Survey of Differentially Expressed Proteins and Genes in Jasmonic

Survey of Differentially Expressed Proteins and Genes in Jasmonic Acid Treated Rice Seedling Shoot and Root at the Proteomics and Transcriptomics Levels Kyoungwon Cho,†,¶ Ganesh Kumar Agrawal,‡,# Junko Shibato,§ Young-Ho Jung,| Yeon-Ki Kim,⊥ Baek Hie Nahm,⊥ Nam-Soo Jwa,| Shigeru Tamogami,O Oksoo Han,† Kimiyoshi Kohda,∇ Hitoshi Iwahashi,§ and Randeep Rakwal*,‡,§ Department of Applied Biotechnology, Agricultural Plant Stress Research Center and Biotechnology Research Institute, Chonnam National University, Gwangju 500-757, Korea, Research Laboratory for Agricultural Biotechnology and Biochemistry (RLABB), Kathmandu, Nepal, Human Stress Signal Research Center (HSS), National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba West, 16-1 Onogawa, Tsukuba 305-8569, Japan, Department of Molecular Biology, College of Natural Science, Sejong University, Seoul 143-747, Korea, Division of Bioscience and Bioinformatics, Myongji University, GreenGene BioTech, Inc., Kyonggido 449-728, Korea, Laboratory of Growth Regulation Chemistry, Department of Biological Production, Akita Prefectural University, Akita 010-0195, Japan, and Hitachi High-Technologies Corporation, Hitachi-naka 312-0057, Japan Received June 9, 2007

Two global approaches were applied to develop an inventory of differentially expressed proteins and genes in rice (cv. Nipponbare) seedling grown on Murashige and Skoog medium with and without jasmonic acid (JA). JA significantly reduced the growth of shoot, root, leaf, and leaf sheath depending on JA concentration (1, 2, 5, 10, 25, and 50 µM) as compared with control. Almost 50% growth inhibition of seedling was observed with 5 µM JA. Shoots and roots of seedlings grown on 5 µM JA for 7 days were then used for proteomics and transcriptomics analyses. Two-dimensional gel electrophoresis revealed 66 and 68 differentially expressed protein spots in shoot and root, respectively, compared to their respective controls. Tandem mass spectrometry analysis of these proteins identified 52 (shoot) and 56 (root) nonredundant proteins, belonging to 10 functional categories. Proteins involved in photosynthesis (44%), cellular respiratory (11%), and protein modification and chaperone (11%) were highly represented in shoot, whereas antioxidant system (18%), cellular respiratory (17%), and defenserelated proteins (15%) were highly represented in root. Transcriptomics analysis of shoot and root identified 107 and 325 induced genes and 34 and 213 suppressed genes in shoot and root, respectively. Except of unknown genes with over 57% of the total, most genes encode for proteins involved in secondary metabolism, energy production, protein modification and chaperone, transporters, and cytochrome P450. These identified proteins and genes have been discussed with respect to the JAinduced phenotype providing a new insight into the role of JA in rice seedling growth and development. Keywords: phytohormone • inhibitory concentration • growth • gel-based approach • mass spectrometry • DNA microarray

1. Introduction The octadecanoid pathway in plant leads to the biosynthesis of oxylipins, which are known to be involved in regulating various aspects of physiological processes involved in plant * To whom correspondence should be addressed. Dr. Randeep Rakwal, HSS, AIST, Tsukuba West, 16-1 Onogawa, Tsukuba 305-8569, Japan. E-mail, [email protected]; fax, +81-29-861-8508. † Chonnam National University. ¶ Present address: Environmental Biology Division, National Institute for Environmental Studies, Tsukuba, Ibaraki 305-8506, Japan. ‡ Research Laboratory for Agricultural Biotechnology and Biochemistry (RLABB). # Present address: University of Missouri-Columbia, Biochemistry Department, 204 Life Sciences Center, Columbia, MO 65211. 10.1021/pr070358v CCC: $37.00

 2007 American Chemical Society

growth and development and abiotic and biotic stresses.1-8 Intriguingly, this pathway is strikingly similar to the mammalian immune system, comprised of the cyclopentanoic fatty acids such as PGEs and leukortriene A4.4,9 Among the oxylipins, the cyclopentanones jasmonic acid (JA) and methyl jasmonate (MeJA) are crucial components that are collectively referred to as jasmonates in plants. The biosynthesis of JA is initiated by 13(S)-hydroperoxydation of linolenic acid by 13-lipoxygenase, and then terminated by the sequential reaction of allene oxide §

National Institute of Advanced Industrial Science and Technology (AIST). Sejong University. ⊥ Myongji University. O Akita Prefectural University. ∇ Hitachi High-Technologies Corporation. |

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research articles synthase and cyclase, 12-oxo-phytodienoic acid reductase, and β-oxidation three times.6,10 It has been shown that JA, the main compound, functions as physiological regulator in a wide range of biological processes, including development (tendril coiling, tuberization, germination, root growth, fertility, fruit ripening, and senescence) and defense (against pathogens and pests, wounding, abiotic stresses, and secondary metabolism).1,2,6,7 We have previously reviewed the progress of octadecanoid pathway in rice.10 Recent advances in high-throughput technologies, such as proteomics11-16 and transcriptomics17,18 and the availability of genome sequence of two flowering model plants Arabidopsis (Arabidopsis thaliana, dicot) and rice (Oryza sativa, monocot), have helped in a better understanding of the role of JA and its regulatory networks in plants. These studies have mainly been conducted in dicot plants, but poorly in monocots. In dicots, though proteomics-scale study is still lacking, the microarray technology has been used for examining the role of JA in Arabidopsis, tomato, and tobacco.19-22 In monocots, though the effect of JA is poorly understood, there are a few targeted studies conducted in rice that reported the separation and identification of proteins from JA-treated rice seedling leaf or stem.23,24 In these studies, the induction of pathogenesis-related (PR) proteins and cellular protectant proteins by 100 or 500 µM JA was shown along with suppression in the amounts of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) in the leaves and stems. Similar information on the induction or suppression of some defense/stress-related genes was obtained by the exogenous application of JA in rice.25 Here, we have applied both proteomics and transcriptomics approaches to systematically identify proteins and genes expressed in rice (cultivar Nipponbare) seedling shoot and root, and profile how their expression is influenced by JA treatment. For this purpose, we have established a rice seedling culture system in which dehusked rice seeds are grown in Murashige and Skoog medium with (treated) and without (control) JA (Figure 1). This systematic proteomics and transcriptomics study has led to the identification of differentially expressed proteins (52 in shoot and 56 in root) and genes (141 in shoot and 538 in root) by JA. These identified proteins and genes are involved in numerous cellular functions, suggesting a multifaceted role of JA in rice. A correlation between these differentially expressed proteins and genes and JA-triggered changes in seedling morphology have been seen and discussed.

2. Materials and Methods 2.1. Plant Material. Rice seeds (cv. Nipponbare; O. sativa L. japonica-type), used in this study were collected from the field and stored at 4 °C. Sterilized (4-fold diluted sodium hypochlorite) dehusked seeds were grown in sterile glass culture tubes having 10 mL of Murashige and Skoog (without sucrose, IAA, kinetin, and agar; Cat No. 2610022; ICN Biomedicals, Inc., Aurora, OH) medium (4.4 g/L in MQ water plus 1% sucrose and 0.25% gellan gum; pH 5.8, and autoclaved) with and without different concentrations of JA (1, 2, 5, 10, 25, and 50 µM) dissolved in methanol.26,27 In the control, the corresponding volume of methanol was added to the medium. Each culture tube carried one seed at the center of the medium and was placed in a growth chamber under white fluorescent light (wavelength 390-500 nm, 150 µmol m-2 s-1, 12 h photoperiod) at 25 °C and 70% relative humidity (RH) for a total period of 15 days. Shoots and roots were collected from 10 independent seedlings at 7 days after seed culture and pooled together, 3582

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Figure 1. Exploring the effect of jasmonic acid (JA) on the expression of proteins and genes in rice seedling’s shoots and roots. In this experiment, rice seeds of cultivar Nipponbare (type japonica) were dehusked, sterilized, and cultured in Murashige and Skoog medium containing different concentrations of JA. Controls were without JA. Changes in morphological parameters such as growth in shoot and root were recorded at 15-day JA post-treatment. Changes in the expression of proteins and genes were investigated by proteomics (2-DGE coupled with LC-MS/ MS) and transcriptomics (DNA microarray chip) using 7-day-old JA-treated shoot and root samples. Differentially expressed proteins and genes were used for a parallel comparative analysis.

called one biological sample. A total of three biological samples were collected for experiments. Samples were immediately frozen in liquid nitrogen and kept at -80 °C. 2.2. Morphological Characterization. Morphological characterization of seedling with or without JA was performed on 15-day-old seedling. Among the morphological parameters studied were total seedling length, length of shoot and roots, and number of leaves. 2.3. Two-Dimensional Gel Electrophoresis. Total protein was extracted using two-step trichloroacetic acid/acetone protein extraction protocol.28 Shoot or root samples (from 7-day-old seedling) were placed in liquid nitrogen and ground thoroughly to a fine powder with a mortar and pestle (precooled). Proteins were precipitated with TCAAEB [acetone containing 10% (w/v) TCA, and 0.07% 2-mercaptoethanol (2ME)] for 1 h at -20 °C, and centrifuged at 15 000 rpm for 15 min at 4 °C. The pellet was washed twice with wash buffer (acetone containing 0.07% 2-ME, 2 mM EDTA, and 2 EDTAfree proteinase inhibitor cocktail tablets (Roche) in a final volume of 100 mL buffer) followed by removal of all the acetone by air-drying the pellet at ambient room temperature (RT). Solubilization of the protein pellet was accomplished in lysis buffer (LB)-TT [7 M urea, 2 M thiourea, 4% (w/v) 3-[(3cholamidopropyl) dimethylammonio]-1-propanesulfonate, 18

