of
the accumulated S2SA, respectively.
The localization of S2SA in transgenic G6-5
seeds was detected by immunological tissue printing (Fig. 6A). The expressed S2SA
9
was found in endosperm, embryo, and aleurone layer of the G6-5 seeds, in accord with
the distribution observed in Fig. 5. Transparency of rice grains harvested from the five
transgenic rice plants, as represented by G6-5 seeds, was substantially reduced when
compared with that of wild-type rice grains (Fig. 6B).
Crude protein content and amino acid composition of transgenic rice seeds
Mature rice grains harvested from one wild-type and the five putative homozygous
transgenic plants were subjected to analyses of crude protein, S-amino acid, and amino
acid composition. Crude protein contents of the five transgenic rice grains were
increased by 0.64-3.54% compared with that of the wild-type rice grains (Table 1). The
increase in total protein content was accompanied by S-amino acid content among the
five transgenic rice grains (Table 2).
In comparison with methionine content (0.21%)
of the wild-type rice, the five transgenic rice grains possessed a considerably higher level
of methionine ranging from 0.27 to 0.37%. Evidently, introduction of S2SA into the
five transgenic plants elevated the methionine content in their seeds by 28-76%.
In
addition, cysteine content was also increased by 31-68% in these five transgenic rice
grains.
Regardless of S-amino acid content, the amino acid contents of transgenic rice
grains, as represented by G6-5 seeds, were unaltered or slightly increased when
compared with those in the wild-type rice grains (Table 3).
10
Discussion
Major crops such as corn, soybean, and rice are low in one or more essential amino
acids, and thus have been engineered to express seed proteins with essential amino acids,
particularly methionine and lysine, at a higher content to increase their nutritional quality.
17)
Glutelin promoter is strongly expressed in the entire seed of maturing rice,
particularly in the aleurone and subaleurone cells of the endosperm. 18) Many proteins
have been successfully expressed in rice grains using glutelin promoters, e.g.,
-phaseolin for enhancing lysine content, 18) ferritin for increasing iron level, 19) and
enzymes in -carotene biosynthesis for accumulating vitamin A. 20) As expected in this
study, S2SA, a sulfur-rich seed storage protein, was successfully expressed and
accumulated in transgenic rice grains under the control of glutelin promoter.
Successful expression of a sulfur-rich protein in transgenic plants does not ensure
the increase of methionine and/or cysteine content in the target sources as a consequence
that the sulfur-rich protein may be synthesized at the cost of reducing other proteins
and/or depriving free S-amino acids without much alternation of their amino acid
composition. 21) Fortunately, in our case, the S2SA specifically accumulated in the
transgenic rice seeds under the control of a rice glutelin promoter enhanced both
methionine and cysteine contents.
In the best (G6-5 line) of our transgenic rice seeds,
76% and 68% increase in methionine and cysteine levels were detected, respectively.
Two other homologous sulfur-rich 2S albumins from Brazil nut and sunflower
have been expressed in various target plants such as canola, 3) narbon bean, 22) lupin, 23)
and potato to improve methionine content. 24) Among these transgenic dicot plants, the
expressed 2S albumin precursor polypeptides were all correctly processed into mature 2S
albumins in which two subunits were linked by disulfide bonds. On the basis of our
11
observation in transgenic rice seeds, it appeared that the dicot S2SA could be also
correctly processed in monocot rice plants.
Since the 2S albumin from Brazil nut has been demonstrated to be an allergen both
in the purified form and in extracts of transgenic seeds, 25) to date, the reported transgenic
plants with higher methionine content are mainly emphasized on their potential
utilization in animal feed. However, sesame has been conventionally used as a food
additive or key ingredient of diverse food products in Asian countries for many years,
e.g., sesame is used in many cookies and commonly sprayed on the top of refreshments
or steamed rice. Apparently, sesame proteins are not allergens, at least, to most people
in Asian countries.
It is assumed that the current transgenic rice seeds with correctly
processed S2SA are edible to human and their domestic animals.
In our experimental
conditions, the phenotype and growth rate of transgenic plants as well as their grain yield
appeared no difference from those of the wild-type control plants (data not shown). A
large scale of field trial will be examined in the follow-up research.
Acknowledgments
The authors thank Dr. Hsin-Kun Wu for providing the glutelin promoter, Ms.
Hsiu-Ya Yang for assistance in rice transformation, Mr. Shih-Chang Jih for
determination of crude protein content, Dr. Jih-Fang Wu for analysis of amino acid
composition, and Professor Chih-Ning Sun for critical reading of the manuscript. The
work was supported by a grant from the National Science Council, Taiwan, ROC (NSC
90-2313-B-005-077 to JTC Tzen).
