Rice Proteomics: A Step Towards Functional Analysis of the Rice Genome
Summary - The technique of proteome analysis with 2D-PAGE has the power to
monitor global changes that occur in the protein expression of tissues, organisms,
and/or under stresses. In this study, proteins extracted from tissues from rice, some
cellular organella of rice, and rice under various kinds of stress were separated by 2D-
PAGE. It was revealed using an image analyzer that a total of 10,589 protein spots
could be detected on 2D-PAGE gels stained by CBB. The separated proteins were
electroblotted onto a polyvinylidene difluoride membrane, and the N-terminal amino
acid sequences of 272 out of 905 proteins were determined. The internal amino acid
sequences of 633 proteins were determined using the protein sequencer or MALDI-
TOF MS after enzyme digestion of the proteins. Finally, a data-file of rice proteins
was constructed which included information on amino acid sequences and sequence
homologies. The major proteins involved in the growth and development of rice can
be identified using the proteome approach. Some of these proteins, including a
calcium-binding protein that turned out to be calreticulin, and a gibberellin-binding
protein, which is RuBisCO activase in rice, have functions in the signal transduction
pathway. The information thus obtained from the rice proteome will be helpful in
predicting the function of the unknown proteins and aid in their molecular cloning.
Key words rice; proteomic; two-dimensional polyacrylamide gel electrophoresis;
plant physiology; protein sequencer; mass spectrometry
Introduction - Rice (Oryza sativa L.) is an important crop in Eastern Asia. A vast
number of rice cultivars as well as wild species of rice are widely grown, and their
genetic and molecular makeup is being actively investigated. Rice is also considered
as a model plant in monocots because of the relatively small size of its genome. The
rice genome conceivably consists of about 430 million base pairs [1], and about 30,000
genes can be expressed in rice plant tissue. High-resolution two-dimensional
polyacrylamide gel electrophoresis (2D-PAGE) is useful for separating complex
protein mixtures [2]. Due to its high resolving power, the technique has been
employed to study alterations in cellular protein expression in response to various
stimuli or as a result of differentiation and development [3]. The latter approach
further allows cDNA cloning from the resultant sequence(s).
Sequencing of a protein separated by 2D-PAGE became possible with the
introduction of protein electroblotting methods that allow the efficient transfer of a
sample from the gel matrix onto a support that is suitable for gas-phase sequencing or
related techniques [4]. Proteins can also be recognized by their amino acid
composition, their exact molecular weight as determined by mass spectrometry (MS),
or partial amino acid sequence. In conjunction with automated gel scanning and
computer-assisted analysis, 2D-PAGE has contributed greatly to the development of a
protein database [5-8].
Gel-separated proteins can be identified rapidly by MS, and if genomic
information is also available, such analyses permit the systematic identification of the
protein complement of a genome, the proteome [9]. In addition, MS is a powerful
tool for the analysis of isoforms, secondary modifications of proteins such as
glycosylation and phosphorylation, and proteolysis, which only require low amounts
(picomoles to attomoles) of proteins [10]. Such systematic analyses of protein
populations are summarized by the term proteomics. Thus, proteomics bridges the
gap between genomic sequence information and the actual protein population in a
specific tissue, cell, or cellular compartment.
Concerning the rice plant, some well-known studies have dealt with the
construction of proteomes from complex origins, such as the leaf, embryo, endosperm,
root, stem, shoot and callus proteome [11-15]. Proteomics studies to date have
mainly focused on those changes in genome expression that are triggered by
environmental factors. Examples of descriptive proteomes include the global
comparison of green and etiolated rice shoots [13], and an analysis of defense-
associated responses in the rice leaf and leaf sheath following a jasmonic acid
treatment [16]. One major advantage of the rice 2D-PAGE database, in which most
known proteins are recorded, is the wealth of new proteins on which experiments can
be conducted at the biochemical and molecular levels. In addition to facilitating the
identification of known proteins, these sequences can be used to prepare
oligodeoxyribonucleotides, which are essential for cloning the corresponding cDNA.
