Differential Expression of Defense/Stress-Related Marker Proteins in
Leaves of a Unique Rice Blast Lesion Mimic Mutant (blm)
Young-Ho Jung,† Randeep Rakwal,*,‡,§ Ganesh Kumar Agrawal,§ Junko Shibato,‡ Jung-A Kim,†
Mi Ok Lee,† Pil-Kyu Choi,† Seung-Hee Jung,† So Hee Kim,† Hee-Jong Koh,| Masami Yonekura,⊥
Hitoshi Iwahashi,‡ and Nam-Soo Jwa*,†
Department of Molecular Biology, College of Natural Science, Sejong University, Seoul 143-747, Korea, Human
Stress Signal Research Center (HSS), National Institute of Advanced Industrial Science and Technology (AIST)
WEST, Onogawa 16-1, Tsukuba, Ibaraki 305-8569, Japan, Research Laboratory for Agricultural Biotechnology
and Biochemistry (RLABB), G.P.O. Box 8207, Kathmandu, Nepal, School of Agricultural Biotechnology,
Seoul National University, Seoul 151-742, Korea, and Food Function Laboratory, School of Agriculture,
Ibaraki University, Ami, Ibaraki 300-0393, Japan
Received March 14, 2006
We analyzed a unique rice (Oryza sativa L.) blast lesion mimic (blm) mutant for differentially expressed
proteins in leaves of one- and two-week-old seedlings manifesting the lesion mimic phenotype. Gelbased one- and two-dimensional electrophoresis (1- and 2-DGE) was performed using leaves (blm and
wild-type, WT) before (stage 1, S1) and after (stage 2, S2) lesion formation. 1-DGE immunoblotting
revealed potent increase in the expression of a key pathogenesis-related (PR) marker biosynthetic
enzyme, naringenin 7-O-methyltransferase, involved in rice phytoalexin sakuranetin biosynthesis, and
three oxidative-stress-related marker proteins, catalase, ascorbate peroxidase (APX), and superoxide
dismutase (SOD) in leaves of the blm mutant. 2-D gel immunoblotting analysis with anti-APX and antiSOD antibodies revealed newly appearing cross-reacting protein spots in blm. 2-DGE analysis detected
50 Coomassie brilliant blue-stained protein spots differentially expressed in blm. A total of 23 and 44
protein spots was excised for analysis by N-terminal amino acid sequencing and nano-electrospray
ionization liquid chromatography mass spectrometry, respectively; 26 nonredundant proteins were
identified. The pathogenesis-related class 5 and 10 proteins, including a new OsPR10d protein, were
significantly induced in blm. The OsPR5 protein spot was stained with Pro-Q Diamond phosphoprotein
gel stain suggesting OsPR5 to be a putative phosphoprotein. Surprisingly, protein spot 20, a leaf
OsPR10b, showed identity to a rice root-specific PR-10 (RSOsPR10). To resolve this discrepancy, we
checked its expression in leaves of blm and WT (S1 and S2), respectively, using gene-specific primers
and reverse transcriptase-polymerase chain reaction; RSOsPR10 mRNA was found to express in the
leaves.
Keywords: Biomarkers • lesion mimic mutant • Oryza sativa • oxidative stress • proteomics
1. Introduction
Rice (Oryza sativa L.) is a staple food crop for more than
one-third of the world’s population accounting for 20% of total
calories consumed globally in 2000 (Word rice statistics from
International Rice Research Institute, http://www.irri.org/science/ricestat/index.asp). The importance of rice in the socio* Authors for correspondence: Dr. Randeep Rakwal, HSS, AIST-WEST,
Onogawa 16-1, Tsukuba, Ibaraki 305-8569, Japan. E-mail: rakwal-68@
aist.go.jp. Tel./fax: +81-29-861-8508. Dr. Nam-Soo Jwa, Department of
Molecular Biology, College of Natural Science, Sejong University, Seoul 143747, Korea. E-mail: [email protected]. Tel./fax: +82-31-378-0486/0485.
†
Sejong University.
‡
National Institute of Advanced Industrial Science and Technology (AIST)
WEST.
§
Research Laboratory for Agricultural Biotechnology and Biochemistry
(RLABB).
|
Seoul National University.
⊥
Ibaraki University.
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Journal of Proteome Research 2006, 5, 2586-2598
Published on Web 08/29/2006
economic fabric of our society makes it one of the most
important crop species in South Asia to study, especially on
its response to stress and disease, the main research focus of
our group. With tremendous advances in new technologies, it
is now possible to get more precise and broad information on
plant response to a wide variety of biotic and/or abiotic
stresses. Rice is a reference plant for the monocots,1 especially
cereal crops, and therefore, it was termed a cornerstone for
functional genomics of crop plants (for review, see ref 2).
Among the “omic pillars” that constitute functional genomics,
proteomics is a rapidly expanding field today. Proteomics is
one of the high-throughput approaches currently being used
to address biological function of plant by studying globally
expressed proteins in a given tissue.2-10
Two-dimensional gel electrophoresis (2-DGE) is the most
commonly used proteomics technology for monitoring changes
10.1021/pr060092c CCC: $33.50
 2006 American Chemical Society
Differential Expression of Defense/Stress-Related Marker Protein
in the expression levels of complex protein mixtures, and is
also the most widely utilized.2,8-10 2-DGE can simultaneously
separate thousands of proteins (and their modified forms) to
homogeneity, deliver high-quality protein resolution and dynamic range, and generate reference maps in a short period
of time, and at a cost “affordable” to proteomics researcher.
Moreover, 2-DGE, in conjunction with immunological detection
techniques, is a very powerful tool to identify a range of
proteins for which suitable antibodies are available. Thus, we
have every reason to believe that 2-DGE remains one of the
most suitable approaches toward the systematic characterization of the plant proteome. Notably, recent use of immobilized
pH gradient (IPG) technology for development of high-quality
2-D gel reference maps with greater spot reproducibility is the
next target in rice proteomics. Embracing IPG technology for
the gel-based approach will make 2-DGE even more attractive
soon. It is also emphasized that 2-DGE is not suitable for highand low-masses (>150 and <10 kDa), highly acidic and basic,
hydrophobic, and low-abundance proteins. Moreover, the
question of spot overlaps in low-quality gels makes it very
difficult for comparative protein profiling and quantitation. To
partially overcome these problems, we undoubtedly need to
apply parallel and complementary approaches (like one-DGE
and multidimensional protein identification technology, MudPIT) to exhaustively investigate the proteomes.11,12
In the present study, a proteomics approach aimed at
examining an important aspect of the rice plant response to
pathogenesis (defense responses to pathogens) was employed
for studying the lesion mimic mutants (LMMs). In LMMs, the
pathogenesis-related (PR) hypersensitive reaction (HR)-like
symptom (cell death) is activated in the absence of a pathogen,
resulting in spontaneous lesion appearance.13-15 The LMMs
have been identified in a variety of plants, such as maize,16
barley,17 rice,18-20 and Arabidopsis.15 Many of the LMMs present
an aberrant regulation of cell death, enhanced resistance to
pathogens, and constitutive expression of defense mechanisms
[autofluorescent phenolic compounds, callose deposition, reactive oxygen species (ROS), PR marker proteins/genes, and
phytoalexins]. It has been speculated that LMMs are affected
in genes of general importance for signaling pathways. The
LMMs may also affect mechanisms that control initiation or
propagation of multiple biochemical events in pathogentriggered R-gene-specific resistance.14 Conversely, the observed
lesion phenotype might be the result of a mutation that alters
cellular homeostasis like those seen in transgenic plants.14 Thus,
not all but a few LMMs are highly valued natural research
materials for elucidating the cell death mechanisms and may
ultimately provide insight into the pathogen-regulated plant
self-defense responses. This gel-based proteomics study reports
the identification of 33 differentially expressed proteins in the
rice LMM mutant (termed blm, blast lesion mimic) leaf over
the wild-type (WT).
2. Materials and Methods
2.1. blm Mutant and Wild-Type. The blm mutant was
derived from rice (O. sativa L.) cv. Hwacheong (WT) treated
with a chemical mutagen N-methyl-N-nitrosourea (MNU).20 For
characterization of blm, an in vitro condition was used exactly
as described previously.20 Briefly, the seeds were grown on 1/2
MS agar (Murashige and Skoog Basal Salt Mixture 2.15 g L-1,
sucrose 15 g L-1, and Phytagel 3 g L-1) medium in a sterilized
glass bottle under white fluorescent light (wavelength 390-500
nm, 150 µmol‚m-2‚s-1, 16 h photoperiod) at 28 °C and 70%
research articles
relative humidity. One-week after germination (stage 1, S1), the
temperature of the growth chamber was changed to 24 °C.