Differentially Expressed Proteins/Genes in JA-Treated Rice

mM Tris-HCl (pH 8.0), 14 mM trizma base, two EDTA-free proteinase inhibitor cocktail tablets, 0.2% (v/v) Triton X-100 (R), and 50 mM dithiothreitol (DTT) to a final volume of 100 mL]. Protein quantification was performed using a Coomassie Plus (PIERCE, Rockford, IL) protein assay kit. For 2-DGE, isoelectric focusing (IEF, pH 3-10) in the first dimension was followed by 15% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension as described previously.14,28 A total of three 2-D gels were run for each shoot and root tissue using total protein (350 µg) derived from three independent biological samples. The 2-D gels were stained with colloidal Coomassie brilliant blue G-250 (24 h) to visualize protein spots. Protein patterns in the gels were recorded as digitalized images using a digital scanner (CanoScan 8000F, resolution 300 dpi), and saved as TIFF files. Downstream image/data analysis of the scanned (300 dpi, 16bit grayscale pixel depth, TIFF file) gels was performed using ImageMaster 2D Platinum imaging software (hereafter called ImageMaster software) ver. 5.0 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) as described previously.29 Differentially expressed protein spots between control and JA-treated samples were visually confirmed, marked, and excised (Gel picker, One Touch Spot Picker, P2D1.5, The Gel Company, San Francisco, CA) for trypsin digestion and protein analysis. 2.4. In-Gel Digestion. The excised protein spots were in-gel digested with sequencing grade modified trypsin (Promega, Madison, WI) as described previously.30 In brief, each protein spot was placed in a polypropylene (Eppendorf) tube and washed 4-5 times (until the gel was clear) with 150 µL of pure water. The gel slices were dried in a Speedvac concentrator, then 50 µL of dithiothreitol (10 mM in 100 mM ammonium bicarbonate, AMBIC) was added and incubated for 45 min at 55 °C, and then 50 µL of iodoacetamide (55 mM in 100 mM AMBIC) was added and incubated for 45 min at RT in the dark. The solution was removed and washed twice with 100 mM AMBIC, and then 50 µL of acetonitrile was added. The solution was removed and was dried in a Speedvac concentrator, and then 10 µL of 50 mM AMBIC, 50 mM CaCl2, and 10% (v/v) acetonitrile containing 50 ng of trypsin was added. After incubation at 37 °C for 16 h, the liquid was transferred to a new tube. Tryptic peptides remaining in the gel matrix were extracted for 40 min at 30 °C with 20 µL of 50% (v/v) aqueous acetonitrile containing 0.1% (v/v) formic acid. The combined supernatants were evaporated in a Speedvac concentrator and dissolved in 8 µL of 5% (v/v) aqueous acetonitrile solution containing 0.1% (v/v) formic acid for MS analysis. 2.5. Identification of 2-D Gel Protein Spots by LC-MS/MS. Trypsin-digested protein spots were subjected to analysis on either nLC Linear-ion trap-time-of-flight mass spectrometer (nLC-IT-TOF-MS/MS, Hitachi High-Technologies Corporation, Hitachi, Japan) or LC/MSD Trap XCT (nESI-LC-MS/MS, Agilent, Palo Alto, CA). 2.5.1. nLC-IT-TOF-MS/MS. The HPLC/ESI-MS instrument used was a Nano Frontier system (Hitachi HighTechnologies) consisting of a capillary HPLC system based on the AT10PV nanoGR generator and an ESI-TOF mass spectrometer. Analytical column was MonoCap for nanoflow (0.05 mm i.d., 150 mm length, GL-Science, Tokyo, Japan). Trap column was monolith (0.2 mm i.d., 5 mm length, Kyoto Monotech, Kyoto, Japan), which was cut off to arbitrary length and was custom-made. A silica tip (tip diameter of 10 µM; New Objective, Woburn, MA) was used as an electrosprayer. The HPLC conditions were the following. (i) The flow rate of the

research articles nanoflow pump was set at 100 nL/min. Solvent A was 2% (v/ v) aqueous acetonitrile containing 0.1% (v/v) formic acid, and solvent B was 98% (v/v) aqueous acetonitrile containing 0.1% (v/v) formic acid. (ii) The composition of solvent B was linearly increased from 2% at 0.0 min to 45% at 60 min, kept at 98% until 80 min, and (iii) then returned to the initial condition of 2%. The nLC-IT-TOF-MS/MS conditions were as follows: ESI voltage, +1.3 kV; curtain (nitrogen) gas flow rate, 1.0 L/min without heating; scan range, m/z 200-2000. Nano Frontier Data Processing P/N:3807051-01(Hitachi High-Technologies) software was used for generating the peaklist. The search engine was the MASCOT (version 2.1, Matrix Science, Inc., London, U.K.; server, www.matrixscience.com), and the nonredundant protein databases were SwissProt and National Center for Biotechnology Information (NCBI). Peptide mass tolerance at 0.5 Da, MS/MS ion mass tolerance at 0.5 Da, allowance of missed cleavage at 1, and charge states (+1, +2, and +3) were taken into account. The cutoff score/ expectation value for accepting individual MS/MS spectra was 53, which indicates identity or extensive homology of probability lower than 0.05 (p < 0.05). The threshold employed was -10 Log(P), where P is the probability that the observed match is a random event; protein scores are derived from ions scores as a non-probabilistic basis for ranking protein hits. For peptides matching to multiple members of a protein family, we focused on the unique peptide of identified proteins. The unique peptide of each protein was manually selected to reduce the multi-matching peptide mistake to regenerate the protein identification list. By eliminating peptides having scores lower than the applied cutoff score of 53, it was possible to have an unambiguous identification of isoforms/individual members of a protein family. Multiple proteins ID having the same accession number were considered as a single nonredundant protein for the protein list provided in this study. 2.5.2. nESI-LC-MS/MS. The nESI-LC-MS/MS was carried out as described previously.29 Briefly, after initial destain and wash processes, the gel pieces were swollen in a digestion buffer containing 10 µg/mL trypsin (Promega, sequencing grade), 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 was analyzed) solution was injected for analysis with an Agilent 1100 NanoLC-1100 system coupled with a LC/ MSD Trap XCT with a nano electrospray interface MS. 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). The spectra from MS and MS/MS data were submitted to the Agilent Spectrum Mill MS proteomics workbench (Agilent) for protein identification (using NCBI and Swiss-Prot databases) as described.29 2.6. Total RNA Extraction and Transcriptomics Analysis. Total RNA was extracted from rice shoot and root samples (from 7-day-old seedling) using the QIAGEN RNeasy Plant Maxi Kit (QIAGEN, MD). The quality of RNA is the single most important factor in determining the outcome of a transcriptomics analysis. For this reason, the yield and RNA purity was determined spectrophotometrically (NanoDrop, Wilmington, DE) and visually confirmed using formaldehyde-agarose gel electrophoresis. A rice 22K custom oligo DNA microarray chip (G4138A, Agilent Technologies) was used that contains 21 475 oligonucleotides synthesized based on the sequence data of the rice full-length cDNA project.18 The slide contained 60-mer probes. Total RNA (400 ng) was labeled with Cy-3 or Cy-5 using Journal of Proteome Research • Vol. 6, No. 9, 2007 3583

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Table 1. Details of Microarray Experiment

replicate

sample

microarray slide no.

Pooled SHOOT MA 1 Biological MA 2 Replicates (×3) Pooled ROOT MA 3 Biological MA 4 Replicates (×3)

labeling Cy3

Cy5

Control JA RT-PCR validation Control JA RT-PCR validation

JA Control JA Control

an Agilent Low RNA Input Fluorescent Linear Amplification Kit. Fluorescently labeled targets of control (minus JA) and treated (plus JA) samples were hybridized to the same microarray slide. A flip labeling (dye-swap or reverse labeling with Cy3 and Cy5 dyes) procedure was followed in the second biological replication to nullify the dye bias associated with unequal incorporation of the two Cy dyes into cDNA.31-33 The scheme of the microarray experiment is schematically depicted in Supplementary Figure 1 in Supporting Information. In our experience, the use of a dye-swap approach provides a very stringent selection condition for changed genes rather than simply doing 2 or 3 replicates, which overlook the dye bias. Although, we do not argue against replications, it must be noted that a minimum of 6-10 replications are necessary for any meaningful statistical analysis that in general is not possible due to the prohibitive cost of microarray chips. The design of the microarray experiment is presented in Table 1. Hybridization and wash processes were performed according to the manufacturer’s instructions, and hybridized microarray slides were scanned using a Gene Pix microarray scanner. Quantification of gene expression was done using the Genepix ver. 4.0 quantitative microarray analysis application program (Axon Instruments, Union City, CA). The ratio of intensity Cy3/Cy5 and Cy5/Cy3 was calculated and normalized [LOWESS (locally weighted linear regression)] with negative control spots. The scatter plots are shown in Supplementary Figure 2 in Supporting Information. All the calculations and normalizations were done using “Chip Cleanser” program.34 To classify the rice genes according to their functional orthologous relationships, we used the database of eukaryotic orthologous groups, which consists of 4852 clusters of orthologous groups (COG) of proteins, including 59 838 proteins from 7 genomes.35 2.7. Confirmatory Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). Pooled total RNA from three replications were used for RT-PCR. Briefly, total RNA samples were DNasetreated 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 StartaScriptTM RT-PCR Kit (Stratagene) according to the protocol provided by the manufacturer using 10 µg total RNA isolated from shoot and root of control and treated plants. 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 from the 3′-UTR regions (forward and reverse primer sequences are provided in Supplementary Table 1 in Supporting Information) of each of the genes used in this study by comparison and alignment with all available 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 3584


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, 10 µL of the reaction was loaded into wells of a 1.5% agarose (Agarose ME, Iwai Chemicals, Tokyo, Japan) gel, and electrophoreses 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 1× TAE buffer) for 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.

3. Results and Discussion 3.1. JA Application Reduces Growth of Shoot and Root in Rice Seedling. JA has been shown to reduce the growth in soybean and Arabidopsis.36,37 However, very little is known on how JA affects growth and development in rice. For this, we established a rice in vitro seed culture system where seeds were cultured in Murashige and Skoog medium containing different concentration of JA for a maximum of 15 days (Figure 2). A 15-day-old seedling was used for measuring the morphological parameters (Figure 3). We selected this stage mainly because at 7-days the growth of seedling was not sufficient for complete measurement of the growth parameters in all the doses checked, in particular at 50 µM JA concentrations. The result revealed that the growth of JA-treated rice seedling is inhibited with increasing (higher) concentration of JA. At a concentration of 5 µM JA, the growth is reduced by 2-fold over the control. Interestingly, the root growth at 1 µM JA is reduced 2-fold compared with control, and the similar reduction in shoot is seen at 10 µM JA. These results may be caused by the direct versus no contact of root and shoot, respectively, with medium containing JA or may arise due to the tissue-specificity. 3.2. Protein Profiling of Developing Shoot and Root Identified 128 Differentially Expressed Proteins Belonging to 10 Functional Categories. Total proteins extracted from shoots and roots were examined by 2-DGE, and the separated proteins were stained with colloidal CBB (Supplementary Figures 3 and 4 in Supporting Information). The 2-D gels revealed a total of 66 and 68 spots (marked by arrows) that were differentially expressed in shoot and root of JA-treated rice seedling, respectively, to their respective controls (minus JA). As a representative of the changed protein expressions, five boxed regions (A-E, marked on the 2-D gel images in Supplementary Figures 3 and 4 in Supporting Information) are enlarged and presented (Figure 4). In the shoot, a total of 27 and 39 protein spots increased and decreased, respectively, over the control. On the other hand, a slightly higher number of protein spots, 27 increased and 41 decreased, were seen in the root over control. The identification (green arrows) of 12 shoot (indicated as S before the spot number throughout the text) and 13 root (indicated as R before the spot number throughout the text) protein spots was performed with the nLC-IT-TOF-MS/MS. The identified proteins are listed in Table 2. The remaining protein spots marked with yellow (decreased) and red (increased) arrows (indicated as small s and r for shoot and root, respectively, before the spot numbers throughout the text) were identified by nESI-LC-MS/MS. The protein IDs for these spots are shown in Tables 3 and 4.