The aim of this study was to separate proteins from rice, to determine their relative
molecular weights and isoelectric points, and to perform N-terminal and internal amino
acid sequence analysis using a protein sequencer and MS. Finally, an attempt was
also made to study the physiological significance of some proteins thus identified from
rice.
1 A part of the proteome research for functional analysis of the rice genome
1-1 An important objective in rice proteome research
The Rice Genome Research Project is a joint project of the National Institute of
Agrobiological Sciences and the Institute of the Society for Techno-innovation of
Agriculture, Forestry and Fisheries. In addition, partial support comes from the
Genome Research Program of Japanese Ministry of Agriculture, Forestry and Fisheries.
The program started in October 1991 and the first phase continued through 1997,
resulting in the establishment of some of the basic tools of rice genome analysis.
Reorganized into a national project in 1998, the Rice Genome Research Project now
intends to completely sequence the entire rice genome and subsequently to pursue
integrated goals in functional genomics, genome informatics and applied genomics.
Some important objectives in rice proteome research are as follows: (1) to determine
whether the cDNA encoding particular proteins from the cDNA library constructed
from rice can be identified by a computer search of an amino acid sequence homology,
and (2) to predict the function of the proteins and study the physiological significance
of functional proteins in rice.
1. 2 Strategy to determine the amino acid sequence in rice proteome analysis
1. 2. 1 Gel electrophoresis
A portion of the rice tissues was removed, homogenized with a lysis buffer [2],
and centrifuged. The supernatant was subjected to 2D-PAGE [2]. Isoelectric
focusing (IEF) or immobilized pH gradient (IPG) was carried out in a glass tube with a
length of 13 cm and a diameter of 3 mm. Sodium dodecyl sulfate (SDS)-PAGE in the
second dimension was performed with 15% separation gels. The isoelectric point and
relative molecular weight of each protein were determined using the 2D-PAGE
standard (Bio-Rad, Richmond, CA, USA). The localization sites of individual
proteins on the gels were evaluated automatically with Image Master 2D Elite software
(Amersham Pharmacia Biotech, Uppsala, Sweden).
1. 2. 2 N-terminal amino acid sequence analysis
Following separation by 2D-PAGE, the proteins were electroblotted onto a
polyvinylidene difluoride (PVDF) membrane (ProBlott; Applied Biosystems, Foster
City, CA, USA) and detected by Coomassie Brilliant blue (CBB) [11]. The spots
were excised from the PVDF membrane and applied to the upper glass block of the
reaction chamber in a gas-phase protein sequencer (Procise; Applied Biosystems).
Edman degradation was performed according to the standard program supplied by
Applied Biosystems.
1. 2. 3 Internal amino acid sequence analysis
The proteins were separated by 2D-PAGE and stained with CBB. Gel pieces
containing protein spots were removed and the protein was electroeluted from the gel
pieces using and an electrophoretic concentrator (ISCO, Lincoln, CA, USA) at 2 W
constant power for 2 h. After electroelution, the protein solution was dialyzed against
deionized water for 2 days and lyophilized. The protein dissolved in the SDS sample
buffer (pH 6.8) was applied to a sample well in SDS-PAGE. The sample solution was
overlaid with a solution containing Staphylococcus aureus V8 protease (Pierce,
Rockford, IL, USA). Electrophoresis was performed until the sample and protease
were stacked in the upper gel and interrupted for 30 min to digest the protein [17].
Electrophoresis was then continued and the separated digests were electroblotted onto
the PVDF membrane and subjected to gas-phase sequencing [18].
1. 2. 4 Homology search of amino acid sequences
The amino acid sequences obtained were compared with those of proteins in the
amino acid sequence database (MPSRCH-pp protein-protein database, University of
Edinburgh, UK).