Lesion mimic phenotype started to appear 3-4 days after
temperature shift (stage 2, S2). Leaves (3rd/4th from bottom)
were harvested from three independent sets of rice seedlings
grown at different time periods in the year 2005 using 10-12
randomly selected seedlings/replication. The harvested leaves
were immediately frozen in liquid nitrogen and stored at -80
°C until further analysis.
2.2. Preparation of Total Crude Protein Extract and 1-DGE.
Ca. 100 mg of rice leaves from 7- (S1) and 14- (S2) days-old
seedlings were used for protein extraction. Leaves were homogenized in a prechilled glass mortar and pestle with 400 µL
of cold 0.2 M Tris-HCl buffer, pH 7.8, containing 5 mM EDTA‚
2Na, 14 mM 2-mercaptoethanol (2-ME), 10% (v/v) glycerol, and
2 EDTA-free proteinase inhibitor tablets (Roche Diagnostics
GmbH, Mannheim, Germany) per 100 mL of buffer solution
in Milli Q (MQ) water. Each sample was homogenized along
with ca. 50 grains of sterilized sea sand and 5 mg of polyvinylpolypyrrolidone (PVPP). The homogenates were centrifuged at 18 500g once for 10 min followed by centrifugation of
the resulting supernatant for another 10 min at 18 500g to
obtain total crude protein extract. Centrifugation steps were
carried out at 4 °C. The protein content was measured by a
standard assay procedure using bovine serum albumin (Sigma,
Fraction V) as the standard.21 Equal amounts (10 µg) of protein
were boiled for 3 min at 95 °C in SDS sample buffer [62 mM
Tris (pH 6.8) containing 10% (v/v) glycerol, 2.5% (w/v) SDS,
and 5% (v/v) 2-ME (2-mercaptoethanol), pH 6.8] and cooled
to room temperature (RT). Electrophoresis was carried on a
model AE-6531 ATTO PAGERUN equipment using precast
12.5% ePAGEL from ATTO (Tokyo, Japan). Five microliters of
the commercially available “ready-to-use” molecular mass
standards (Precision Plus Protein Standards, Dual Color, BioRad, Hercules, CA) was loaded in the well adjacent to the
samples. Electrophoresis was carried out at RT, at a constant
current of 20 mA for ca. 83 min or until the lowest marker (10
kDa) reached the bottom of the gel.
2.3. Total Protein Extraction for 2-DGE. For extraction of
total protein for 2-DGE, ca. 300 mg of leaves was homogenized
in 700 µL of lysis buffer [a 10 mL solution in MQ water contains
4.8 g of urea (ICN Biomedicals, Aurora, OH), 0.2 mL of NP-40
(Nacalai Tesque, Kyoto, Japan), 0.2 mL carrier ampholyte (pH
3.5-10, Amersham Biosciences, Uppsala, Sweden), 0.5 mL of
2-mercaptoethanol (WAKO, Osaka, Japan), 0.5 g of PVP-40
(Sigma, St. Louis, MO)] with sea sand using a prechilled glass
mortar and pestle. The homogenates were centrifuged at
18 500g for 10 min at 4 °C. The resulting supernatant was
recentrifuged (18 500g) for a further 10 min (4 °C); the final
supernatant was used as the total soluble protein extract.
Protein concentration was measured as described in Section
2.2. Equal amounts (300 µg) of protein were subjected to
2-DGE, following O’Farrell’s method.22
2.4. Two-Dimensional Gel Electrophoresis. 2-DGE [isoelectric focusing (IEF) in first-dimension, and SDS-PAGE in second
dimension] was performed utilizing the hand-cast Nihon Eido
2-DGE system exactly as described.2 The IEF gel (12 cm)
composition was as follows: 12 M urea, 5% (w/v) acrylamide,
3% (v/v) Nonidet P-40 (NP-40), 3.8% (v/v) ampholine (pH 3.510 and 5-8), 0.022% (v/v) ammonium persulfate (APS), and
0.15% (v/v) N,N,N′,N′-tetramethylethylenediamine (TEMED) in
MQ water. Ca. 350 µg of total protein extract was loaded onto
the basic end. A total of 10 2-D gels was run using total soluble
Journal of Proteome Research • Vol. 5, No. 10, 2006 2587
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protein extracts prepared from leaves of blm and WT. Three
gels were used for image analysis, one gel was used for
immunoblotting (Section 2.5), and six gels were used for
N-terminal amino acid sequencing. The basic and acidic buffers
were 1% TEMED and 0.02 N H3PO4 (phosphoric acid), respectively, and electrophoresis was performed at a constant voltage
of 200 V (30 min), 400 V (16 h), and 600 V (1 h) at RT. After
completion of the focusing step, the IEF tube gels were carefully
removed with a syringe (containing MQ water), transferred to
glass vials containing 5 mL of SDS-sample buffer followed by
incubation with gentle agitation at RT for 15 min (×2 times).
The IEF tube gels were placed onto acrylamide gels (15%
separation and 5% stacking gels) and overlaid with 1% (w/v)
agarose solution (in SDS-sample buffer minus 2-ME). Composition of the 15% separation [15% (v/v) acrylamide, (30% (w/
v) separation acrylamide, and 0.135% (w/v) bisacrylamide), 1×
(v/v) separation buffer (1 M Tris, pH 8.8 containing 0.27% (w/
v) SDS), 0.07% APS, and 0.1% TEMED], and 5% stacking [5%
(v/v) acrylamide (29.2% (w/v) stacking acrylamide and 0.8%
(w/v) bisacrylamide), 1× (v/v) stacking buffer (0.25 M Tris, pH
6.8 containing 0.2% (w/v) SDS), 0.05% APS, and 0.32% TEMED]
gels, was in MQ water. Ten microliters of the molecular mass
standards (Precision Plus Protein Standards) was loaded next
to the acidic end of the IEF tube gel. For phosphoprotein
visualization, PeppermintStick standards were used by treating
as above in SDS loading buffer. The PeppermintStick standards
carry two phosphorylated (ovalbumin and bovine β-casein of
45.0 and 23.6 kDa, respectively) and four nonphosphorylated
(β-galactosidase, bovine serum albumin, avidin, and lysozyme
of 116.25, 66.2, 18.0, and 14.4 kDa, respectively) proteins.
Electrophoresis was carried out at a constant current of 35 mA
for 2-1/2 h or until the dye (250 µL BPB; 0.1% (w/v) in 10%
(v/v) glycerol in MQ water) reached the bottom of the gel.
2.5. Immunoblotting. Electrotransfer of proteins on gel to
a polyvinyldifluoride (PVDF) membrane (NT-31, 0.45 µM pore
size; Nihon Eido) was carried out at 1mA/cm2 for 80 min at RT
using a semi-dry blotter (Nihon Eido) as described.23,24 The antinaringenin-7-O-methyltransferase (NOMT) polyclonal antibody
was produced in adult female rabbits by standard procedures
at the School of Agriculture, Ibaraki University (Japan). The
anti-ascorbate peroxidase (APX, spinach AP8, Mouse monoclonal) was a kind gift from Drs. Akihiro Kubo and Hikaru Saji,
National Institute for Environmental Studies (NIES), Tsukuba,
Japan. The anti-catalase (CAT, rabbit polyclonal) and antisuperoxide dismutase (SOD, rabbit polyclonal) antibodies were
commercially obtained (Abcam Ltd.. Cambridgeshire, U.K.).
The ECL+plus Western Blotting Detection System protocol for
blocking, primary and secondary antibody (anti-Mouse/Rabbit
IgG, Horseradish peroxidase linked whole antibody; from
donkey) incubation was followed exactly as described (Amersham Biosciences, Little Chalfont, Buckinghamshire, U.K.).
Immunoassayed proteins were visualized on an X-ray film (XOMAT AR, Kodak, Tokyo, Japan) using an enhanced chemiluminescence protocol according to the manufacturer’s directions (Amersham).
2.6. Protein Visualization. The 1- and 2-D gels were fixed
in 20% (w/v) trichloroacetic acid (TCA) for 30 min followed by
staining with Coomassie brilliant blue [CBB 0.1% (w/v) (Fluka
Chemie GmbH, Buchs, Switzerland); 50% (v/v) methanol
(MeOH), 10% (v/v) acetic acid] for 30 min. The detection limit
for CBB is in the range of 10-100 ng;25 the dye provides a linear
response with protein amount over a 10- to 30-fold range of
concentration. Destaining of the gels was carried out for 2 h (4
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Journal of Proteome Research • Vol. 5, No. 10, 2006
Jung et al.
× 30 min) with a gel-destaining solution (36.67% (v/v) MeOH,
and 10% (v/v) acetic acid). All solutions were prepared in
distilled water. All procedures were carried out at RT. The gels
were dried on Whatman No. 2 filter paper using a Model 583
Bio-Rad gel vacuum gel dryer operating at a temperature of 80
°C. The phosphoproteins on 2-D gels were detected with Pro-Q
Diamond phosphoprotein gel stain (Pro-Q DPS; Molecular
Probes, Inc., Eugene, OR).26 Total proteins were stained with
SYPRO Ruby (Molecular Probes).