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Figure 2. JA treatment inhibits growth of rice seedlings in a dose-dependent manner. Dehusked rice seed was cultured in Murashige and Skoog medium containing 0, 1, 2, 5, 10, 25, and 50 µM concentrations of JA. (A) The phenotype of 7-day-old seedlings; (B) the phenotype of enlarged root portion of 7-day-old seedlings; and (C) the phenotype of 15-day-old seedlings. The first (1st), second (2nd), third (3rd), and fourth (4th) leaves are marked by arrows. The scale indicates the size of seedlings. The seedling exposed to 5 µM JA is boxed. Journal of Proteome Research • Vol. 6, No. 9, 2007 3585

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Figure 3. The effect of JA on the growth of root is more dramatic than on that of shoot. JA caused reductions in seedling growth. Phenotypic observation was performed with 15-day-old seedling. (A) Shoot length (SL) and (B) main root (MR). Average lengths in mm are given on the x-axis. For each treatment, 30 individual seedlings per replicates were used for measuring shoot and root lengths; a total of three biological replications were performed. The JA concentration (1, 2, 5, 10, 25, and 50 µM) is shown on the y-axis.

LC-MS/MS analysis of these differentially expressed proteins identified 128 protein spots out of 134 excised spots from 2-D gels of both shoot and root. The identified proteins represented 52 and 56 nonredundant proteins in shoot and root, respectively. Surprisingly, only 10 proteins were common between these two data sets (Figure 5). The identified proteins belonged to 10 functional categories depending on their function (Figure 6). The majority of the shoot proteins could be classified into photosynthesis, cellular respiration, and protein modification and chaperone, representing 66% of the total identified proteins in shoot. In the root, antioxidant, cellular respiration, defenserelated proteins, protein modification and chaperone, and cell wall and membrane biogenesis represented 74% of the total root proteins. Photosynthesis was the main functional category (44%) in the shoot, and not surprisingly constituted a low number (3%) of identified proteins in the root. On the other hand, defense-related proteins (15%) and proteins involved in cell wall and membrane biogenesis (12%) in root showed low representation in the shoot. These proteins have been discussed in relation to JA-mediated seedling growth. 3.2.1. Photosynthesis. 3.2.1.1. Carbon Dioxide (CO2) Assimilation. Four spots were identified as the RuBisCO large subunit. Spots S1and s42 were decreased and spots S4 and s19 were increased in the shoot. Four spots (S2, s25, s26, and s41), 3586


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which were decreased in shoot, were identified as RuBisCO small subunit (S2) and RuBisCO activase large isoform precursor (s25, s26, and s41). RuBisCO, which consists of 8 large subunits and 8 small subunits, catalyzes the carboxylation of ribulose 1,5-bisphospate in CO2 fixation reaction as well as the oxidative fragmentation of the pentose substrate in the photorespiration process in chloroplast.38 RuBisCO activase activates RuBisCO by mediating the release of substrate from RuBisCO active site and the carbamoylation of Lys201.38 RuBisCO activase alpha, which has 95% positive homology with spot s25, s26, or s41, maintains the active form of RuBisCO.39 Results suggest that the decrease of RuBisCO activase and RuBisCO subunits may result in the inactivation of RuBisCO and thus cause decrease in the CO2 assimilation, the absolute step in carbohydrate biosynthesis by photosynthesis. Spot s30 identified as chloroplastic fructose 1,6-bisphosphate (FBP) aldolase was decreased in the shoot. Two protein spots (s8 and s35) found to be increased in the shoot showed identity to a putative transketolase. In CO2 assimilation process, FBP aldolase catalyzes the reversible conversion of glyceraldehydes 3-phosphate (GAP) and dihydroxyacetone phosphate (diHAP) into fructose 1,6-bisphosphate and the interconversion of diHAP and erythrose 4-phosphate into sedoheptulose 1,7bisphosphate.38 Transketolase catalyzes the reversible transfer of a two-carbon ketol group from fructose 5-phosphate to GAP to form xylulose-5-phosphate and erythrose 4-phosphate, and the interconversion of GAP and sedoheptulose 7-bisphosphate into ribose 5-phosphate and xylulose-5-phosphate.38,40 Interestingly, 2 spots, putative transketolase (r6) and FBP aldolase (r22), were decreased in the root. The differential regulation of aldolase and transketolase in JA-treated root and shoot may provide a cue to the fact that the growth rate of root is more reduced than that of shoot. 3.2.1.2. Photorespiration. The glycine decarboxylase multienzyme complex catalyzes the reversible cleavage of glycine to form 1 molecule each of methylene-tetrahydrofolate, CO2, and NH3 in mitochondrial photorespiratory carbon recovery process.41,42 This complex consists of 4 proteins, such as P (spots s2, s3, and s4), H (spot s44), L (spot s36, putative dihydrolipoamide dehydrogenase), and T (spot s37) proteins.43-47 The spot s3 was increased, whereas 5 spots s2, s4, s36, s37, and s44 were decreased. In addition to this, chloroplast glutamine synthetase (s24) and putative glycine (serine) hydroxymethyltransferase (s50) were found to be decreased. Chloroplast glutamine synthetase plays a potential role of photorespiration and the protection against salt stress in rice plants.48 Glycine (serine) hydroxymethyltransferase, a component of the photorespiratory pathway, catalyzes the reversible interconversion of serine and glycine with tetrahydrofolate and pyridoxal phosphate.49 The cooperation of glycine (serine) hydroxymethyltransferase with the mitochondrial glycine decarboxylase complex, where 2 molecules of glycine are converted into serine, CO2, and NH3, is considered to be the exclusive source of photorespiratory CO2 release.50 These results suggest that the correlative decrease of glycine decarboxylase multienzyme complex and glycine (serine) hydroxymethyltransferase might cause decrease of photorespiration in shoot. 3.2.1.3. Photosystems I and II. Thirteen protein spots in shoot were identified as components of the electron-transfer chain in photosystems I and II (PSI and PSII), where H2O in the presence of light is converted to nicotinamide adenine dinucleotide phosphate (NADP+), in the thylakoid membrane of chloroplast.51 Among these spots, 8 proteins are involved in


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Figure 4. Dynamic variations of shoot and root protein profiles by 2-DGE. Five different regions (boxed in Supplementary Figures 3 and 4 in Supporting Information) of a 2-D gel from both shoots and roots were selected to display the dynamic variations of proteins in these tissues. Total soluble protein (ca. 350 µg) was separated by 2-DGE. Protein spots were visualized by staining with colloidalCBB G-250. The selected regions are marked on the 2-D gel in Supplementary Figures 3 and 4 in Supporting Information; for further details on the 2-DGE experiment, see the legends to these supplementary figures.

electron flow in photosynthesis process. Four spots (s28, s29, s32, and s38) belong to PSII oxygen (O2)-evolving complex.52,53 Spots s28 and s29 are identical to putative 33 kDa O2-evolving protein of PSII, but the former was increased and the latter was decreased. Two remaining proteins (s32 and s38) were identified as probable PSII O2-evolving complex protein 2 precursor and were also decreased. Furthermore, a chloroplast plastocyanin precursor (spot s45) which participates in electron transfer between P700 and the cytochrome b6-f complex in PSI was decreased.54 The plastocyanin in Hordeum vulgare ssp. vulgare, which has 90% positive homology with spot s45, is expressed in photosynthetic tissue by a developmentally regulated manner and light.55 Moreover, 1 increased (S7) and 2 decreased (s33 and s39) spots were identified as ferredoxin-NADP reductase and putative ferredoxin-NADP(H) oxidoreductase, respectively. Ferredoxin-NADP reductase, which catalyzes reduced ferredoxin and NADP into oxidized ferredoxin and NADPH, plays crucial roles for an efficient electron flux in photosynthesis.51,56,57 Therefore, the decrease of protein spots described above may cause the reduction of electron-transfer efficiency and the delay of the formation

of proton gradient for ATP synthesis in chloroplast. Finally, it will result in the decreased ATP productivity in photosynthesis. 3.2.1.4. ATPase Synthase. Three protein spots (s15, s16, and s18), identified as ATP synthase, were differentially expressed in shoot. The decreased spots s15 and s16 were identical to plastid ATP synthase beta subunit, whereas the increased spot s18 was identified as chloroplast ATP synthase alpha subunit. ATP synthase can be divided into 2 components, the catalytic core (F1) involved in ATP synthesis, and the membrane integral channel (F0) implicated in proton pumping. ATP synthase alpha and beta subunits are parts of the catalytic core.51,58,59 These results indicate that the decrease of proteins involved in electron flow and ATP synthesis may prevent efficient ATP production by photophosphorylation in chloroplasts, thus, playing a curial role in the decrease of growth rate by JA treatment. In addition, mitochondrial ATP synthase subunit alpha (spots s17 and r13), a regulatory subunit of ATP synthase that synthesizes ATP from proton gradient induced by the oxidation of biological fuels such as glucose and fatty acid,51 is increased in shoot and decreased in root.60 This result suggests Journal of Proteome Research • Vol. 6, No. 9, 2007 3587

research articles

Cho et al.

Table 2. Rice Protein Identification with nLC-IT-TOF-MS/MSa spot no. 1

protein name RuBisCO large subunit

expression

queries matched

620

70

2

RuBisCO small subunit

DOWN

P18567

129

13

Methionine synthase

UP

gi|886471

291

12

4

RuBisCO large subunit

UP

P12089

406

27

5

Unnamed protein product

UP

gi|28317

283

9

6

Dehydroascorbate reductase

UP

gi|55168334

415

20

7

Ferredoxin-NADP reductase, leaf isozyme

UP

P41344

111

4

8 9

Triosephosphate isomerase, Proteasome component C2

DOWN UP

P48494 P52428

55 234

2 8

UP

gi|50899388

381

25

11

Putative plastid-specific ribosomal protein 2 precursor Beta-3 tubulin

UP

P46265

570

39

12

Calreticulin precursor

UP

Q9SLY8

412

21

13

Lipoxygenase L-2

UP

P29250

717

47

14

Probable glutathione S-transferase GSTF2 Putative pathogenesisrelated protein Phenylalanine ammonia-lyase