1. 2. 5 Analysis using mass spectrometry
The CBB stained protein spots were excised from a gel, washed with 25% (v/v)
methanol and 7% (v/v) acetic acid for 12 h at room temperature, and destained with 50
mM NH4 HCO3 in 50% (v/v) methanol for 1 h at 40¡î. The proteins were reduced
with 10 mM DTT in 100 mM NH4 HCO3 for 1 h at 60¡î and incubated with 40 mM
iodoacetamide in 100 mM NH4 HCO3 for 30 min at room temperature. The gel pieces
were minced and allowed to dry and then rehydrated in 100 mM NH4 HCO3 with 1
pmol trypsin at 37¡î overnight. The digested peptides were extracted from the gel
slices with 0.1 % trifluroacetic acid (TFA) in 50% (v/v) acetonitrile/water 3 times.
The peptide solution thus obtained was dried up and reconstituted with 30 µL of 0.1%
TFA in 5% acetonitrile/water, and then desalted by Zip Tip C18TM pipette tips
(Millipore, Bedfold, MA, USA). Matrix-assisted laser desorption ionization
(MALDI)-MS was performed using a Voyager Elite XL time-of-flight mass
spectrometer (Applied Biosystems, Framingham, MA, USA). The above peptide
solution was mixed with the matrix solution, the supernatant of a 50% acetonitrile
solution saturated with α-cyano-4-hydroxycinnamic acid, and then air-dried on the flat
surface of a stainless steel plate. Calibrations were carried out using a standard
peptide mixture [19]. The mass spectra were subjected to a sequence database search
with Mascot software (Matrix Science Ltd, London, UK).
1. 3 Enlargement of the rice protein data-file
The technique of proteome analysis with 2D-PAGE has the power to monitor
global changes that occur in the protein expression of tissues and organisms whether or
not they are under stress. In this study, proteins extracted from endosperm, embryo,
root, callus, green shoot, etiolated shoot, leaf sheath and panicle, some cellular
organella of rice, and rice under various kinds of stress were separated by 2D-PAGE
(Fig. 1).
It was revealed using an image analyzer that a total of 10,589 protein spots could
be detected on 2D-PAGE gels stained by CBB. The separated proteins were
electroblotted onto a PVDF membrane, and the N-terminal amino acid sequences of
272 out of 905 proteins were determined. The N-terminal regions of the remaining
proteins could not be sequenced, and it was concluded that they had a blocking group
at the N-terminus. The internal amino acid sequences of 633 proteins were
determined using the gas-phase protein sequencer or MS after enzyme digestion of
proteins. Finally, a data-file of rice proteins was constructed which included
information on amino acid sequences and sequence homologies (Table 1). The rice
cDNA catalog contains about 39.6% of genes in the entire genome. The cDNA
encoding particular proteins could be screened with a 40% probability using a
computer search for sequence homology.
Fig. 1. Strategy to determine the amino acid sequence in rice proteome analysis.
2 Comparison of some techniques for the analysis of genome function
2. 1 Comparison of proteome and cDNA microarray techniques to monitor changes
in protein and gene expression
Brassinosteroids (BRs) are a group of naturally occurring plant steroids with
structural similarities to insect and animal steroid hormones [21]. Exogenous
application of BRs to plant tissues evokes various growth responses such as cell
elongation, proliferation, differentiation, organ bending and a number of other
physiological processes [22]. It is believed that BR affects plant growth through the
regulation of gene expression. However, only a few BR-regulated genes have been
identified so far. We are now systematically analyzing the changes of gene
expression caused by BR in rice seedlings using a combination of proteome and cDNA
microarray approaches. The bending of the second leaf and its leaf sheath in rice
seedlings is very sensitive to BRs and this unique characteristic of rice leaves has been
used as a quantitative bioassay for BRs [23]. We adopted this model system and
found that a 1 µM BL treatment caused the maximum bending under these
experimental conditions (Fig. 2).