2.7. Image and Data Analysis. Image/data analysis of the
scanned (300 dpi, 16-bit gray scale pixel depth, TIFF file) gels
was performed using ImageMaster 2D Platinum imaging
software ver. 5.0 (hereafter called ImageMaster software; GE
Healthcare Bio-Sciences AB, Tokyo, Japan). Protein abundance
was expressed as relative volume according to the normalization method provided by ImageMaster software that compensates for gel-to-gel variation in sample loading, gel staining,
and destaining.27,28 To compensate for subtle differences in
sample loading, gel staining, and destaining, the volume of each
spot (i.e., spot abundance) was normalized as relative volume.
The 2-D gel protein patterns between the blm and the WT were
compared, and the changed protein spots were marked. The
CBB-stained protein spots were selected for comparative
profiling only if they were confirmed in three independent
sample sets.
2.8. Edman Sequencing. Electrotransfer of proteins on gel
to a PVDF membrane was carried out as described in Section
2.5. A total of five to six individual PVDF membranes corresponding to the same number of gels were used for excision
of CBB-stained protein spots. The transfer efficiency is ca. 99%
for almost all low molecular mass proteins below ca. molecular
masses of 100 kDa; the transfer efficiency was also checked by
staining the gels after transfer with CBB, which revealed no
proteins spots left on gel except for a slightly stained standard
marker protein of 250 kDa. N-Terminal amino acid sequencing
of proteins on the PVDF membranes was carried out on an
Applied Biosystems 494 protein sequencer (Perkin-Elmer; Applied Biosystems, Foster City, CA).23,24 Proteins were identified
by using the Swiss-Prot and EST databases from NCBI (ftp://
ftp.ncbi.nih.gov/blast/db/).
2.9. Mass Spectrometry. The protein spots were excised from
the CBB-stained 2-D gels using a gel picker (One Touch Spot
Picker, P2D1.5 and 3.0, The Gel Company, San Francisco, CA),
and transferred to sterilized eppendorf tubes (1.5 mL). Gel
pieces were incubated in 0.2 M NH4HCO3 (pH 7.8) for 20 min,
shrunk by dehydration in acetonitrile, which was then removed,
followed by washing with vortexing in the same volume of
acetonitrile and 0.1 M NH4HCO3 (pH 7.8). The solution was
removed, and gel pieces were dehydrated with vortexing by
addition of acetonitrile and swelled by rehydration in 0.1 M
NH4HCO3 (pH 7.8). The dehydration process was repeated
twice, and the gel pieces were completely dried in a vacuum
centrifuge. Gel pieces were swollen in a digestion buffer
containing 10 µg/mL trypsin (Promega, sequencing grade) on
ice. The digestion buffer was removed after 45 min incubation
and replaced with 20-30 µL of 50 mM NH4HCO3 (pH 7.8). Gel
pieces were incubated at 37 °C for 8-12 h. The supernatant
was desalted through a C18 ZipTip (Millipore, Bedford, MA)
according to the manufacturer’s protocols, and a 2-8 µL
(minimum 1 pg by standard chemical; dilution of each sample,
1, 5, and 8 µL were analyzed) solution was injected for analysis
with nano-electrospray ionization-liquid chromatographytandem mass spectrometry (nESI-MS/MS; Agilent, Palo Alto,
Differential Expression of Defense/Stress-Related Marker Protein
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Figure 1. Phenotype of the blm mutant leaves. (A) Lesion symptoms in leaves of the blm mutant versus no such lesions in the wildtype (WT) leaves. S1 (stage before lesion formation; 7-days-old) and S2 (stage after lesion formation; 14-days-old) refer to the seedling
stages used for the sampling of leaves. (B and C) Cross-reacting secondary metabolite-(SM, naringenin-7-O-methyltransferase, NOMT)
and oxidative stress (OS, catalase, CAT; ascorbate peroxidase, APX; superoxide dismutase, SOD)-related marker proteins induced in
the blm mutant leaves. Leaves from WT and blm mutant were sampled at stages mentioned above each lane. Molecular weights (kDa)
of protein markers (Precision Plus Protein Standards, Bio-Rad) are indicated at the left. Approximate molecular weights of the
immunostained polypeptide bands are given in kDa on the right-hand-side. Immunoassaying was carried out as described in Section
2.5.
CA). The nLC was performed with an Agilent 1100 NanoLC1100 system combined with a microwell-plate sampler and
thermostated column compartment for preconcentration (LC
Packings, Agilent). Samples were loaded on the column (Zorbax
300SB-C18, 150 mm × 75 µm, 3.5 µm) using a preconcentration
step in a microprecolumn cartridge (Zorbax 300SB-C18, 5 mm
× 300 µm, 5 µm). Four micriliters of the sample was loaded on
the precolumn at a flow rate of 15 µL/min. After 5 min, the
precolumn was connected with the separating column, and the
gradient was started at 300 nL/min. The buffers used were 0.1%
HCOOH in water (A) and 0.1% HCOOH in acetonitrile (B). A
linear gradient from 2 to 70% B for 25 min was applied. A single
run took 75 min, which included the regeneration step. A LC/
MSD Trap XCT with a nano-electrospray interface (Agilent) was
used for MS. Ionization (2.0 kV ionization potential) was
performed with a liquid junction and a noncoated capillary
probe (New Objective, Cambridge, MA).
For tandem mass spectrometry, peptide ions were analyzed
by the data-dependent method to collect ion signals from the
peptides in a full mass scan range (m/z 300-2200). After
determination of the charge states of an ion on zoom scans,
an MS/MS spectrum was recorded to confirm the sequence of
the precursor ion using collision-induced dissociation (CID)
with a relative collision energy dependent on molecular weight.
The individual spectra from MS and MS/MS data were submitted to the Agilent Spectrum Mill MS proteomics workbench (Agilent) for protein identification. Proteins were
identified by database search against the Updating Data-
bases Swiss-Prot (ftp://www.expasy.ch/databases/sp_tr_nrdb/
fastasprot.fas.gz) and NCBI (ftp://ftp.ncbi.nlm.nih.gov/blast/
db/FASTA/nr.gz) using default parameters. Modifications of
methionine and cysteine, peptide mass tolerance at 2 Da, MS/
MS precursor mass tolerance of 2.5 Da, product mass tolerance
of 0.7 Da, allowance of missed cleavage at 2, and charge states
(+1, +2, and +3) were taken into account. For all protein
assignments, a minimum of two unique peptides was required.
Only significant hits as defined by the probability analysis were
considered initially.
2.10. Reverse Transcriptase-Polymerase Chain Reaction
(RT-PCR). Total RNA was isolated from ca. 100 mg of leaves
using QIAGEN RNeasy Plant Mini Kit (QIAGEN, MD). Total RNA
from three independent leaf samples was pooled for the next
step. Briefly, total RNA samples were DNase-treated with an
RNase-free DNase (Stratagene, La Jolla, CA) prior to RT-PCR.
First-strand cDNA was synthesized in a 50 µL reaction mixture
with a StartaScript RT-PCR Kit (Stratagene) according to the
protocol provided by the manufacturer, using 10 µg of total
RNA isolated from leaves of blm and WT. The 50 µL reaction
mixture (in 1× buffer recommended by the manufacturer of
the polymerase) contained 1.0 µL of the first-strand cDNA
from above, 200 mM dNTPs, 10 pmol of each primer set, and
0.5 U of taq polymerase (TaKaRa Ex Taq Hot Start Version,
TaKaRa Shuzo Co. Ltd., Shiga, Japan). Specific primers were
designed form the 3′-UTR regions (forward and reverse primer
sequences are provided in Figure 5) of each of the genes used
in this study by comparison and alignment with all available
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Jung et al.
related genes in the databases, NCBI, and KOME (http://
cdna01.dna.affrc.go.jp/cDNA/). Thermal-cycling parameters
were as follows: after an initial denaturation at 97 °C for 5 min,
samples were subjected to a cycling regime of 25 cycles at 95
°C for 45 s, 55 °C for 45 s, and 72 °C for 1 min. At the end of
the final cycle, an additional extension step was carried out
for 10 min at 72 °C (TaKaRa PCR Thermal Cycle Dice, Model
TP600, Tokyo, Japan). After completion of the PCR, the total
reaction mixture was mixed with 2.0 µL of 10× loading buffer
and vortexed, and 10 µL was loaded into wells of a 1.5% agarose
(Agarose ME, Iwai Chemicals, Tokyo, Japan) gel, and electrophoresis was performed for ca. 30 min at 100 V in 1× TAE
buffer, using a Mupid-ex electrophoresis system (ADVANCE,
Tokyo, Japan). The gels were stained (20 µL of 50 mg/mL
ethidium bromide in 100 mL of 1× TAE buffer) for ca. 10 min,
and the stained bands were visualized using an UV-transilluminator (ATTO, Tokyo, Japan). The intensity of each band
(area) was calculated using the ATTO lane and spot analyzer
ver 6.0 (ATTO, Japan).