UP

O82451

167

7

UP

gi|50933915

60

4

UP

gi|82496

70

3

FEILEAITK, LAGIEGGFFTLNPK

17

Putative phenylalanine ammonia-lyase

UP

gi|50910709

593

35

18

Putative malate dehydrogenase

UP

gi|50932771

357

21

19 20

Cationic peroxidase Putative Chitinase

UP UP

gi|2425101 gi|55168113

54 265

2 45

21

Prb1

UP

gi|33440014

205

13

22 23

ND Probenazole-induced protein

UP UP

ND gi|7442204

ND 300

ND 11

24 25

ND ND

UP UP

ND ND

ND ND

ND ND

15

a

functional category

peptides

3

16

P12089

score

LGLSAK, ASVGFK, LEDLR, IPPTYSK, EGNEIIR, AMHAVIDR, SQAETGEIK, ALRLEDLR, DTDILAAFR, DDFIEKDR, DNGLLLHIHR, EMTLGFVDLLR, AIKFEFEPVDK, FEFEPVDKLDS, LTYYTPEYETK, LEDLRIPPTYSK, DDENVNSQPFMR, MSGGDHIHAGTVVGK, TFQGPPHGIQVER, MSGGDHIHAGTVVGK, TFQGPPHGIQVERDK, GGLDFTKDDENVNSQPFMR VGFVYR, SPGYYDGR, YWTMWK, IIGFDNVR, AYPDAFVR, KAYPDAFVR, VGFVYRENHR TLDLIK, LLSVFR, QMADAGIK, VVEVNALAK, SWLAFAAQK, YLFAGVVDGR, IPSTEEIADR, IPSTEEIADRINK LGLSAK, IPPTYSK, EGNEIIR, AMHAVIDR, SQAETGEIK, LRLEDLR, DTDILAAFR, DDFIEKDR, LTYYTPEYETK, LEDLRIPPTYSK, DDENVNSQPFMR, MSGGDHIHAGTVVGK, TFQGPPHGIQVER, MSGGDHIHAGTVVGK LASYLDK, VLDELTLTK, VTMQNLNDR, DAEAWFNEK, SLLEGEGSSGGGGR, SQYEQLAEQNR, QSLEASLAETEGR KVPYEMK, VLLTLEEK, LYHLQVALEHFK, ALLTELQALEEHLK, LIDVQNKPDWFLK, WIPDSDVITQVIEEK, YPTPSLVTPPEYASVGSK, IPEDLTNVHAYTEALFSR, AHGPFINGQNISAADLSLAPK VDYAVSR, LYSIASSALGDFGDSK, LYSIASSALGDFGDSK, DPNANIIMLATGTGIAPFR FFVGGNWK, SLLGESNEFVGDK TFLER, LFQVEYAMEAVK, NQYDTDVTTWSPAGR, FEGYNDYTPEQLIK, RFEGYNDYTPEQLIK AEVMFDK, VYVGNLAK, SVTTEMLK, LYVGNIPR, GEVLSATVSR, KLYVGNIPR, VNVTESFLPNIDR, SAPEPEPVFVDSQYK, IREEYPDR, LAVNLIPFPR, FPGQLNSDLR, FPGQLNSDLRK, YSGDSDLQLER, RVSEQFTAMFR MMLTFSVFPSPK, EVDEQMLNVQNK, LHFFMVGFAPLTSR, AVLMDLEPGTMDSVR, LHFFMVGFAPLTSR, NSSYFVEWIPNNVK, ALTVPELTQQMWDAK, MASTFIGNSTSIQEMFR, FWEVICDEHGIDHTGK AAFDEAEK, EIPDPDAK, LLGGDVDQK, WNGDPEDK, FEDGWESR, TLVLQFSVK, GIQTSEDYR, LLGGDVDQKK, EYIPDPEDK, KPEGYDDIPK, FYAISAEYPEFSNK, QSGSIYEHWDILPPK, KPEDWDDKEYIPDPEDK, AGEDDDDLDDEDAEDEDKADEK GSLVLMR, GDAAGLSAR, LTEIESR, EYDELAR, LLLPHYR, DTPEWTSDAK, DATWWPEMK, LPSIPALEELR, AWMTDDEFAR, HSSDEVYLGQR, LSLLENIYVPR, VFFSNDTSLPSK, EYDELARDPEK, LPSIPALEELRK, FGVTFEWEVEK, EILAGVNPMVIAR, GDDQQGPYQEHDR, TYVDLTPGEFDSFK, YGDQTSTITAAHVER, DWSFADQALPDDLVK, QMQAIVGISLLEILSK, QTLINGGGIFEMTVFPR, TYVDLTPGEFDSFKDILK, VGPTNFPYTLLYPNTSDLK VASLMKPPA, VVEENLEK, VLEVYEAR, LYGSTLSWNVTR, MAPMKLYGSTLSWNVTR GALLECGHYTQVVWR

10

DOWN

accession

MVAQFR, IQGATLR, FEEELR, VFLGISQGK, KVDAAEAFK, VGQVAAVAQAK, VNEMDPLLKPK, KVNEMDPLLKPK, TKDGPALQVELLR, VLTMNPTGDLSSAR, LAGIEGGFFTLNPK, VLTMNPTGDLSSAR, KVLTMNPTGDLSSAR, TSPQWLGPQIEVIR, VNSVNDNPVIDVHR, ALHGGNFQGTPIGVSMDNAR, AVLVEHALTSGDAEPEASVFSK, HLNAGIFGTGSDGHTLPSETVR EGLENLK, TQDGGTEVVEAK, RTQDGGTEVVEAK, VAILGAAGGIGQPLALLMK, NGVEEVLGLGQLSEFEK, DIGIAAGLVR, DVPVNSVTQELDVR VFGDGR, WMAAYPQSR, LQAALSTGLFSR, VLVGVVASPEADR, LPNYGGIMVWNR, DLYYDVLQFINK, LDISGHTVSAVGPDIK AGDCALIHSGSWEK, VEGVGEVVWDDAVAAYAENYAAER, RVEGVGEVVWDDAVAAYAENYAAER, AGYGENLFGGSGSEWTAADAVNSWVGEK AFMDASTLPK, DIVDGYYGMLK, EKDIVDGYYGMLK, MIEDYLVAHPAEYA, VEYELEDGSSLSPEK

Photosynthesis

Photosynthesis Amino acid metabolism Photosynthesis

Unknown Antioxidant system

Photosynthesis Cellular respiration Protein modification and chaperone Transcriptional and translational protein Cell wall and membrane biogenesis

Protein modification and chaperone

Defense-related protein

Antioxidant system Protein modification and chaperone Secondary metabolite biosynthesis Secondary metabolite biosynthesis

Cellular respiration Antioxidant system Defense-related protein Defense-related protein

Unknown Defense-related protein Unknown Unknown

ND, not determined. These proteins are indicated with S (for shoot; spots 1-12) and R (for root, spots 13-25) throughout the text.

3588


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Differentially Expressed Proteins/Genes in JA-Treated Rice Table 3. Rice Shoot Protein Identification by nESI-LC-MS/MSa

spot no.

protein name

distinct summed MS/MS % AA distinct search NCBI coverage peptides score accession

pI/MW (Da)

peptide sequence

Putative Aconitate hydratase

20

13

181.21

2

Glycine dehydrogenase P protein

26

15

243.6

3

Glycine dehydrogenase P protein Putative glycine dehydrogenase P protein

11

6

80.6

17

13

194.76

5

OSJNBa0020P07.3

16

10

156.31

6

OSJNBa0039C07.4

34

22

352.01

7

Heat shock-related protein Putative transketolase

17

4

60.85

39

20

322.57

9

Phenylalanine ammonia-lyase

26

17

235.81

10

Putative oxalyl-CoA decarboxylase

29

16

217.3

11

DNAK-type molecular chaperone hsp70

40

22

367.94

12

DNAK-type molecular chaperone hsp70 Putative RuBisCO subunit bindingprotein alpha subunitprecursor Putative chaperone 60 beta

5

2

33.74

14

8

121.25

31193919 5.36/61400

VVNDGVTIAR/TNDSAGDGTTTASVLAR/LGLLSVTS GANPVSIKK/GILNVAAIK/LSGGVAVIKVGAATETELE DR/GADIIQK


54

29

486.26

34897924 5.60/64085


15

ATP synthase CF1 beta chain

73

25

412.45

11466794 5.47/54014

16

ATP synthase CF1 beta chain

26

7

106.11

11466794 5.47/54014

17

ATP synthase alpha chain, mitochondrial

30

13

180.96

114409 5.85/55281

LADLVGVTLGPK/IVNDGVTVAREVELEDPVENIGAK/ TNDLAGDGTTTSVVLAQGLIAEGVKVVAAGANPVQIT R/ALVEELKKLSKEVEDSELADVAAVSAGNNYEIGNM IAEAMSK/GVVTLEEGRSSENNLYVVEGMQFERGYI SPYFVTDSEK/DLINVLEEAIR/TQYLDDIAILTGATVIRD EVGLSLDK/ESTTIVGDGSTQEEVTKR/NLIEAAEQEY EKEK/LAGGVAVIQVGAQTETELKEK/LRVEKALNATK/ VDAIKDNLENDEQKVGAEIVR/FGYNAATGQYEDLMA AGIIDPTK/TFLTSDVVVVEIKEPEPAPVTNPMDNSGYGY TNPTTSRPGVSTIEEK/LPYIYNALVVK/AVAMSATDGL MRGMEVIDTGAPLSVPVGGATLGRIFNVLGEPVDNL GPVDTSATFPIHRSAPAFIELDTKLSIFETGIK/IGLFGG AGVGKTVLIMELINNIAKAHGGVSVFGGVGERTREGN DLYMEMK/VALVYGQMNEPPGAR/VGLTALTMAEYFR DVNKQDVLLFIDNIFRFVQAGSEVSALLGRMPSAVGY QPTLSTEMGSLQER/GSITSIQAVYVPADDLTDPAPATT FAHLDATTVLSR/GIYPAVDPLDSTSTMLQPRIVGNE HYETAQR/YKELQDIIAILGLDELSEEDRLTVAR/GFQL ILSGELD IFNVLGEPVDNLGPVDTSATFPIHR/IGLFGGAGVGKT VLIMELINNIAK/DVNKQDVLLFIDNIFRFVQAGSEVSA LLGR/ELQDIIAILGLDELSEEDRLTVAR/GFQLILSGEL DGLPEQAFYLVGNIDEASTK AAELTTLLESRMTNFYTNFQVDEIGRVVSVGDGIARV YGLNEIQAGEMVEFASGVK/TGSIVDVPAGK/VVDALG VPIDGK/KSVHEPMQTGLK/TAIAIDTILNQK/DNGMHA LIIYDDLSK/EAFPGDVFYLHSR/QPQYEPLPIEK/A