First, proteins were extracted from lamina joints treated with 1 µM BL for 48 h
or water as a control and analyzed by 2D-PAGE. A systematic comparison of protein
patterns showed that 6 proteins were increased, when compared to the water control, in
the lamina joint treated with BL (Fig. 2). Sequence analysis revealed that 2 protein
spots (LJ258 and LJ262) were homologous to the RuBisCO large subunit (LSU), and
one protein (LJ133) showed homology to glyceraldehyde-3-phosphate dehydrogenase,
which is a key enzyme in glycolysis. The other 3 protein spots (LJa, LJb and LJ195)
did not display any significant homology to proteins in the databases researched.
Second, a cDNA microarray containing 1,265 independent rice genes randomly
selected from 9,600 ESTs was used to analyze differential gene expression caused by
BL. The arrays were hybridized with Cy5 fluorescent-labeled probes of lamina joint
sample treated with 1 µM BL or water control (Fig. 2). Fluorescent signal differences
greater than two-fold between the control and BL-treated samples were considered to
be significant. Data analysis showed that the expression of 12 genes was enhanced by
the BL treatment (Fig. 2). Among them, 5 genes had homologies based on a search in
the GenBank database using the BLAST program. A vacuolar H+-transporting
ATPase homologue (Element 0145) showed higher expression in the BL-treated
lamina joint. Element 0550 was homologous to Brmheadia finlaysonia mRNA for
extensin suggesting a role in BR-mediated cell expansion. Elements 0250 and 1029
showed homologies to the Arabidopsis ubiquitin-conjugating enzyme and Helianthus
annuus mRNA for the ACC oxidase-related protein, respectively. Element 0654 was
a rice photosystem II oxygen-evolving complex protein I. The other 7 had no
significant homologies in the database.
By using proteome analysis of differential protein expression and cDNA
microarray analysis of differential gene expression, we identified some changes at the
transcription and translation levels caused by the BL in the lamina joint. However,
we did not find any overlaps in the results of the two approaches in the present study.
This can be explained in the following manner: (1) the amount of some proteins is far
beyond the detection sensitivity of CBB staining on 2D-PAGE, and (2) the cDNA
microarray we used in the present study contains 1,265 genes, which account for only
about 4% of the total number of genes predicted in rice, and those changed proteins
detected in proteome are not contained in the cDNA microarray used.
Fig. 2.
2. 2 Identification of protein sequences using protein sequencer and mass
spectrometry
Fifty-four proteins of leaf sheath from rice seedling were analyzed by Edman
sequencing and MS. For Edman sequencing, most of the proteins were N-terminally
blocked. Using MS, all proteins were identified by matching the protein from rice
and other species (Table 2). The similar proteins are spot LS083, homologous to
calreticulin, a calcium-binding protein located in the endoplasmic reticulum (ER) [24];
spot LS261, matched to Bowman Birk trypsin inhibitor (BBTI); spot LS317, identified
as a superoxide dismutase (SOD) (Cu/Zn) 1, a cytoplasmic protein that destroys
radicals that are normally produced within the cells and are toxic to biological systems
[25]; spot LS322, found to be a C97454 rice callus cDNA clone; spot LS332, identified
as a chloroplast SOD (Cu/Zn) [26]; spot LS346, matched to RuBisCO small subunit
(SSU) from chloroplast protein, which catalyzes the first reaction in the Calvin cycle
[27].
The N-terminal sequences of three proteins, spots LS327, LS328 and LS329,
were identical to each other and to the sequence of SOD (Cu/Zn) 2 (AC: P28757) from
rice [28]. Using MALDI-TOF MS, these proteins were identified from a family
protein of SOD; spots LS327 and LS328 were homologous to SOD (Cu/Zn) 2; and spot
LS329 was homologous to SOD (Fe) (AC: JG0179). Spots LS347, LS349 and LS351
had the same N-terminal sequences and were homologous to salt stress protein [29].
In MS analysis, the three proteins displayed a different peptide mass fingerprint and
were matched to different salt-induced proteins. Two proteins (LS347 and LS351)
were matched to the mannose-binding rice lectin (MRL) (AC: BAA25369), and
another (LS349) was matched to a salt gene-product protein (AC: AAB53810).