3. Results and Discussion
3.1. Spontaneous Lesion Appearance on Leaves of Rice
Seedlings Is Correlated with Accumulation of Secondary
Metabolite- and Oxidative Stress-Related Proteins. A previous
physiobiochemical characterization of the blm mutant revealed
lesion resemblance with the blast fungal disease symptom.
Therefore, blm was referred to as an initiation-type LMM.20
Spontaneous lesion formation was found to be associated with
development under long-day condition and temperature shift
from 28 to 24 °C. The blm mutant also showed resistance
against fungal pathogens along with activation of a variety of
hallmark features of defense mechanisms that were tightly
linked with the stage of lesion progression in leaves. Moreover,
a preliminary 2-DGE analysis of protein changes associated
with the lesion leaf phenotype in blm led to the finding of two
PR10s, one PR5, and multiple cross-reacting SOD proteins by
immunoblotting.20 This result prompted us to conduct a
detailed investigation of the blm mutant with respect to stage
1 (S1, just before appearance of lesion) and stage 2 (S2, when
lesions are apparent) leaf phenotype (Figure 1A). The aim was
to identify protein components associated with these two
defined stages in blm lesion development using 1- and 2-DGE.
Knowing well induction of HR-associated secondary metabolites in S2 blm leaves over WT,20 we produced an antibody
against NOMT, the key biosynthetic enzyme in sakuranetin
production (Rakwal, R. et al., unpublished results). Results
revealed that the expression of NOMT is strongly induced in
leaves of the blm mutant over the WT, providing a first
phytoalexin-related protein antibody marker in rice (Figure 1B).
Another HR-associated symptom is the production of ROS29,30
causing oxidative burst upon pathogen infection or under stress
conditions. In a series of reaction, ROS is efficiently scavenged
by SOD, APX, glutathione reductase, and CAT, which exist in
different subcellular location.31,32 In our initial report, we had
speculated that blm has most probably acquired elevated levels
of antioxidant enzymes.20 To support our hypothesis, antibodies
against three enzymes involved in antioxidant systems such
as CAT, APX, and SOD were used to show that oxidative stress
signaling and protection are activated in blm (Figure 1C). All
these three proteins were induced to various degrees in leaves
of the blm mutant. As the APX and SOD are known to be part
of multigene families having many isoforms, a large number
of cross-reacting protein bands were expected on SDS-PAGE.
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Journal of Proteome Research • Vol. 5, No. 10, 2006
Figure 2. Induction of APX and SOD proteins in the blm mutant.
Equal protein amounts (300 µg) were analyzed by 2-DGE (details
are mentioned in Sections 2.3 and 2.4) followed by immunoassaying as described in Figure 1. Newly appearing cross-reacting
APX (A) and SOD (B) protein spots are circled (dotted) in the blm
mutant. Molecular weights (kDa) of protein markers (Precision
Plus Protein Standards) are indicated at the left, and leaf stages
are indicated on the right.
To further resolve these overlapping proteins, we carried out
2-DGE in conjunction with immunoblotting. Results revealed
clearly defined newly cross-reacting protein spots with different
isoelectric points on 2-D gels in blm S1 and S2 leaves; these
newly appearing spots are marked by dotted line circles (Figure
2B,C). The induction of a relatively large number of APX and
SOD proteins (or isoforms) in blm indicates that enzymes of
antioxidant systems exist at high levels in and around the
lesions, and are also present before the lesion appearance in
leaves. The induction of antioxidant enzymes/genes in Arabidopsis LMMs has also been reported.15 It is noteworthy that
our database search of available full-length rice cDNAs (KOME)
indicates the presence of at least 18 and 15 predicted APX and
SOD genes. This number corresponds well with the number
of cross-reacting spots in blm. Results show 3 and 10 new APX
and SOD cross-reacting proteins, respectively, which may be
newly synthesized in the leaves of the blm mutant during the
lesion formation. However, the question remains as to whether
these proteins are products of a single gene or of different
Differential Expression of Defense/Stress-Related Marker Protein
research articles
Figure 3. Representative CBB-stained 2-D gel protein profiles of leaves (stage 1 and 2) from the blm mutant and WT. 2-DGE (equal
protein amounts ca. 350 µg were applied) was carried out as described in Figure 2. Ca. 295/314 and 400/414 CBB-stained spots were
detected by ImageMaster software in the WT and blm S1/S2 gel profiles, respectively. Numbered arrows (spots 1-50) show proteins
selected for N-terminal amino acid sequencing (black arrows and letters) and mass spectrometry (blue arrows; these spots are color
coded: red, increased and yellow, decreased.), respectively. The positions of the proteins are marked in almost the same direction
and position for clarity, in each of the gels. The RuBisCO LSU (spot no. 1) and SSU (spot no. 2) also serve as reference proteins.
genes, and whether they (APX/SOD) constitute important
factors involved in imparting resistance to blm against oxidative
stress.
3.2. Proteomes of blm Leaves and Differential Expression
of Proteins. To investigate the temporal changes of protein
profiles during the S1 and S2 blm leaves over the WT, 2-DGE
analysis of total proteins from three biological replicates was
performed. The representative CBB-stained 2-D gels are shown
in Figure 3. Quantitative image analysis using the ImageMaster
software revealed a total of 50 protein spots changed their
intensities (equal to or greater than 1.5-fold over the WT spot)
significantly. These changed spots were verified by visually
checking the gel images under high-resolution. It must be
emphasized here that our 2-D gel profile (Figure 3) is “clean”
as also reported in previous studies.2,3,23,24,33 Moreover, the
hand-cast IEF and SDS-PAGE gel electrophoresis system is
highly reproducible in our hands.
Out of a total of 50 changed proteins spots in blm, 12 protein
spots showed increased expression, whereas 38 protein spots
showed reduced expression; these spots are color-coded red
(induced) and yellow (suppressed) in 2-D gel number 4 (blm
S2) of Figure 3. The marked protein spots were excised and
analyzed by either N-terminal amino acid sequencing (23 spots,
Table 1) or nESI-MS/MS (44 spots, Table 2). A total of 13 (out
of 23 spots) and 20 (out of 44 spots) proteins was unambigu-
ously identified; 26 proteins were nonredundant. An increased
induction or suppression of protein spot levels in the S2 over
S1 blm leaves indicated that the changes in proteins are
associated with the progression of the lesion stage. The
identified proteins are discussed below based on their functional categories:
3.2.1. Photosynthesis. Among the differentially expressed
proteins, most of the proteins were found to be related to the
photosynthesis category. Many proteins were related to the
major photosynthetic enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) large subunit (LSU) and its
fragments. RuBisCO, the major leaf protein, plays bifunctional
roles as a carboxylase for mediating photosynthetic CO2 assimilation and as an oxygenase for catalyzing the first step of
the photorespiratory pathway in plants. We identified 20
proteins (spots 1, 8, 13, 14, 17, 19, 23, 28-30, 35, 37, 40, 42, 43,
45, 48, 49) corresponding to the RuBisCO LSU and SSU (small
subunit, spot 2). All these proteins showed a decrease in the
leaves of the blm mutant. We also identified two other proteins
related to RuBisCO, namely, RuBisCO activase large isoform
precursor (spots 6, 34, and 44) and RuBisCO subunit binding
protein R subunit precursor (spot 26), all of which were reduced
in blm. We identified two proteins (spots 11 and 36) corresponding to the photosystem II (PSII) oxygen evolving complex,
namely, a putative 33 kDa oxygen evolving protein (OEP) of
Journal of Proteome Research • Vol. 5, No. 10, 2006 2591
research articles
Jung et al.
Table 1. Amino Acid Sequence and Homology of Proteins Affected in the blm Mutanta
spot
no.
MW
(kDa)
sequence
homologous protein
accession
number
homology
(%)
blm
S1
leaf
S2
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
49
14
53
52
52
40
37
37
35
34
33
28
27
27
24
23
20
39
15
18
17
19
10
N-blocked; I-TGEIKGX
N-FQVWPIEGIK
N-EVFFQEWFED
N-F/MRTNPTTSRP
N-MRTNPTTSRPGVSTIEEKST
N-blocked
N-XLXVAINGFG
N-QDETMFVDAN
N-GAYDDELVXT
N-blocked
N-EGVPRPLTFD
N-QDETMFVDAN
N-LSAKNYGRAX
N-LSAKNYFMAXYEP
N-APNYPVVSAE
N-VTTVALPDLP
N-TKETETKDTDVL
ND
N-SPILAGFRTS
N-APAXVSDEHAVAVSVERLXKV
N-APVSISDERA
N-ATFTITNRXS
N-RARGIFFTQD
RuBisCO LSU
RuBisCO SSU
Calreticulin precursor
ATP synthase beta chain
ATP synthase beta chain
?