8

13

14

NILTTLPKPGGGEYGKFYSLPALNDPR/IIDWENTSP K/SPNAVQSNMELEFK/FVEFYGEGMGK/SDETVAM IEAYLR/MFVDYNEPQTER/FDFHGQPAELK /YLLQSGLQEYLNK/SEGHDTIVLAGAEYGSGSSR/ GPMLLGVK/AGEDADSLGLTGHERYTIDLPTNVSEI RPGQDITVTTDNGK 42416979 6.03/96948 FDAGFTESEMIEHMQR/NLMENPAWYTQYTPYQAEIA QGR/AAGFDLNVVVADAK/FGVPMGYGGPHAAFLATS QEYK/IIGVSVDSSGKPALR/KLGTVTVQELPFFDTVK/ VVDATTITVAFDETTTLEDVDKLFK/ITGFDSFSLQPNA GASGEYAGLMVIR/GNINIEELRK/GVNGTVAHEFIIDL R/TTAGIEPEDVAK/LMDYGFHAPTMSWPVPGTLMIE PTESESK/ADVNNNVLK 42416979 6.03/96948 FDFKAGFTESEMIEHMQR/AAGFDLNVVVADAK/IIGV SVDSSGKPALR/LGTVTVQELPFFDTVK/VVDATTITVA FDETTTLEDVDKLFK/EEIAEIES 34910498 6.51/111428 FDAGFTESQMIDHMQR/NLMENPAWYTQYTPYQAEI AQGR/AAGFDLNVIVADAK/IIGVSVDSSGKPALR/LGT VTVQELPFFDTVK/VVDATTITVAFDETTTLEDVDKLFK /GNINIEELRK/IAILNANYMAK/GVNGTVAHEFIIDLR/TT AGIEPEDVAK/ADVNNVLK/EYAAFPAAWLR 38344860 5.85/93974 STGISLFYEMSDESLK/VIENANVIMATYEDTLLGDVQV YPEK/LWGENFFDPATK/YRVENLYEGPLDDVYATAIR/L YMEARPLEEGLAEAIDDGR/ILSEEFGWDKDLAK/GV QYLNEIK/EGALAEENMR/EQMTPLSDFEDKI 38347158 5.79/98497 VIMLAQEEAR/GSGFVAVEIPFTPR/VLELSLEEAR/VLE SLGADPNNIR/MVGESTEAVGAGVGGGSSGQKMPTL EEYGTNLTKLAEEGKLDPVVGR/TAIAEGLAQR/VPEP TVDETIQILR/AIDLIDEAGSR/AQITAIIDK/AETESGEVG PLVTEADIQHIVSSWTGIPVEK/IIGQDEAVK/ALAAYYF GSEEAMIR/LIGSPPGYVGYTEGGQLTEAVR/NTLLIMT SNVGSSVIEK/IGFDLDYDEKDTSYNR/LDEMIVFR/DID LQVTEK/LLEDSLAEK/EGDSAIVDVDSEGKVIV 29367425 5.02/44985 DEGIDLLK/MELSTLSQTNISLPFITATADGPK/LSVDNL DEVILVGGSTR/SEVFSTAADGQTSVEINVLQGER 11875175 6.12/80029 AAAVETLEGQAATGALLEK/LIAFYDDNHISIDGDTEIA FTEDVSAR/AVTDKPTLIKVTTTIGFGSPNKANSYSVH GSALGTKEVEATRENLGWPYEPFFVPEDVK/HVPQG AAFEADWNAK/KYPEDAATLKSIVSGELPAGWADALP K/VVPGLLGGSADLASSNMTLLKMFGDFQKDTPEER/ RPSVLALSR/LAQLPGTSIEGVEKGGYIVSDNSTGNKP DFIVMSTGSELEIVAK/ESVLPEAVTARVSLEAGSTLG WQK/AIGIDKFGASAPAGKIYQEYGITAENVIATA 48716258 6.06/75498 VGQVAAVAQAKDAAGVAVELDEEARPR/TKDGPALQV ELLRHLNAGIFGTGSDGHTLPSETVR/FEILEAITK/KV DAAEAFK/KVNEMDPLLKPK/TSPQWLGPQIEVIR/LDY GFK/KTLEAVDILK/KVLTMNPTGDLSSAR/NLLTAIDR/A VLVEHALTSGDAEPEASVFSKITKFEEELR 13359052 5.95/60826 VDGSALAGR/GDFQELDQIAATKPFIK/ATTIADIPR/AD AIWK/KPHVGIVGDAK/VVELINR/DVVPFNFLTPLRIIRD AILAEGNPAPVVVSEGANTMDVGRAVLVQNEPR/SPD EITGPYKDDPAPTSFVPAAGYHK/GYLVETPDELKSAL SESFR/KPAVINVIIDPYAGAESGR 1076746 5.13/71113 VEIIANDQGNRTTPSYVGFTDSER/NQVAMNPINTVFD AK/FSDASVQSDIK/QFAAEEISSMVLIKMREIAEAYLG TTIKNAVVTVPAYFNDSQR/DAGVIAGLNVMRIINEPTA AAIAYGLDKK/ATAGDTHLGGEDFDNRMVNHFVQEFK/ ARFEELNMDLFR/SSVHDVVLVGGSTR/NINPDEAVAY GAAVQAAILSGEGNEK/KEQVFSTYSDNQPGVLIQVY EGER/NALENYAYNMR/K 1076746 5.13/71113 QFAAEEISSMVLIK/EQVFSTYSDNQPGVLIQVYEGER

Cellular respiration

1

4

40253814 5.67/98083

functional category

Photosynthesis

Photosynthesis Photosynthesis

Transcriptional and translational protein Protein modification and chaperone

Protein modification and chaperone Photosynthesis

Secondary metabolite metabolism Cellular respiration



Photosynthesis

Photosynthesis


Journal of Proteome Research • Vol. 6, No. 9, 2007 3589

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Table 3. (Continued)

spot no.

protein name

distinct summed MS/MS search NCBI % AA distinct score accession coverage peptides

pI/MW (Da)

18

ATP synthase alpha chain

44

18

306.33

11466784

5.95/55665

19

RuBisCarboxylase

16

9

114.87

11466795

6.22/52881

20

S-adenosylmethionine synthetase 1

31

10

142.86

1170937

5.74/43220

21

OSJNBa0011P19.5

39

11

168.05

34894718

5.68/42900

22

Putative glutamate-1semialdhyde 2,1-aminomutase

36

13

195.45

42761379

6.48/50237

23

Translational elongation factor Tu

50

17

293.93

17225494

6.19/50414

24

Glutamine synthetase, chloroplast

45

14

252.68

121343

5.96/46643

25

RuBisCO activase large isoform precursor

44

15

250.64

8918359

5.43/51454

26


49

18

289.33

8918359

5.43/51454

27

Putative fructokinaseII

50

16

255.31

16566704

5.02/35516

28

Putative 33 kDa oxygen evolvingprotein of photosystem II Putative 33 kDa oxygen evolvingprotein of photosystem II

42

11

177.65

34914480

6.09/34861

45

13

207.18

34914480

6.09/34861

30

Fructosebisphosphate aldolase

31

10

146.99

3913018

7.59/42148

31

Cytoplamic malate dehydrogenase

36

8

133.57

37535388

5.75/35569

32

Probable photosystem II oxygen-evolving complex protein 2 precursor Putative ferredoxinNADP(H) oxidoreductase Cytosolic glyceraldehyde 3-phosphate dehydrogenase Putative transketolase

56

12

195.33

34899580

8.66/26939

31

9

146.45

41052914

7.98/38748

45

11

176.33

38346061

6.34/36773

13

10

134.78

11875175

6.12/80029

36

Putative dihydrolipoamide dehydrogenase precursor

36

18

282.26

34894800

7.20/52643

37

OSJNBa0053K19.11

17

8

118.01

38344172

8.53/43968

29

33 34

35

3590


peptide sequence VGIENIGRVVQVGDGIARIIGLGEIMSGELVEFAEGTR GIALNLESKNVGIVLMGDGLMIQEGSFVK/IAQIPVSEA YLGR/LIESPAPGIISR/SVYEPLQTGLIAIDSMIPIGR/TA VATDTILNQK/ERHTLIITDDLSK/EAYPGDVFYLHSR/Q SQANPLPVEEQIATIYIGTRGYLDSLEIGQVK/FLDELR K/DTKPQFQEI LTYYTPEYETKDTDILAAFR/GGLDFTKDDENVNSQP FMR/DNGLLLHIHR/EMTLGFVDLLRDDFIEKDR/FEFE PVDKLDS TNMVMVFGEITTK/TQVTVEYR/VHTVLISTQHDETVT NDEIAADLKEHVIKPVIPEQYLDEKTIFHLNPSGRFVIG GPHGDAGLTGR/SIVASGLAR/IPDKEILK/TAAYGHFGR DDPDFTWEVVKPLK TNMVMVFGEITTK/GIGFVSDDVGLDADR/TQVTVEYL NDAGAMVPVRVHTVLISTQHDETVTNDEIAADLKEHVI KPVIPDKYLDEKTIFHLNPSGRFVIGGPHGDAGLTGR/ SIVASGLAR/IPDKEILK/TAAYGHFGREDPDFTW AYTVEKSEEIFNAAKELMPGGVNSPVR/SVGGQPIV FDSVK/AGSGVATLGLPDSPGVPKGATSETLTAPYN DVEAVKK/DLTKQDGALLVFDEVMTGFRLAYGGAQE YFGITPDVSTLGKIIGGGLPVGAYGGR/LMEPGTYDY LDK/GMLEEGVYLAPSQFEAGFTSLAHTSQDIEK TTLTAALTMVLASVGGSAPKKYDEIDAAPEER/GITINT ATVEYETETR/NMITGAAQMDGAILVVSGADGPMPQT K/KDQVDDEELQLVELEVRELLSSYEYDGDEVPIVAG SALKALENLMANPAIKRGDDEWVDGIFSLIDSVDNYIP VPQRQTDLPFLLAVEDVFSITGR/VGDTVDIVGIR/TMD DAMAGDNVGLLLR/GMVLAKPASITPHTKFDAVVYVL K/TVGAGVINTIL MEQLLNMDTTPFTDKIIAEYIWVGGTGIDLR/TISK[VE DPSELPKWNYDGSSTGQAPGEDSEVILYPQAIFKDPF R/AAQVFSDPKVVSQVPWFGIEQEYTLLQR/SMREDG GFEVIKKAILNLSLRHDLHISAYGEGNERRLTGLHETA SIDNFSWGVANR/RPASNMDPYVVTALLAETTILWEP TLEAEVLAA GLAYDISDDQQDITR/GFVDSLFQAPTGDGTHEAVLS SYEYLSQGLRTYDFDNTMGGFYIAPAFMDKLVVHISK NFMTLPNIK/MGINPIMMSAGELESGNAGEPAK/YREA ADIIK/VPIIVTGNDFSTLYAPLIR/IVDSFPGQSIDFFGAL R/WVSDTGVENIGKR/EGPPEFEQPK/LMEYGYMLVK/ RVQLAEQY GLAYDISDDQQDITR/GFVDSLFQAPTGDGTHEAVLS SYEYLSQGLRTYDFDNTMGGFYIAPAFMDKLVVHIS KNFMTLPNIKVPLILGIWGGK/MGINPIMMSAGELESG NAGEPAK/YREAADIIK/VPIIVTGNDFSTLYAPLIR/TDN VPDEDIVKIVDSFPGQSIDFFGALR/KWVSDTGVENIG KR/EGPPEFEQPK/LMEYGYMLVK/RVQLAEQYLSEAA LGDANSDAM LGGSSAFVGKFGDDEFGHMLVDILKK/TALAFVTLK/A DMLLTEAELNLDLIRR/LPLWPSEDAARAGILSIWK/VS DDEVAFLTQGDANDEKNVLSLWFDGLKLLIVTDGEK/ GSVPGFSVNTVDTTGAGDAFVGSLLVNVAKDDSIFH NEEK/KGAIPALPTVAVAQELISK RLTFDEIQSK/LTYTLDEIEGPLEVSSDGTIKFEEKDGID YAAVTVQLPGGER/NLVATGKPESFGGPFLVPSYR/GG STGYDNAVALPAGGRGDEEELAKENVKNASSSTGNI TLSVTKSKPETGEVIGVFESVQPSDTDLGAK RLTFDEIQSK/LTYTLDEIEGPLEVSSDGTIKFEEKDGI DYAAVTVQLPGGERVPFLFTIKNLVATGKPESFGGPF LVPSYR/GRGGSTGYDNAVALPAGGRGDEEELAKEN VKNASSSTGNITLSVTKSKPETGEVIGVFESVQPSD TDI AGAYDDELVK/LASIGLENTEANR/TLLVTAPGLGQYIS GAILFEETLYQSTVDGKKIVDILTEQK/EAAWGLARYAA ISQDNGLVPIVEPEILLDGEHGIDR/DRATPEQVSDYTL K/ANSLAQLGK GVMLGADQPVILHMLDIPPATESLNGLKMELVDAAFPL LK/VLVVANPANTNALILK/LNVQVTDVKNAIIWGNHSS TQYPDVNHATVK/ELVADDEWLNTEFISTVQQR/KMDA TAQELSEEK TNTEFIAYSGEGFK/EREFPGQVLRYEDNFDANSNVS VIINPTTKKTITEFGSPEEFLAQVDFLLGKQAYSGKTD SEGGFESDAVATANILESSAPVVGGKQYYSVTVLTRTA DGDEGGKHQLITATVNDGK/KFVESAASSFSV ITGDDAPGETWHMVFSTDGEIPYREGQSIGVIPDGID K/LYSIASSAIGDFADSK/LVYTNDQGEIVK/MFFEEHDD YK/MAEYKDELWELLK/GIDDIMIDLAAKDGIDWLDYKK VALQSDDVELVAVNDPFITTDYMTYMFKYDTVHGQW K/NPEEIPWGETGAEFVVESTGVFTDKDK/KVVISAPS KDAPMFVVGVNEK/VPTVDVSVVDLTVRLEKPASYDQ IK/GILGYVEEDLVSTDFQGD/AGIALNDNFVKLVS FLAIDAVEK/NPYWFNRAVTDKPTLIKVTTTIGFGSPN K/KYPEDAATLK/YTPESPADATR/MFGDFQKDTP/QKL AQLPGTSIEGVEK/ESVLPEAVTA ALLHSSHMYHEAK/FSNLEVDLPAMMAQKGIEGLFKK/ LASPSEVSVDLSDGGSTVVKGKNIIIATGSDVKSLPGV TIDEKK/LGSEVTVVEFAPDIVPSMDGEVRK/TKVVGV DTSGDGVK/VPYTAGIGLESVGVETDKAGR/EGHVDY DTVPGVVYTHPEVASVGK/VGKFPLLANSR/AIDDAE GLV DGTGTLTVFTNDRGGAIDDSVVTK/TGYTGEDGFEIS VPSENAVDLAK/KAEGGFLGADVILK/VGLLSQGPPPR