Twenty-seven proteins were identified by both methods, and 19 of them
(19/27=70%) had a homology similar to the rice databases (Table 2). The N-terminal
sequences are important because the N-termini of mature proteins that often differ from
those predicted from open-reading frames of DNA sequences are shown. MALDI-
TOF MS analysis not only provides the likely identities of proteins but can also
confirm whether the protein species analyzed are post-translational modifications or
isoforms. Sometimes, an MS-identified result needs to be further confirmed by
sequence tags. However, with the combination of these two techniques, it is possible
to identify the corresponding genes.
3 Identification of functional proteins using a proteome approach
3. 1 A calcium-binding protein, which turned out to be calreticulin
Plant-cultured cells are useful for transformation and recombination for in crop
improvements. The capacity for regeneration is thus essential for plant-cultured cells.
Short-term cultured cells of rice can regenerate, but they do so to a lesser extent in
long-term cultures (Fig. 3.). A phospho-protein involved in the regeneration of rice-
cultured cells was identified using an in vitro phosphorylation assay (Fig. 3.). This
protein was purified, and the N-terminal and internal amino acid sequences were
determined [30].
Using the proteome data-file of rice cultured cells, this protein was identified as
a calcium-binding protein, which is homologous to calreticulin of maize. Degenerate
PCR primers, designed on the basis of the amino acid sequence of the protein, were
used for PCR. A cDNA library was constructed in a Uni-ZAP XR vector using
mRNA prepared from rice suspension-cultured cells. Approximately 20,000 plaques
forming units of the rice cDNA library were screened using the PCR fragment as a
probe. Positive clones were isolated and analyzed by Southern blot. A full-length
cDNA insert, CRO1, was sequenced [24].
In order to know precisely the function of calreticulin in rice tissues, the full-
length cDNA for calreticulin was introduced into rice cells in the sense and antisense
orientation under the control of the cauliflower mosaic virus (CaMV) 35S promoter in
the pIG121-Hm vector (a gift from Dr. K. Nakamura) by means of Agrobacterium (a
gift from Dr. E. Hood)-mediated transformation [31]. In our previous research,
calreticulin negatively affected the rice callus regeneration and growth [30]. In the
present study, the over-expression of calreticulin inhibited plant regeneration from
callus and seedling growth (Fig. 3.). These results suggest that calreticulin is a
negative repressor in differentiation system and development. These stages of rice are
affected by plant hormones. Calreticulin might play significant role in plant hormone
signal transduction.
Fig. 3.
3. 2 A gibberellin-binding protein, which is RuBisCO activase
Gibberellin (GA) is a class of plant hormones which regulates growth and
development, stem elongation, flowering and seed germination (Fig. 4.). The cellular
responses to GAs are thought to be mediated by GA receptors. A few GA-binding
proteins have been identified as candidates for GA receptors by using a variety of
techniques. A GA-binding protein in rice was identified using a ligand-binding assay
[32]. The GA-binding protein was purified, and N-terminal and internal amino acid
sequences were determined.
Using the proteome data-file of rice leaf sheath and leaf, this protein was
identified as RuBisCO activase in barley. Degenerate PCR primers, designed on the
basis of the amino acid sequence of RuBisCO activase, were used for PCR. A cDNA
library was constructed in an Uni-ZAP XR vector using mRNA prepared from a leaf
sheath and a leaf of rice. Approximately 20,000 plaques forming units of the rice
cDNA library were screened using the PCR fragment as a probe. Positive clones
were isolated and analyzed by Southern blot. Two full-length cDNA inserts, OsrcaA1
and OsrcaA2, were sequenced [33].