Glyceraldehyde 3-phosphate dehydrogenase
Unknown
Fructose-bisphosphate aldolase
?
Oxygen evolving enhancer protein
Unknown
RuBisCO LSU
RuBisCO LSU
Ascorbate peroxidase
Superoxide dismutase
RuBisCO LSU
?
P6 protein
OsPR10b
OsPR10a
Thaumatin-like protein precursor (OsPR5)
RuBisCO LSU (fragment)
P30828
P05347
Q9SP22
P12085
P12085
?
CAA30152
?
Q40677
?
Q49079
?
O63190
AAG09793
P93404
P41978
P31195
?
P11128
Q9LKJ9
Q9LKJ8
P31110
P12089
90.00
100.00
90.00
100.00
100.00
?
?
?
?
?
90.00
?
100.00
75.00
90.00
100.00
75.00
?
70.00
90.00
70.00
90.00
100.00
o
+
o
o
o
o
o
+
+
o
+
+
+
+
-
+
+
+
+
+
+
+
+
+
-
a
N, N-terminal amino acid sequence; I, internal amino acid sequence; ND, not determined/detected; +/-, induction/suppression over the respective controls,
and bold symbol indicates a strong increase (+) or decrease (-); o, no change.
PSII and probable PSII OEP complex protein 2, respectively.
These two proteins showed slight increase and decrease,
respectively, in blm. These proteins are involved in oxygen
evolution and PSII stability34 and were previously found to be
suppressed by ozone and sulfur dioxide23,24 but induced by
salt.35 In general, photosynthesis-related proteins are suppressed, indicating a general down-regulation of photosynthesis
in blm.
We further identified two protein spots related to the
photorespiratory pathway that were down-regulated in blm.
Three proteins were glycine dehydrogenase P protein (spot 1)
and putative glycine dehydrogenase (spot 2). This family
consists of glycine cleavage system P-proteins EC:1.4.4.2 from
bacterial, mammalian, and plant sources. The P protein is part
of the glycine decarboxylase multienzyme complex EC: 2.1.2.10
(GDC) also annotated as glycine cleavage system or glycine
synthase. GDC consists of four proteins P, H, L, and T. These
proteins catalyze the interconversion of glycine-serine, an
integral part of photorespiratory pathway.36 It was reported that
environmental stresses remarkably inhibited GDC from isolated
pea mitochondria, suggesting that GDC inhibition may be an
initial marker of oxidative damage in photosynthetic cells.37
Reductions in the protein amounts in blm indicate damage to
the photorespiratory pathway during lesion formation, which
is morphologically apparent by leaf color and lesion presence.
3.2.2. Energy Metabolism. The second largest category was
related to energy and metabolism. Two protein spots (4 and
5) were identified as ATP synthase CF1 β chain. Surprisingly,
spot 4 was highly abundant in WT, compared to its potent
reduction in blm, whereas the opposite was true for spot 5.
ATP synthase catalyzes the formation of ATP from ADP, and Pi
in the presence of protein gradient across the membrane. As
this protein plays a central role in energy transduction in
bacteria, chloroplasts, and mitochondria, its suppression or
induction may be easy to explain under stress; however, a
suppression of one “isoform” (spot 4) over induction of the
other “isoform” (spot 5) in blm is a mystery. A genetic basis
2592
Journal of Proteome Research • Vol. 5, No. 10, 2006
for this “protein change” may be one reason for the blm mutant
carrying one form of ATP synthase over the other. This remains
to be investigated in future studies. Two protein spots, 6 and
32, were identical to phosphoribulokinase (PRK, EC 2.7.1.19)
precursors and were found to be reduced in blm. PRKase plays
an important role in regulating the flow of sugar through the
Calvin cycle. A RT-PCR analysis revealed that OsPrk gene was
expressed in all rice tissues tested and that the OsPrk transcript
level could be dramatically boosted by light illumination.38
Interestingly, it was also found in the above study that OsPrk
expression was down-regulated by externally applied methyl
JA (MeJA). We have found some LMMs showing constitutive
lesion formation to have high levels of JA, and its key biosynthetic enzyme, such as OsOPR1 (Rakwal, R. et al., unpublished
data). Does it mean that the lesion formation correlates with
increased JA/MeJA levels and is thus regulated by these
signaling molecules in leaves of the blm mutant? This remains
an interesting topic for further investigation. Since blm rice is
smaller than WT,20 both ATPase and OsPrk down-regulation
in leaf may help explain the blm mutant phenotype.
Three protein spots identified belonged to the glycolysis and
glyconeogenesis/Calvin cycle energy subcategory; spots 7 and
33 are similar to glyceraldehyde-3-phosphate dehydrogenase,
a tetrameric NAD-binding enzyme, whereas protein spot 9 was
identified as fructose-bisphosphate aldolase (FBP aldolase)
chloroplast precursor; these three proteins were reduced in
blm. Protein spot 31 was identified as glutamine synthetase
(GS) shoot isozyme, chloroplast precursor, which is a lightmodulated chloroplast enzyme, encoded by a nuclear gene, and
expressed primarily in leaves. GS is responsible for the reassimilation of the ammonia generated by photorespiration. The
GS activity has been shown to be decreased by exogenous
application of H2O2 in rice leaves.39 Reductions of their
enzymatic levels in blm point to a general reduction in energy
levels and/or efficiency therein during lesion formation.
3.2.3. Oxidative Stress. Two redox proteins were identified,
one induced (spot 15 was L-APX, EC 1.11.1.11) and the other,
research articles
Differential Expression of Defense/Stress-Related Marker Protein
Table 2. Protein Identification by MSa
% AA
coverage
distinct
peptides
distinct
summed
MS/MS
search score
NCBI
accession
pI/MW
(Da)
Glycine
dehydrogenase
P protein
12
10
143.55
42416979
6.03/96948
82
Putative glycine
dehydrogenase
10
9
129.87
34910498
6.51/111428
26
57
12
6
95.95
31193919
5.36/61400
27
53
Putative rubisco
subunit bindingprotein alpha
subunit precursor
ESTs
C99033
46
20
334.98
34897924
5.60/64085
ESTs
C99033(E4350),
C99032(E4350),
D46006(S10372)
53
24
415.7
34897924
5.60/64085
spot
no.