functional category Photosynthesis

Photosynthesis Amino acid metabolism Amino acid metabolism Secondary metabolite metabolism Transcriptional and translational protein

Amino acid metabolism

Photosynthesis

Photosynthesis


Photosynthesis

Photosynthesis

Photosynthesis

Cellular respiration Photosynthesis

Photosynthesis Cellular respiration Photosynthesis Photosynthesis

Photosynthesis

research articles

Differentially Expressed Proteins/Genes in JA-Treated Rice Table 3. (Continued)

spot no. 38

39 40

protein name Probable photosystem II oxygen-evolving complex protein 2 precursor Putative ferredoxin-NADP(H) oxidoreductase Unknown protein

distinct summed MS/MS % AA distinct search NCBI coverage peptides score accession 54

11

182.91

10

2

32

34.03

41052914 7.98/38748 ITGDDAPGETWHMVFSTDGEIPYR/ GIDDIMIDLAAK

Photosynthesis

4

59.02

42407675 6.85/25510 DAAEYVYEVPEGWKER/GTNGTDSEFFNPR/ALAPVG AVLDNLALSDVGLQDQIASADGVLSTER/LYAHFVTAP NPEWSR 8918359 5.43/51454 TYDFDNTMGGFYIAPAFMDKLVVHISKNFMTLPNIK/M GINPIMMSAGELESGNAGEPAK/YREAADIIK/FYWAPT RDDR/TDNVPDEDIVK/WVSDTGVENIGKR/EGPPEFE QPK/LMEYGYMLVK/RVQLAEQYLSEAALGDA 11466795 6.22/52881 LTYYTPEYETKDTDILAAFR/TFQGPPHGIQVER 21741225 4.75/17266 VELVVEVK/VVSYSVVDGELVSFYK/VTLQVTPKGGAA APAADGAVVSWTMDFDKASEEVPDPDVIKETAAKTFH DLDDYLLKN 37536058 4.92/17367 YSSSHEWVK/LSETPGLINSSPYEDGWMIKVKPSSPS ELDALLDPAK

Unknown

31

13

174.32

42 43

RuBisCarboxylase OSJNBb0048E02.12

6 52

3 6

42.42 84.56

44

Putative glycine decarboxylase H subunit Plastocyanin, chloroplast precursor Putative r40c1 protein

28

3

48.16

20

2

25.92

42

11

168.5

47

r40g2 protein

32

8

123.38

48

Putative ribosomal protein L12 Putative catalase

26

4

65.66

25

12

159.54

50

Putative glycine hydroxymethyltransferase

25

13

210.69

51

Putative catalase A

26

11

145.01

52

Catalase A

11

3

42.27

53

Putative ribonucleoprotein

16

2

36.42

54

Dehydroascorbate reductase

44

6

82.14

49

a

functional category Photosynthesis


46

peptide sequence

34899580 8.66/26939 TNTEFLAYSGEGFK/EREFPGQVLRYEDNFDANSNVS VIINPTTKKTITEFGSPEEFLAQVDFLLGK/TDSEGGFE SDAVATANILESSAPVVGGKQYYSVTVLTRTADGDEG GKHQLITATVNDGK/KFVESAASSFSVA

41

45

pI/MW (Da)

Photosynthesis

Photosynthesis Defense-related protein Photosynthesis

21903451 5.61/15576 SGETITFKNNAGFPHNVVFDEDAVPSGVDVSK

Photosynthesis

34902150 6.26/41693 EGNVVLAPTNPRDEHQHWYK/IKDEEGNPAFALVNK/ LAPFNPEYPDESVLWTESGDVGK/LNFDAFHGDKDH GGVHDGTTIVLWEWAK/ILPWGDEAYAGGSANAPR/A DEGFSVTVR/EYQHWIK/HSNSIKDEEGYPAFALVNR/L VPYNPGYQDESVLWTESR 34394517 6.80/38528 DSAVVLAPVNPK/VKDGEGMPAFALVNK/LVPFNPEYE DASVLWTESK/LNLDAFHGDKDHGGVR/DEEGYPAFA LVNK/LVPYNPEYQDESVLWTESK/MVNNIYLNFDAFH GDK 34912074 5.36/18590 VLELGDAIAGLTLEEARGLVDHLQER/TEFDVVIEEVPS SAR/DLIEGLPK


20503000 8.21/62576 GPILLEDYHLVEK/APGVQTPVIVRFSTVIHER/HMDGS GVNTYTLVNR/LFIQTIDPDHEDRFDFDPLDVTKTWPE DIVPLQPVGR/LGPNYLLLPPNAPK/DEEVDYFPSR/YP IPSATLTGR/WIDALSDPR/SIWLSYWSQADF 31126793 8.82/61354 SGVTWPKQLNAPLEEVDPEIADIIEHEK/YSEGYPGAR /LDESTGLIDYDQMEK/LIVAGASAYAR/VLENVHIAANK NTVPGDVSAMVPGGIR/GFVEEDFAKVADFFDAAVNL ALK/LKDFVATLQSDSNIQSEIAKLRHDVEEYA 41052594 6.52/56698 TTTTNAGAPVWNDNEALTVGPRGPILLEDYHLIEK/SP GAQTPVIV/LFVQVIDPEEEERFDFDPLDDTK/VFAYAD TQR/LGPNYLMLPVNAPK/DEEVDYYPSR/QNDFKQP GER/FAGELAHPK/LNVKPS 11375424 6.75/56646 TTTTNAGAPVWNDNEALTVGPRGPILLEDYHLIEK/LF VQVIDPEEEERFDFDPLDDTK 34913270 4.75/28099 VNSGPPPPRDDFAPR/GFGFVTYGSAEEVNNAISNLD GVDLDGR 28192425 5.81/23570 VLLTLEEK/WIPDSDVITQVIEEKYPTPSLVTPPEYASV GSK/ALLTELQALEEHLKAHGPFINGQNISAADLSLAP K/IPEDLTNVHAYTEALFS

Defense-related protein Transcriptional and translational protein Antioxidant system Amino acid metabolism Antioxidant system Antioxidant system Transcriptional and translational protein Antioxidant system

These proteins are indicated with small s (for shoot) throughout the text.

that the capability of ATP synthesis by oxidation of biological fuels in shoot might be better than that in the root. Most of the identified proteins mentioned under photosynthesis are decreased in shoot. These are involved in CO2 assimilation, photorespiration, and photophosphorylation (PSI/ II and ATP synthesis) of photosynthesis in chloroplast. Therefore, it is logical not to expect the expression of these proteins in the root due to their tissue specificity, particularly chloroplast specificity. In addition, photophosphorylation in plant produces energy-rich NADPH and ATP from light, which are subsequently used in the carbon assimilation reaction that reduces CO2 to form trioses, or are used in phosphorylation of glycerate into 3-phosphoglycerate during photorespiration in chloroplast. Trioses produced from carbon assimilation reaction or photorespiration can be converted into more complex compounds such as sucrose, starch, cellulose, and various cellular metabolic intermediates for plant growth.38,51 Therefore, our results show that the inhibition of growth in JA-treated rice may result from the reduction of energy productivity, CO2 assimilation, and photorespiration in the chloroplast.