In order to know the precise function of the RuBisCO activase in rice tissues, the
full-length cDNA for RuBisCO activase was introduced into the rice cells in the sense
and antisense orientation under the control of the CaMV 35S promoter in the pIG121-
Hm vector by means of Agrobacterium-mediated transformation [31]. The over-
expression of OsrcaA1 promoted seedling growth better than the over-expression of
OsrcaA2 and vector control-transformed rice. Plant regeneration from callus and
seedling growth was repressed in antisense Osrca transgenic rice. It is known that
light activation of RuBisCO is one of the first processes adversely affected by elevated
temperature. In spinach, the larger form of RuBisCO activase is more thermostable
than the smaller form [34]. In Arabidopsis, at physiological ratios of ADP/ATP, the
larger isoform has minimal ATP hydrolysis and RuBisCO activation activity in
comparison with the smaller isoform [35]. Recently, the function of the RuBisCO
activase larger isoform has been identified. However, the precise function of the
smaller isoform of RuBisCO activase is still not clear. The above results suggest that
the small isoform might have a unique role in the regulation of leaf sheath elongation
(Figure 4-II). GA3 ligand-binding assay shows that RuBisCO activase is a receptor
for GA signal transduction pathway(s). The antisense tranasformant was repressed
stem and leaf growth, suggesting that the GA signal pathway is essential for rice
development.
Fig. 4.
4 Concluding remarks
2D-PAGE separation and analysis provides a convenient way to study the
various proteins that are present or induced in rice plants under different growth
conditions, i.e., normal and under stress. Knowing which proteins are being
synthesized in specific tissues and at different developmental stages of rice under a
variety of physiological conditions, can lead to identifying the roles for these proteins.
The study of partial amino acid sequence analysis of plant proteins will contribute
greatly to the field of molecular biology for the identification of proteins through
homology search. The information thus obtained from amino acid sequence of these
proteins will be helpful in predicting the function of the proteins and aid their
molecular cloning in future experiments.
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Figure legends
Fig. 1. Strategy to determine the amino acid sequence in rice proteome analysis.
The proteins were separated by 2D-PAGE with IEF in the first dimension and SDS-
PAGE in the second dimension according to their isoelectric point (pI) and then
according to their molecular mass, resulting in a two-dimensional gel. The spots were
then visualized by CBB staining and then scanned for image analysis. To obtain
sequence tags by Edman sequencing, we stained gels with CBB before blotting to
increase the sensitivity and to allow easier matching of the gels. The spots contained
5 to 20 pmol. For MS, individual protein spots were then selected, excised from the
gel, and digested with the site-specific protease trypsin, resulting in a set of tryptic
peptides. The peptides were extracted, and their masses were measured by MALDI-
TOF MS. The list of measured peptide masses was compared with the masses of the
predicted tryptic peptides for each entry in the sequence database.
Fig. 2. Comparison of proteome (A) and cDNA microarray (B) techniques to monitor
changes in protein and gene expression caused by brassinolide in rice lamina joint.
A. Proteome analysis of protein expression after brassinolide treatment on lamina joint.
B. Microarray analysis of gene expression after brassinolide treatment on lamina joint.
Fig. 3. Identification of a calcium-binding protein, calreticulin, and effects of sense
over-expression and antisense suppression of calreticulin on the growth of rice plant.
I: Time-course changes of rice-cultured cells after they were transferred to a
regeneration medium. The proteins from rice-cultured cells corresponding to each
stage were extracted, phosphorylated in vitro, and separated by 2D-PAGE. Spot C
shows the position of calreticulin.
II: Rice was transformed with Agrobacterium tumefaciens strain EHA101 containing
binary vector pIG121-Hm harboring the rice calreticulin gene either in the sense or
antisense direction under the control of a CaMV35S promoter.
Fig. 4. Identification of the gibberellin-binding protein, which is RuBisCO activase,
and effects of sense over-expression and antisense suppression of RuBisCO activase on
the growth of rice plant.
I: Growth of rice seedlings after a gibberellin treatment. GA-binding protein was
identified using a ligand-binding assay using 3H-labeled GA3 and 2D-PAGE after an in
vitro phosphorylation assay. Arrow heads indicate the position of RuBisCO activase.
II: Rice was transformed with Agrobacterium tumefaciens strain EHA101 containing
binary vector pIG121-Hm harboring the rice RuBisCO activase gene either in a sense
or antisense direction under the control of a CaMV35S promoter.
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