MW
(kDa)
24
88
25
27
protein
name
4
51
ATP synthase
CF1 beta chain
74
25
422.81
11466794
5.47/54014
5*
51
ATP synthase
CF1 beta chain
57
24
397.96
11466794
5.47/54014
1
46
20
9
124.31
11466795
6.22/52881
28
46
22
11
154.21
11466795
6.22/52881
29
46
24
12
153.75
11466795
6.22/52881
30
46
23
11
149.56
11466795
6.22/52881
31*
42
27
9
145.24
121343
5.96/46643
33
38
Ribulose1,5bisphosphate
carboxylase
Ribulose1,5bisphosphate
carboxylase
Ribulose1,5bisphosphate
carboxylase
Ribulose1,5bisphosphate
carboxylase
Glutamine
synthetase
shoot isozyme
EST
C74302(E30840)
corresponds to
a region of the
predicted gene
Aspartate
aminotransferase
34
13
198.62
6063542
6.22/47110
28
10
141.45
29468084
5.90/45874
RuBisCO
activase large
isoform
precursor
48
18
286.24
8918359
5.90/45874
Phosphoribulokinase
precursor
32
9
136.59
23198434
5.68/44865
Phosphoribulokinase
precursor
Ribulose1,5bisphosphate
carboxylase
27
11
169.08
23198434
5.68/44865
19
8
109.06
11466795
6.22/52881
33
6
38
6
32
38
8
37
peptide sequence
NAS/AAGFDLNVVVADAK/KLGTVTVQELPFFDTVK/
VVDATTITVAFDETTTLEDVDKLFK/NFT/NAT/GNINIE
ELRK/GVNGTVAHEFIIDLR/TTAGIEPEDVAK/EEIAEI
ESGKADVNNNVLK
AAGFDLNVIVADAK/LGTVTVQELPFFDTVK/VVDATTITV
AFDETTTLEDVDKLFK/GNINIEELRK/TTAGIEPEDVAK/E
EIAEIESGKADVNNNVLK/EYAAFPAA WLR
VVNDGVTIAR/TNDSAGDGTTTASVLAR/LGLLSVTSGAN
PVSIK/LNVAAIK/VGAATETELEDR/LGADIIQK
LADLVGVTLGPK/IVNDGVTVAREVELEDPVENIGAK/
TNDLAGDGTTTSVVLAQGLIAEGVKVVAAGANPVQIT
R/EVEDSELADVAAVSAGNNYEIGNMIAEAMSK/GVVT
LEEGRSSENNLYVVEGMQFERGYISPYFVTDSEK/DLI
NVLEEAIR/TQYLDDIAILTGATVIR/ESTTIVDGGSTQEE
VTKR/NLIEAAEQEYEKEK/LRVEDALNATK/VDAIKDNL
ENDEQK/FGYNAATGQYEDLMAAGIIDPTK/CC/TFLTSD
VVVVEIKEPEPAPVTNPMDNSGYGY
LADLVGVTLGPK/IVNDGVTVARENIGAK/TNDLAGDGT
TTSVVLAQGLIAEGVKVVAGANPVQITR/EVEDSELADV
AAVSAGNNYEIGNMIAEAMSK/GVVTLEEGRSSENNLY
VVEGMQFERGYISPYFVTDSEK/LLLVDKK/DLINVLEEA
IR/TQYLDDIAILTGATVIRDEVGLSLDK/ESTTIVGDGSTQ
EEVTKR/NLIEAAEQEYEKEK/LAGGVAVIQVGAQTETEL
KEK/LRVEDALNATK/VDAIKDNLENDEQK/FGYNAATGQ
YEDLMAAGIIDPTK/TFLTSDVVVVEIKEPEPAPVTNPMDN
SGYGY
TNPTTSRPGVSTIEEK/LPYIYNALVVK/NVTC/AVAMSAT
DGLMRGMEVIDTGAPLSVPVGGATLGRIFNVLGEPVDN
LGPVDTSATFPIHRSAPAFIELDTKLSIFFTGIK/LGLFGGA
GVGKTVLIMELINNIAKAHGGVSVFGGVGERTREGNDLY
MEMK/VALVYGQMNEPPGAR/VGLTALTMAEYFRDVNKQ
DVLLFIDNIFRFVQAGSEVSALLGRMPSAVGYQPTLSTEM
GSLQER/GSITSIQAVYVPADDLTDPAPATTFAHLDATTVLS
R/GIYPAVDPLDSTSTMLQPRIVGNEHYETAQR/YKELQDI
IAILGLDELSEEDRLYVAR/GFQLILSGELDGLPEQAFYLVG
NIDEASTKAINLEEENKLK
LPYIYNAKVVK/NVTC/AVAMSATDGLMRGMEVIDTGAPLS
VPVGGATLGRIFNVLGEPVDNLGPVDTSATFPIHRSAPAFI
EDLTKLSIFETGIK/GLFGGAGVGKTVLIMELINNIAKAHGG
VSVFGGVGERTREGNDLYMEMK/VALVYGQMNEPPGAR/
VGLTALTMAEYFRDVNKQDVLLFIDNIFRFVQAGSEVSALL
GRMPSAVGYQPTLSTEMGSLQER/IYPAVDPLDSTSTML
QPRIVGNEHYETAQR/YKELQDIIAILGLDELSEEDRLTVA
R/AINLEEENKLK
LTYYTPEYETKDTKILAAFR/TFQGPPHGIQVER/GGLDFT
KDDENVNSQPFMR/MSGGDHIHAGTVVGK/EMTLGFVDL
LRDDFIEK/AIKFEFEPVDKLDS
LTYYTPEYETKDTKILAAFR/TFQGPPHGIQVER/GGLDFT
KDDENVNSQPFMR/AMHAVIDR/MSGGDHIHAGTVVGK/
EMTLGFVDLLRDDFIEK/AIKFEFEPVDKLDS
LTYYTPEYETKDTKILAAFR/TFQGPPHGIQVER/GGLDFT
KDDENVNSQPFMR/DNGLLLHIHRAMHAVIDR/MSGGDH
IHAGTVVGK/EMTLGFVDLLRDDFIEK/AIKFEFEPVDKLDS
LTYYTPEYETKDTDILAAFR/TFQGPPHGIQVER/GGLDF
TKDDENVNSQPFMR/DNGLLLHIHRAMHAVIDR/MSGGD
HIHAGTVVGK/EMTLGFVDLLRDDFIEK/FEFEPVDKLDS
MEQLLNMDTTPFTDKIIAEYIWVGGTGIDLRTISKPVEDP
SELPKWNYDGSSTGQAPGEDSEVILYPQAIFKDPFRAAQ
VFSDPKSMREDGGFEVIKAILNLSLRHDLHISAYGEGNER
ENSPLEVVVV/GGVRNAS/YDSMLGTFK/IVDDQTISVDG
K/KVIITAPAK/NAS/ILDEEFGIVKGTMTTTHSYTGDQR/AA
ALNIVPTSTGAAKAVALVLPQLK/VPTPNVSVVDLVINTVK
TGITADDVNAAFRK/VVAWYDNEWGYSQRVVDLAHLVASK
FEGVPMAPPDPILGVSEAFK/TEELQPYVLNVVK/GENK
EYLPIEGLAAFNKATAELLFGADNPVLK/LAAAFIQR/IADV
IQEK/GLEVFVAQSYSKNLGLYAER/IVANVVGDPTMFGE
WK/NKT/NMT/NVS
GLAYDISDDQQDITR/GFVDSLFQAPTGDGTHEAVLSSY
EYSQGLRTYDFDNTMGGFYIQPAFMDKLVVHISKNFMTL
PNIK/MGINPIMMSAGELESGNAGEPAK/YREAADIIK/VPIIV
TGNDFSTLYAPLIR/FYWAPTRDDR/TDNVPDEDIVKIVDSF
PGQSIDFFGALR/KWVSKTGVENIGKR/EGPPEFEQPK/LM
EYGYMLVK/RVQLAEQYLSEAALGDANSDAMK
LTSVFGGAAEPPK/GVTALDPR/AIEKPIYNHVTGLLDPPE
LIQPPK/KPDFDAFIDPQK/C/C/FAYGPDTYFGHEVSVLEM
DGQFDRLDELIYVESHLSNLSTKFYGEVTQQMLK/NGT/IR
DLYEQIIAERAGAPTEAAKV
RLTSVFGGAAEPPK/GVTALDRRANDFDLMYEQVK/IFVIE
GLHPMFDER/KPDFDAFIDPQK/LDELIYVESHLSNLSTKF
YGEVTQQMLK/IRDLYEQIIAERAGAPTEAAKV
LTYYTPEYETKDTKILAAFR/TFQGPPHGIQVER/GGLDFT
KDDENVNSQPFMR/AMHAVIDR/MSGGDHIHAGTVVGK/
EMTLGFVDLLRDDFIEK
Journal of Proteome Research • Vol. 5, No. 10, 2006 2593
research articles
Jung et al.
Table 2. (Continued)
spot
no.