3.2.2. Cellular Respiration. 3.2.2.1. Acetyl-CoA Production. Three spots (s34, r20, and r40) were identified as cytosolic GAP dehydrogenase that catalyzes GAP, phosphate, and NAD into 1,3-bisphosphoglycerate and NADH, and 2 spots (s27 and r27) were putative fructokinase II that catalyzes the conversion of fructose to fructose-6-phosphate.61 The Zea mays cytosolic GAP dehydrogenase having 97% positive homology with spots s34, r20, and r40 is strongly transcripted in shoots and roots by heat and anaerobic stresses,62 and is increased during endosperm development and pollination.63 Moreover, fructokinase II was also reported to play important roles in developing rice grains.64 Interestingly, spots s34 and s27, and spots r20, r27, and r40, were found to be increased and decreased in shoots and roots, respectively. In shoots, cytosolic triosephosphate isomerase (S8) which catalyzes reversibly diHAP into GAP was decreased.61,65 In roots, putative 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (r5) that catalyzes the interconversion of 3-phosphoglycerate and 2-phosphoglycerate,66 and enolase (r11) that catalyzes the reversible dehydration of 2-phosphoD-glycerate to phosphoenolpyruvate,61 were also decreased. The Journal of Proteome Research • Vol. 6, No. 9, 2007 3591

research articles

Cho et al.

Table 4. Rice Root Protein Identification by nESI-LC-MS/MSa

spot no.

protein name

1

Putative aconitate hydratase

2

Putative aconitate hydratase Putative aspartate-tRNA ligase

3

distinct summed MS/MS % AA distinct search NCBI coverage peptides score accession 20

15

222.37

1

2

26.39

28

12

184.23

4

DnaK-type molecular chaperone hsp70

38

19

318.44

5

Putative 2,3-bisphosphoglycerateindependent phosphoglycerate mutase

34

15

246.59

6

Putative transketolase

20

10

146.28

7

Beta-tubulin R2242

27

9

131.68

8

Calreticulin precursor

21

8

116.03

9

Gamma-glutamylcysteine synthetase

20

8

111.74

10

Mitochondrial chaperonin-60

37

16

257.23

11

Enolase

49

14

237.66

12

UDP-glucose pyrophosphorylase

37

14

219.34

13

ATP synthase alpha chain, mitochondrial

36

13

195.06

15

Cytosolic glutamine synthetase isozyme 1-1 Putative caffeic acid 3-O-methyltransferase

24

8

113.09

41

12

177.34

17

Cytoplasmic malate dehydrogenase

33

7

118.62

18

Reversibly glycosylated polypeptide Guanine nulceotidebinding protein beta subunit-like protein Cytosolic glyceraldehyde 3-phosphate dehydrogenase

22

7

110.83

31

9

141.27

51

12

196.62

Putative r40c1 protein - rice

38

12

189.45

16

19

20

21

3592


pI/MW (Da)

peptide sequence

40253814 5.67/98083 NILTTLPKPGGGEYGKFYSLPALNDPR/ILLESAI R/IIDWENTSPK/VLLQDFTGVPAVVDLAAMR/SP NAVQSNMELEFKR/FVEFYGEGMGK/SDETVAMIEA YLR/MFVDYNEPQTER/FDFHGQPAELK/YLLQSGL QEYLNK/TVHVPTGEK/GPMLLGVK/YTIDLPTNV SEITPGQDITVTTDNGK 40253814 5.67/98083 ILLESAIR/STYEAITK 41052719 5.99/60862 QQQQQPQADADDPFAANYGDVPVEEIQSK/VWTEI GGLDEAAAGR/GAAQAIRPVSK/MAFVVLR/ESIV DVEGVVSLPK/ATTQQVEIQVR/AIPTLPINLEDA SR/TPANQAIFR/LIAGSSEGGAAVFK/LTYEEGI QMLK/YGTEFFILYR/GEEIISGAQ 1076746 5.13/71113 VEIIANDQGNRT/NQVAMNPINTVFDAK/FSDASV QSDIK/QFAAEEISSMVLIK/EIAEAYLGTTIKNA VVTVPAYFNDSQR/DAGVIAGLNVMRIINEPTAAA IAYGLDKK/ATAGDTHLGGEDFDNRMVNHFVQEFK /ARFEELNMDLFR/SSVHDVVLVGGSTR/NINPDE AVAYGAAVQAAILSGEGNEK/KEQVFSTYSDNQPGV LIQVYEGER/NALENYAYNMR/KIEDAIDQAIQWL DGNQLAEADEFDD 15128394 5.53/60996 TVALVVLDGWGEANADK/LVDLALASGKIYDGEGF NYIK/LDQVQLLLK/DVLDGSSVGFVETLESDLSQ LR/YENDWDVVKRGWDAQVLGEAPYKFQNAVEAVK /SVGPVVDGDAVVTFNFR/ALEYADFDKFDR/YAG MLQYDGELKLPSHYLVSPPEIER/FGHVTFFWNGN RS/IILDAIEQVGGIYLVTADHGNAEDMVK 11875175 6.12/80029 ENLGWPYEPFFVPEDVK/VPQGAAFEADWNAK/KY PEDAATLKSIVSGELPAGWADALPK/LAQLPGTSI EGVEKGGYIVSDNSTGNKPDFIVMSTGSELEIVAK /ESVLPEAVTARVSLEAGSTLGWQK/IYQEYGIT AENVIATA 1076738 4.73/49864 YSGDSDLQLER/AVLMDLEPGTMDSVR/GHYTEGA ELIDSVLDVVR/FPGQLNSDLRK/LHFFMVGFAPL TSR/ALTVPELTQQMWDAK/EVDEQMLNVQNKNSS YFVEWIPNNVK/VSEQFTAMFR Q9SLY8 4.47/48309 GIQTSEDYRFYAISAEYPEFSNKDK/QSGSIYEHW DILPPK/APMIDNPDFKDDPYIYAFDSLKYIGIE LWQVKSGTLFDNFLITDDPELAI 23343600 5.30/50337 FDWDKIVEENNVIGLK/AVGEEMGIGFLGIGFQPK WALSDIPIMPK/VGSLGLDMMFR/GMLPFVFDDSF GFER/DLAEEILQLSK/LLNLYETK 37535140 5.71/60850 GVEELADAVK/SVAAGMNAMDLR/GISMAVDAVVT NLK/EIGELIAK/EGVITIAD/GNTLYNELEVVEGMK/ GYISPYFVTNPK/VSNLHAVVKVLELALK/QRPLLI VAEDVESEALGTLIINK/KANLQDLAILTGGEVITEEL GMNLEK/SAIELSTSDYDKEK/LLEQDNTDLGYDAA KGEYVDMVKAGIIDPLK/TALVDAASVSSLMTTTES 33113259 5.41/47972 AAVPSGASTGVYEALELR/AVDNVNSVIAPALIGK DPTSQAELDNFMVQQLDGTK/LAMQEFMILPTGAAS FK/MGVEVYHNLK/YGQDATNVGDEGGFAPNIQEN KEGLELLK/VVIGMDVAASEFYNDKDKTYDLNFKEE NNDGSQK/SFVSEYPIVSIEDPFDQDDWEHYAK/V NQIGSVTESIEAVK/SGETEDTFIADLAVGLATGQIK/ 15823775 5.43/51682 AATDKLKQISENEKSGFISLVSRYLSGEAEQIEWS K/IVTEDFLPLPSK/DGWYPPGHGDVFPSLNNSGK /EYVFVANSDNLGAIVDIK/VQLLEIAQVPDEHV NEFK/VLQLETAAGAAIR/TNPSNPSIELGPEFK/ VANFLAR/SIPSIVELDTLK/SGKLEIP 114409 5.85/55281 AAELTTLLESRMTNFYTNFQVDEIGRVVSVGDG IARVYGLNEIQAGEMVEFASGVKGIALNLENENVG IVVFGSDTAIK/TGSIVDVPAGK/VVDALGVPIDG K/KSVHEPMQTGLK/TAIAIDTILNQK/NGMHALI IYDDLSK/EAFPGDVFYLHSR/EVAAFAQFGSDLDA ATQALLNR/AILSTINPELLI 121349 5.51/39201 IIAEYIWIGGSGMDLR/TLSGPVTDPSKLPKWNYD GSSTGQAPGEDSEVILYPQAIFK/DIVDSHYK/ND GGYEIIK/HKEHISAYGEGNER 37805861 5.41/39749 NAIELGLLETLQSAAVAGGGGKAALLTPAEVADKL PSKANPAAADMVDR/LLASYNVVR/WLTPNEDGVS MAALALMNQDKVLMESWYYLKDAVLDGGIPFNKAY GMTAFEYHGTDAR/GGDAILMK/EQGVFHVDMIML AHNPGGK/AAGFTGF 37535388 5.75/35569 GVMLGADQPVILHMLDIPPATESLNGLKMELVDAA FPLLK/VLVVANPANTNALILK/NAIIWGNHSSTQ YPDVNHAYVK/ELVADDEWLNTEFISTVQQR/KMDA 34915190 5.83/41348 DELDIVIPTIR/VPEGFDYELYNR/NALEQHIK/Y VDAVMTVPK/DLIGPAMYFGLMGDGQPIGR/TGLP YIWHSKASNPFVNLK 34911282 5.97/36231 DKSLLVWDLTNPVQNVGEGAGASEYGVPFR/LWDL STGVTTR/FVGHDKDVLSVAFSVDNR/FSPNTFQP TIVSGSWDR/DGVTLLWDLAEGKR/HIVQDLKPEI PVSK 38346061 6.34/36773 VALQSDDVELVAVNDPFITTDYMTYMFKYDTVHGQ WK/NPEEIPWGETGAEFVVESTGVFTDKDK/KVV ISAPSKDAPMFVVGVNEK/FGIVEGLMTTVHAITA TQK/VPTVDVSVVDLTVRLEKPASYDQIK/GILGY VEEDLVSTDFQGDNRS/AGIALNDNFVKLVSWYD NEWGYSSR 34902150 6.26/41693 EGNVVLAPTNPR/IKDEEGNPAFALVNKATGLAIK/ LAPFNPEYPDESVLWTESGDVGK/LNFDAFHGDK/ ILPWGDEAYAGGSANAPR/ADEGFSVTVR/HSNS IKDEEGYPAFALVNR/LVPYNPGYQDESVLWTESR/ DGTTVALWK

functional category Cellular respiration

Cellular respiration Amino acid metabolite



Photosynthesis

Cell wall and membrane biogenesis Protein modification and chaperone Antioxidant system Protein modification and chaperone


Cell wall and membrane biogenesis Cellular respiration

Amino acid metabolite Secondary metabolite biosynthesis

Cellular respiration Cell wall and membrane biogenesis Transcriptional and translational protein Cellular respiration


research articles

Differentially Expressed Proteins/Genes in JA-Treated Rice Table 4. (Continued)

spot no.

protein name

distinct summed MS/MS NCBI % AA distinct search coverage peptides score accession