12
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15
Table 1. Crude protein content (%) of rice seeds from one wild-type and five transgenic
plants
Transgenic lines (T2)
Wild-type
9.310.02
G1-5
G3-4
G3-5
G5-2
G6-5
9.480.01
9.410.02
9.390.00
9.370.02
9.640.04
(N=2)
16
Table 2. Content (%) of S-amino acid in rice seeds from one wild-type and five
transgenic plants (extracted from 50 seeds for each sample)
Transgenic lines
Amino acid
a
Wild-type
G1-5
G3-4
G3-5
G5-2
G6-5 a
Met
0.21
0.33
0.32
0.27
0.28
0.37
Cys
0.16
0.28
0.27
0.21
0.23
0.27
The contents of the best transgenic line, G6-5 were determined twice with identical
values.
17
Table 3.
Amino acid composition (%) of rice seeds from one wild-type and one
transgenic (G6-5) plants (extracted from 50 seeds for each sample)
____________________________
Wild-type
G6-5
____________________________
Asx
0.95
1.01
Glx
1.88
1.96
Thr
0.35
0.34
Ser
0.51
0.52
Gly
0.49
0.50
Ala
0.53
0.55
Val
0.64
0.67
Ile
0.37
0.40
Leu
0.88
0.92
Phe
0.61
0.63
His
0.25
0.25
Lys
0.39
0.38
Arg
0.84
0.88
Tyr
0.50
0.59
Met
0.21
0.37
Cys
0.16
0.27
____________________________
18
Figure legends
Figure 1.
Southern blot analysis of genomic DNA extracted from seedlings of one
wild-type (WT) and four transgenic rice plants.
Each lane was loaded with 15 g of genomic DNA completely digested with Hind
III-EcoR I (A) or Bgl I (B).
After blotting, the membrane was hybridized with a
32
P-labeled probe containing the coding sequence of S2SA.
(C) Northern blot analysis
of total RNA extracted from maturing rice seeds of one wild-type (WT) and four
transgenic rice plants.
Each lane was loaded with 10 g of total RNA extracted from
maturing rice seeds 25 days after pollination. After blotting, the membrane was
hybridized with a 32P-labeled probe containing the coding sequence of S2SA. Only the
portion of the membrane corresponding to the visible hybridized RNA is shown.
Figure 2.
SDS-PAGE and Western blotting of the total proteins in ten transgenic rice
grains.
Along with sesame total seed proteins (50 g), the total proteins (50 g) extracted
from rice grains of one wild-type (WT) and ten transgenic plants (G1-G10) were
resolved in SDS-PAGE in the absence (A) or presence (B) of -mercaptoethanol.
(C) A
duplicate gel in the presence of -mercaptoethanol was transferred onto nitrocellulose
membrane and then subjected to immunodetection with antibody against the large
subunit of S2SA (anti-2SL).
The locations of the extra 14 kDa mature S2SA (a minor
band in panel A) and its 9 kDa large subunit (a diffuse band in panel B) were indicated
by stars between G1 and G2 lanes.
19
Figure 3.
SDS-PAGE and Western blotting of the total proteins in T1 and T2 rice
grains.
Along with sesame total seed proteins, the total proteins extracted from wild-type
(WT), T1 transgenic (G6), and T2 transgenic (G6-5) rice grains were resolved in
SDS-PAGE (left panel).
A duplicate gel was transferred onto nitrocellulose membrane
and then subjected to immunoblotting detected by anti-2SL (right panel).
Labels on the
right indicate the molecular mass of the large subunit of S2SA.
Figure 4.
(A) Northern blot analysis of total RNA extracted from diverse tissues of a
transgenic rice plant. Each lane was loaded with 10 g of total RNA extracted from
root, stem, leaf, and maturing seed of a transgenic rice plant, G6-5.
After blotting, the
membrane was hybridized with a 32P-labeled probe containing the coding sequence of
S2SA. Only the portion of the membrane corresponding to the visible hybridized RNA
is shown. (B) SDS-PAGE and Western blotting of proteins extracted from various
tissues of a transgenic rice plant. Total proteins extracted from root, stem, leaf, and
seed of the transgenic rice, G6-5 were resolved in SDS-PAGE (upper panel). A
duplicate gel was transferred onto nitrocellulose membrane and then subjected to
immunoblotting detected by anti-2SL (lower panel).
molecular mass of the large subunit of S2SA.
20
Labels on the right indicate the
Figure 5.
SDS-PAGE and Western blotting of proteins extracted from milled rice and
rice bran of a transgenic rice plant.
Along with total proteins of a wild-type (WT) rice grain, the total proteins extracted
from whole grain, milled rice, and rice bran of the transgenic rice, G6-5, were resolved
in SDS-PAGE (left panel). A duplicate gel was transferred onto nitrocellulose
membrane and then subjected to immunoblotting detected by anti-2SL (right panel).
Labels on the right indicate the molecular mass of the large subunit of S2SA.
Figure 6.
(A) Immunological tissue printing of S2SA in transgenic rice seeds.
Rice
grains from a wild-type and two transgenic rice seeds (G6-5) were cut in half and
subjected to immuno-staining in a manner similar to Western blot.
from a wild-type and the transgenic rice, G6-5.
21
(B) Rice grains