MW
(kDa)
7
36
7
9
33
10
32
10
11*
29
34
30
14
27
13
26
35
24
36
22
15*
22
37
20
39*
18
17
18
38
19
40
17
41
16
20*
15
20*
43
14
42
15
44
15
45
13
19
14
46
14
47
14
2594
% AA
coverage
distinct
peptides
distinct
summed
MS/MS
search score
NCBI
accession
pI/MW
(Da)
OSJNBa0036B21
.24
36
11
180.22
21740910
7.61/42716
Glyceraldehyde
3-phosphate
dehydrogenase,
cytosolic
Fructosebisphosphate
aldolase,
chloroplast
precursor (ALDP)
Putative nucleic
acid-binding
protein
Putative nucleic
acid-binding
protein
Putative 33 kDa
oxygen evolving
protein of
photosystem II
RuBisCO
activase large
isoform precursor
Ribulose1,5bisphosphate
carboxylase
Ribulose1,5bisphosphate
carboxylase
Ribulose
1,5-bisphosphate
carboxylase
Probable
photosystem II
oxygen-evolving
complex protein 2
L-ascorbate
peroxidase
Ribulose1,5bisphosphate
carboxylase
Hypothetical
protein
Ribulose1,5bisphosphate
carboxylase
Ribulose1,5bisphosphate
carboxylase
Ribulose1,5bisphosphate
carboxylase
Putative ribosomal
protein L12
Pathogenesisrelated protein
PR-10a
Root specific
pathogenesisrelated protein 10
Ribulose1,5bisphosphate
carboxylase
Ribulose1,5bisphosphate
carboxylase
RuBisCO
activase large
isoform precursor
Ribulose1,5bisphosphate
carboxylase
Ribulose1,5bisphosphate
carboxylase
Putative glycine
decarboxylase
subunit
Putative
thioredoxin
peroxidase
34
7
120.83
3023816
6.61/36492
26
10
145.94
3913018
7.59/42148
AGAYDDELVK/NATC/RLASIGLENTEANR/KIVDILTEQK/T
VVSIPNGPSELAVKEAAWGLARYAAISQKNGLVPIVEPEIL
LDGEHGIDR/TWGGQPENVK/ANSLAQLGK
22
5
70.91
42407939
4.36/33257
GFGFVTMSTIEEADKAYVGNLPWQVDDSRNAGFGFVS
MASKEELDDAISA LDGQELDGRPLRVNVAAERPQR
19
4
62.72
42407939
4.36/33257
GFGFVTMSTIEEADK/AYVGNLPWQVDDSR/NA/GFGFV
SMASKEELDDAIS ALDGQELDGRPLR
41
13
192.65
34914480
6.09/34861
24
11
163.58
8918359
5.43/51454
17
9
126.22
11466795
6.22/52881
RLTFDEIQSK/LTYTLDEIEGPLEVSSDGTIKFEEKDGIDYA
AVTVQLPGGERVPFLFTIK/GSSFLDPKGRGGSTGYDNA
VALPAGGRGDEEELAKENVKNASSSTGNITLSVTKSKPET
GEVIGVFESVQPSDTDLGAK
NFMTLPNIKMGINPIMMSAGELESGNAGEPAKYREAADI
IKFYWAPTRTDNVPDEDIVKKWVSDTGVENIGKPPEFEQ
PKLMEYGYMLVKRVQLAEQYLSEAALGDANSDAMK
DTDILAAFR/GGLDFTKDDENVNSQPFMR/MSGGDHIHA
GTVVGK/EMTLGFVDLLRDDFIEK/AIKFEFEPVDKLDS
16
8
126.76
11466795
6.22/52881
16
7
94.13
11466795
6.22/52881
40
6
97.27
34899580
8.66/26939
TNTEFIAYSGEGFK/EFPGQVLRYEDNFDANSNVSVIINP
TTK/TDSEGGFESKAVATANILESSAPVVGGK/TADGDEG
GKHQLITATVNDGK/KFVESAASFSVA
26
5
71.07
7489542
5.42/27155
11
5
70.35
11466795
6.22/52881
NYPVVSAEYQEAVEKTGGPFGTMKTPAELSHAANAGLDI
AVRMSGFEGP WTRALLSDPAFRPLVEK
LTYYTPEYETKDTDILAAFR/TFQGPPHGIQVER/EMTLGF
VDLLR/FEFEPVDKLDS
39
6
75.82
32352148
5.19/19725
9
5
72.47
11466795
6.22/52881
8
5
69.82
11466795
6.22/52881
LTYYTPEYETKDTDILAAFR/ALRLEDLR/TFQGPPHGIQV
ER
7
4
54.93
11466795
6.22/52881
LTYYTPEYETKDTDILAAFR/ALRLEDLRIPPTYSK
30
5
79.7
34912074
5.36/18590
40
6
100.65
9230755
4.96/16657
VLELGDAIAGLTLEEARGLVDHLQER/TEEDVVIEEVPSS
AR/ALTNLALK/DLIEGLPK
AFMDASALPLNPAAGVGSTYKLKVEYELEDGSSLSPEKD
IVDGYYGMLKM IEDYLVAHPAEYA
35
5
71.54
38678114
4.88/16900
LNPA/SEVLDVPAGSK/INVEYELEDGGSLSPEKEK/MIED
YLVAHPTEYA
5
3
36.72
11466795
6.22/52881
NAT/EMTLGFVDLLR/AUKFEFEPVDKLDS
4
2
30.91
11466795
6.22/52881
LTYYTPEYETKDTDILAAFR/NA
20
7
106.29
8918359
5.43/51454
4
2
28.74
11466795
6.22/52881
GLAYDISDDQQDITR/TYDFDNTMGGFYIAPAFMDK/NFM
TLPNIK/NAT/NPT/VPIIVTGNDFSTLYAPLIR/LMEYGYMLV
K/RVQLAEQYLSEAALGDANSDAMK
LTYYTPEYETKDTDILAAFR/NA
4
2
29.02
11466795
6.22/52881
LTYYTPEYETKDTDILAAFR
28
3
46.22
37536058
4.92/17367
YSSSHEWVK/LSETPGLINSSPYEDGWMIKVKPSSPSEL
DALLDPAK
44
6
90.87
46389828
6.15/23179
LPDATLSYFDPADGELKTVTAELTAGR/ESLGLGDADVLL
LSDGNLELTRALGVEMDLSDKPMGLGVR/YALLADDGV
VKVLNLEEGGAFTTSSAEEMLK
protein
name
Journal of Proteome Research • Vol. 5, No. 10, 2006
peptide sequence
NATGDSSPLDVIAINDTGGVKYDSTLGIFDAFVKPVGDNA
ISDGKKVLITAPGKTMTTTHSYTGDQRAAALNIVPTSTGA
AKAVALVLPNLVPTPNVSVVDLVVQVSKKTLAEEVNQAFR
VIAWYDNEWGYSQRVVDLADIVANQWK
VALQSEDVELVAVNDPFITTDYMTYMFK/TLLLGEKPVTVF
GIRNPDEIPWAEAGAEYVVESTGVFTDKEK/VPTVDVSVV
DLTVR/GIIGYVEEDLVSTDFVGDSR/AGIALNDNFVK
GGLDFTKDDENVNSQPFMR/DNGLLLHIHRAMHAVIDR/
MSGGDHIHAGTVVGK/EMTLGFVDLLRDDFIEK/FEFEPV
DKLDS
DTDILAAFGGLDFTKDDENVNSQPFMRDNGLLLHIHRMS
GGDHIHAGTVVGKMTLGFVDLLRAIKFEFEPVDKLDS
AIDWEGMAKFSQEPQPIDWEYYREAYESIEIPKYVDTVT
PQYKPKFDALLVEL KTMTADEYFAK
LTYYTPEYETKDTDILAAFR/LEDLRIPPTYSKTFQGPPHG
IQVER/NAT/NTS/C
research articles
Differential Expression of Defense/Stress-Related Marker Protein
Table 2. (Continued)
spot
no.
MW
(kDa)
protein
name
% AA
coverage
distinct
peptides
distinct
summed
MS/MS
search score
NCBI
accession
pI/MW
(Da)
48
14
12
6
81.46
11466795
6.22/52881
LTYYTPEYETKDTDILAAFR/MSGGDHIHAGTVVGK/EMT
LGFVDLLR/AIKFEFEPVDKLDS
49
14
7
4
56.46
11466795
6.22/52881
DTDILAAFR/MSGGDHIHAGTVVGK/AIKFEFEPVDKLDS
2
13
22
5
65.24
132081
8.25/19468
KFETLSYLPPLTVEDLLK/VGFVYR/YWTMWK/IIGFDNVR
23
12
2
2
31.78
11466795
6.22/52881
AIKFEFEPVDKLDS
50*
19
Ribulose1,5bisphosphate
carboxylase
Ribulose1,5bisphosphate
carboxylase
Ribulose1,5bisphosphate
carboxylase
small chain
Ribulose1,5bisphosphate
carboxylase
Probenazoleinduced protein
10
1
193.74
7442204
4.88/16688
LKVEYELEDGSSLSPEK
a
peptide sequence
Asterisks indicate the induced protein spot numbers in Figure 3, and proteins marked in bold indicate N-terminal amino acid sequence.
Figure 4. Putative phosphoproteins detected on 2-D gels by Pro-Q DPS staining in blm. The phosphoproteins on 2-D gels were detected
by Pro-Q DPS (upper panels) followed by staining with SYPRO ruby gel stain for detection of total protein (lower panels). The
phosphoprotein spots are circled. The bold arrow indicates spot number 22 (see Figure 3, blm). The RuBisCO LSU and SSU also
stained with Pro-Q DPS, and are numbered by matching to corresponding spots in Figure 3 blm S2 gel image for comparison.
a putative thioredoxin peroxidase (spot 47), which was suppressed in blm. APX, which converts H2O2 to water and O2, is
a key enzyme of H2O2-detoxification system in chloroplasts and
cytosol. APX also constitutes an important component of the
ascorbate-glutathione cycle. The ROS, highly toxic byproducts
of O2 metabolism, are carefully regulated metabolites capable
of signaling and communicating key information to the genetic
machinery of the cell.40 ROS production is also provoked by a
variety of natural and stress stimuli that can seriously disrupt
normal metabolism through oxidative damage to cellular
components.40 Interestingly, we observe the induction of APX
isoforms by immunoblotting (Figure 2A), indicating its importance in lesion-associated oxidative stress responses. It should
be noted that, though we could observe specific changes in
CAT and SOD proteins by immunoblotting, we did not find
any corresponding protein spot on 2-D gels. This may be due
Journal of Proteome Research • Vol. 5, No. 10, 2006 2595
research articles
Jung et al.
Figure 5. RT-PCR confirmation of a previously reported root-specific PR10 protein in the leaves of the blm mutant. (A) Alignment of
the amino acid sequences of proteins corresponding to hits obtained upon blasting the MS data of spots 50 and 20. (B) Specific primer
design for these two different genes (AK071613, spot 50; AK061606, spot 20) followed by RT-PCR reveals not only their expression in
leaves but also a significant induction in their mRNA expression levels in blm over WT.
to very low amounts of CAT and SOD proteins in the total
protein extracts. The putative thioredoxin peroxidase belongs
to the alkyl hydroperoxide reductase (AhpC)/thiol-specific
antioxidant (TSA) family, is found uniquely in the leaf, and is
important to the process of photosynthesis. The AhpC and TSA
define a family of more than 25 different proteins where at least
eight of the genes encoding AhpC/TSA-like polypeptides are
found in proximity to genes encoding other oxidoreductase
activities; the expression of several homologues has been
correlated with pathogenicity.41 It remains to be seen whether
the putative thioredoxin is a part of the defense/stress response
mechanisms altered in the blm mutant.
3.2.4. Others. Spot 10, composed of three proteins arranged
in parallel, showed a slight decrease in blm over WT. This
protein was related to an RNA-binding protein, possessing
putative NAD-dependent epimerase/dehydratase family and
3-β hydroxysteroid dehydrogenase/isomerase family signatures.
Their role in plant remains unknown. Spot 33 also showed
homology with an aspartate aminotransferase involved in
amino acid transport and metabolism, whereas spot 41 was
identified as a putative ribosomal protein L12; these proteins
also showed decreased levels in blm. The OsPR5 and OsPR10
protein families belonging to the category cell rescue/defense
and virulence and identified prominently in leaves of the blm
mutant are discussed separately below.
3.3. Putative Phosphoprotein Detection. Proteins, once
synthesized on the ribosomes, are subject to a multitude of
modification steps, referred to as post-translational modifications (PTMs), such as amino- or carboxy-terminal cleavages,
glycosylation, phosphorylation, and sulfation. Consequently,
there are many more proteins in the proteome than there are
genes in the genome. PTMs control a variety of processes in
any organism, including plants, and occur in networks that
unite different cellular processes. Recently, a phosphostaining
dye called Pro-Q DPS that selectively detects phosphoserine-,
phosphothreonine-, and phosphotyrosine-containing proteins
directly in 1- and 2-D gels, and on membranes, such as PVDF
2596
Journal of Proteome Research • Vol. 5, No. 10, 2006
and nitrocellulose, has been shown to detect phosphoproteins
at levels as low as 1 ng on a single electrophoresis gel.26,28 When
1-DGE was used, Pro-Q DPS stained numerous protein bands
in blm over WT (data not shown). This positive result led to
stain 2-D gels with Pro-Q DPS to detect potential phosphoproteins. Although a laser-based scanning protocol is most
suitable for visualizing phosphoproteins (Molecular Probes),
it is also possible to detect the stained phosphoproteins by
short-wave UV light, albeit at reduced detection levels. By this
improvised method, we could detect only a small percentage
of protein spots (Figure 4). Nevertheless, by comparing and
matching with the subsequently SYPRO Ruby-stained 2-D gels
(and with CBB-stained 2-D gel profiles in Figure 3), we could
identify the RuBisCO LSU or fragments (spots 1, 13, 14, 17, 23,
37, and 48) and SSU (spot 2), which are marked by arrows, and
some other proteins spots, including RuBisCO activase large
isoform (spot 6), GAPDH (spot 7), FBP aldolase (spot 9), GS
shoot isozyme (spot 31), probable PSII OEC protein 2 (spot 36),
and a putative thioredoxin (spot 47) as putative phosphoproteins (Figure 4).
A potentially novel finding was the staining of protein spot
22 by Pro-Q DPS, which was identified by both N-terminal
amino acid sequencing and MS as an OsPR5 (Tables 1 and 2).
Thus, we can call OsPR5 as a putative phosphoprotein (Figure
4; marked in bold). The PR5 family accumulates in plant in
response to stress conditions, for example, high salt concentrations, wounding or pathogen attack; in vitro bioassays have
shown their antifungal activity.42,43 Constitutively, they are
thought to play a significant role in protecting seeds against
fungal attack during storage or germination.42,44 The presence
of a low molecular weight (19 kDa) OsPR5 protein was first
reported in leaves of mature rice (cv. Hitomebore) plants
treated with jasmonic acid (JA), heavy metal copper, and shortwave UV-irradiation (UV-C), using 2-DGE.45,46 Detection of
OsOPR5 in leaves of the blm mutant strongly emphasizes its
importance in the defense/stress response pathways in rice.
At present, the functional significance of a phosphorylated
research articles
Differential Expression of Defense/Stress-Related Marker Protein
OsPR5 remains to be elucidated. Moreover, we need to identity
and map the phosphorylation sites of the OsPR5 protein in
order to finally confirm its phosphoprotein identity.
3.4. Three OsPR10 Proteins Are Induced in Leaves of the
blm Mutant, Including a New Highly Acidic PR10-like Protein.
Three protein spots were once again identified as members of
the OsPR10 family, spot 20, 21, and 18/50 (Figure 3 and Tables
1 and 2) confirming our preliminary results.20 The PR10s are a
ubiquitous class of intracellular (in contrast to the extracellular
nature of most PR proteins) defense-related proteins first
described in cultured parsley cells upon elicitor treatment.47
PR10s have been attributed a “ribonuclease-like function” due
to sequence homology with ginseng ribonucleases.48,49 Interestingly, PR10s also share amino acid sequence similarity to pollen
allergens of trees50 and the major food allergen of celery,51
suggesting their diverse functions. The first rice PR10 gene was
identified by treatment with probenazole (PBZ), and hence
termed PBZ1.52 Later, using 2-DGE and N-terminal amino acid
sequencing, we identified three OsPR10 proteins, OsPR10a-c,
ranging in size from 18 to 20 kDa, from stressed rice
plants.33,45,53,54 Here, we also identify for the first time, a
potentially new OsPR10 protein (named OsPR10d), of ca. pI
3.9 and MW 19 kDa from blm mutant leaves (spot 18/50, Figure
3 and Table 2). Like the OsPR5 proteins, OsPR10s may consitute
an important line of defense in combating pathogenesis or
stress in rice.
Interestingly, protein spot 20 showed high homology to both
the originally identified OsPR10a (PBZ152) and a recently
reported root-specific PR10 gene from rice (RSOsPR1055), which
was found to be homologous to OsPR10a,b.45,53 The RSOsPR10
mRNA induction was shown almost exclusively in roots and
was shown to accumulate rapidly upon drought, salt (sodium
chloride), JA, PBZ, and by infection with the rice blast fungus.55
However, these authors55 failed to provide any concrete data
showing its absence in above-ground photosynthetic tissues.
Our present MS analysis results strongly suggested the presence
of RSOsPR10 protein in leaves of the blm mutant. Therefore,
we designed 3′-UTR gene-specific primers for both (OsPR10a
and RSOsPR10) of the full-length cDNA clones in the KOME
database and examined their mRNA abundance in the leaves
of blm and WT (Figure 5). Results revealed differential expression of both the OsPR10a and RSOsPR10 genes in the leaves of
blm and WT, clearly demonstrating the presence of RSOsPR10
in leaves, and contrary to as previously reported. This result
also suggests that the protein spot 20 may constitute a mixture
of proteins, in this case two closely related OsPR10s.
4. Conclusions
Comparative proteomics is a powerful tool for identifying
changes in the expression of proteins patterns in response to
changes in the environmental conditions. We report the
presence of 26 nonredundant differentially expressed proteins
in leaves of the blm mutant using 2-DGE coupled with
N-terminal amino acid sequencing and nESI-LC-MS/MS for
protein identification. These proteins could be grouped into
four main functional categories, broadly correlating with the
changes in protein profiles during lesion formation; especially,
decreased photosynthesis and energy metabolism were found
to be linked with increase in oxidative stress and growth
retardation in blm. Four proteins related to the rice plant
defense/stress responses were also identified; these proteins
may serve as potential markers for LMMs. 1- and 2-DGE
immunoblotting also revealed the presence of newly appearing
cross-reacting protein markers in blm. Because of the need for
high-resolution 2-D gel proteome maps, use of linear 24 cm
IPG strips (pH 4-7) and large-size pre-cast polyacrylamide gels
(ExcelGel XL 12-14% gradient) for a detailed time-course
expression profiling of lesion development in blm mutant
leaves is planned. It is a fact that gel-based proteomics has its
limitations in the degree of comprehensive coverage of the
proteome. Therefore, a complementary MudPIT proteomic
approach for deep proteome measurements in the blm mutant
is being actively considered. With these ongoing studies, our
aim will be to develop lesion-related protein biomarkers in rice.
Acknowledgment. This research is funded by a grant
from the Plant Signaling Network Research Center, Korea,
Science and Engineering Foundation, Korea, and the Crop
Functional Genomics Center of the 21st Century Frontier
Research Program (Grant No. CG2260). The nESI-LC-MS/MS
was carried out at the Carbohydrate Bioproduct Research
Center in Sejong University, Korea. This work was also supported by institutional funds from the HSS, AIST-Tsukuba
WEST, Japan. We appreciate Drs. Akihiro Kubo and Hikaru Saji
(NIES, Tsukuba, Japan) for the kind gift of the anti-APX
monoclonal antibody.
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