22

Fructose 1,6-bisphosphate aldolase

43

15

228.12

23

r40g2 protein

25

6

100.99

24

pI/MW (Da)

peptide sequence

41398198 6.96/38863 YKDELIKNAAYIGTPGKGILAADESTGTIGK/FAS INVENVEENRR/TKDGKPFVDVLK/VDKGTIEV AGTEK/IGPNEPSQLAIDLNAQGLAR/KVSPEVIA EYTVR/TVPAAVPAIVFLSGGQSEEEATLNLNAMN K/ALQQSTLK/GDAVLGEGASESLHV 34394517 6.80/38528 DSAVVLAPVNPK/VKDGEGMPAFALVNK/LVPFNP EYEDASVLWTESK/LNLDAFHGDK/DEEGYPAFALVN K/LVPYNPEYQDESVLWTES 34909798 5.77/23977 LYGSTLSWNVTRC/NPFGQVPALQDGDLFLWESR/ VVEENLEK/KVLEVYEAR 7489542 5.42/27155 NYPVVSAEYQEAVEK/TGGPFGTMKTPAELSHAAN AGLDIAVR/EDKPAPPPEGR/QVFGAQMGLSDQDI VALSGGHTLGR/SGFEGPWTRNPLQFDNSYFTELL SGDKEGLLQLPSDKALLSDPAFRRLVEK/AFFEDY KEAHL 7489542 5.42/27155 NYPVVSAEYQEAVEK/TGGPFGTMKTPAELSHAAN AGLDIAVR/QVFGAQMGLSDQDIVALSGGHTLGR/ SGFEGPWTR/EGLLQLPSDKALLSDPAFRPLVE 16566704 5.02/35516 LGGSSAFVGKFGDDEFGHMLVDILKK/TALAFVTL K/ADMLLTEAELNLDLIRRLPLWPSEDAARAGILS IWKEADFIKVSDDEVAFLTQGDANDEKNVLSLWFD GLKLLIVTDGEK/DDSIFHNEEK/KGAIPALPTVA VAQELISI 29367547 5.57/40232 LNNAILAEEKHLPMYDELASKGNVEYIAGGATQNS IR/KPENWALVEK/VLPFVDYIFGNETEAR/VRGW ETENVEEIALKISQLPLASGK/IAVITQGADPVVV AEDG 34394950 4.98/58545 DASNLIDPPEVVKLLNANSITMVRIYDTDPTVLNA LANTGIK/DLASAGADVGSATNWVK/VSTPIAFD ALDVSFPPSDGR 16580747 5.51/32539 SPEVVLEWPKK/YDIGAGFGHFAIATEDVYK/YTI AMLGYADEDK/GNAYAQVAIGTEDVYKSAEAVELV TK/IASFLDPDGWKVVLVDNADFLK 34394950 4.98/58545 DASNLIDPPEVVK/DLASAGADVGSATNWVK

functional category Photo synthesis


Glutathione S-transferaseII peroxidase

22

5

75.72

25

L-ascorbate

56

11

165.83

26

L-ascorbate

peroxidase

40

7

100.9

27

Putative fructokinase II

43

16

252.43

28

Adenosine kinase-like protein

29

9

156.96

29

Putative beta-1,3-glucanase

14

5

86.94

30

Glyoxalase I

31

8

121.31

31

Putative beta-1,3glucanase

5

2

30.92

32

Citrate synthase

23

7

107.03

33

DnaK-type molecular

18

8

120.97

34

Ascorbate peroxidase

52

10

138.17

35

Putative actin depolymerizing

34

3

44.43

36

Superoxide dismutase [Cu-Zn]1 Probable germin protein 4 DnaK-type molecular

16

2

35.75

11066954 7.71/52228 GMTGMLWETSLLDPDEGIR/DGEPLPEGLLWLLLT GKVPTKEQVDALSK/TIAADNALDYAANFSHMLGF DDPK/SVIGETGSDVTTDQLK/SIGIGSQLIWDRALGL 1076746 5.13/71113 VEIIANDQGNRT/NQVAMNPINTVFDAK/FSDASV Protein modification QSDIK/QFAAEEISSMVLIK/DAGVIAGLNVMRI and chaperone INEPTAAAIAYGLDK/ATAGDTHLGGEDFDNR/E QVFSTYS 11094301 5.21/27117 SYPTVSDEYLAAVGK/TGGPFGTMKNPGEQSHAAN Antioxidant system AGLDIAVR/QDKPEPPPEGRLPDATQGSDHLRQVF SAQMGLSDKDIVALSGGHTLGR/EGLLQLPSDKAL MADPAFRPLVEKYAADEDAFFADYAEAHLK 29124123 5.72/15946 FAIYDFDFLTAEDVPK/MLYASSNER/ELNGIQLEVQ Cell wall and ATDAGEISLDALK membrane biogenesis 134595 5.71 AVVVLGSEIVK/AVVVHADPDDLGK Antioxidant system

17

3

46.64

34902528 7.76

11

5

64.93

39

Putative disulfideisomerase precursor

33

9

130.62

40

Putative glyceraldehyde 3-phosphate dehydrogenase

34

11

165.5

41

Actin

25

7

110.64

42

Putative glutathione S-transferase

37

7

104.68

43

Probable superoxide dismutase 2 precursor

39

8

139.28

44

58

6

102.08

45

Translationally controlled tumor protein homologue Glycine-rich protein

24

4

61.11

46

Glycine-rich protein

16

2

36.13

47

Tau class GST protein 4

36

9

139.19

48

Putative UDP glucose dehydrogenase

38

13

221.07

37 38

IDYAPGGQNPPHTHPR/TVTAGEVFVFPR/VDVPQVD AIK 1076746 5.13/71113 VEIIANDQG/NQVAMNPINTVFDAK/QFAAEEISSM VLIK/IINEPTAAAIAYGLDK/ATAGDTHLGGEDFDNR 34897646 6.58/39912 TAEALAEYVNSEAATNVKIAAVPSSVVVLTPETFD SVVLDETK/HLAPIYEK/QDEGVVIANLDADKHTA LAEKYGVSGFPTLK/AGEDYDGGRELDDFVK/GQL TSEAGIVESLAPLVK/ADEFVIK 29150193 6.41/42052 YDTVHGQWK/NPEEIPWGETGAEFVVESTGVFTD KDK/KVVISAPSKDAPMFVVGVNEK/VPTVDVSVV DLTVRLEKPASYDQIKA/GILGYVEEDLVSTDFQG DNRSSIFDAKAGIALNDNFVKLVSWYDNEWGYSSR 37535954 5.31/41680 AGFAGDDAPR/YPIEHGIVSNWDDMEKIWHHTFY NELRVAPEEHPILLTEAPLNPK/GYSFTTSAER/L AYVALDYEQELETAK/SYELPDGQVITIGAER 37536360 5.82/25656 SLPYEYVEENLGDKSDLLLASNPVHK/FWAAYV/G KPFFGGDGVGFVDVVLGGYLGWFTAIDK/TPALAA WEER/GVVPDDADKLLEFR 7433347 6.50/24999 HHATYVANYNKALEQLDAAVAKGDAPAIVHLQSAI K/LGWAIDEDFGSFEALVK/KLSVETTANQDPLVT K/NVRPDYLSNIWK/YAGEVYENATA 549063 4.51/18946 MLVYQDLLTGDELLSDSFPYREIENGILWEVDGKW VVQGAIDVDIGANPSAEGGGDDEGVDDQAVKVVDI VDTFRLQEQPPFDK/DGATDPTFLYFSHGLK 2331131 7.80/15872 LEAAFSTYGEILDSK/VNEAQSR/GGGYGGDSGG NWRN 2331131 7.80/15872 SLEAAFSTYGEILDSK/VNEAQSR 33304610 4.97/25591 WPSPFVTR/GLSYEYVK/ELLLASNPVHK/QLLAA VETLEGALK/APLLAAWAQRFGELDVAEKVLPDV DGVVEFAK/LAEAAAAAAAASKN 13236672 5.79/52899 IDAWNSEQLPIYEPGLDEVVK/HVAEADIIFVSVN TPTK/AADLTYWESAAR/GINYQILSNPEFLAEGTA IDDLFKPDR/SVYAHWVPEDRIITTNLWSAELSKL AANAFLAQR/VVSSMFNTVSGK/IAVLGFAFK/AQ ISIYDPQVTEDQIQR/QVSVVWD AYEATK/IYDN MQKPAFVFDGRNVVDPEKLR

Antioxidant system Antioxidant system

Antioxidant system Cellular respiration

Transcriptional and translational protein Cell wall and membrane biogenesis Defense-related protein Cell wall and membrane biogenesis Cellular respiration

Defense-related protein Protein modification and chaperone Protein modification and chaperone Celluar respiration

Cell wall and membrane biogenesis Antioxidant system Antioxidant system Defense-related protein Transcriptional and translational protein Transcriptional and translational protein Antioxidant system Cell wall and membrane biogenesis


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Cho et al.

Table 4. (Continued)

spot no.

protein name

distinct summed MS/MS % AA distinct search NCBI coverage peptides score accession

pI/MW (Da)

peptide sequence

49

Putative leucine aminopeptidase

27

14

225.27

47497219

8.29/61819

50

Methylmalonate semi-aldehyde dehydrogenase

32

12

198.08

34394614

5.99/57246

51

Putative glutathion S-transferase Glutamine synthetase root

24

5

76.76

37536312

5.71/25332

20

6

90.27

121332

5.73/39258

53

ESTs D24970, AU031961

35

14

202.68

34897870

5.85/52721

54

GF14-c protein

48

10

156.31

7435022

4.78/28826

55

Elongation factor 1-beta

42

8

126.43

6166140

4.36/24862

52

a

DVEFSEWKGDILAIAVTENDLVK/FENAVLKKLDG QLGGLLSEASAEEDFTGK/GIGESVASVAK/QVDL IGFGSGPEVDQK/YANDLSSGVIFGK/GLTFDSGGY NIK/FDMGGSAAVFGAA/TIEVNNTDAEGR/EVAA ASEISGEK/MPLEESYWESMKS LLIGGEFVESRADEHVDVTNPATQEVVSRIPLTTA DEFR/LAENITTEQGK/DAWGDVFR/DPGAAMMLA ELAMEAGLPK/ASSLVVNSGMASDADLGPVISK/DI VVPNFENGNFVGPTLLADVK/EEIFGPVLLLMKAES LDDAIQIVNR/YGNGASIFT GLSYEYVEEDLKNKSELLLTSNPVHK/FWAAYIDDK/ ETFAAVANLEAAFK/SPLLDAWLDR TVKGPITDVSQLPKWNYDGSSTGQAPGEDSEVIL YPQAIFK/DIVDAHYK/HKEHIAAYGEGNER/HET ADINTFK GFPISVY/GLLYLGMGVSGGEEGAR/YIEDILLK/ MVHNGIEYGDMQLISEAYDVLK/LTNSELQQVFSEW NK/FLSGLKDER/VFQGDFSSNLPVDKAQLIEDVR /GWSLNLGELAR/NSDLANLLVDPEFAQEIMDR/D RLPANLVQAQRDYFGAHTYERV LAEQAERYEEMVEYMEK/TVDVEELTVEER/GKIE AELSK/LLDSHLVPSSTAAESK/AAQDIALADLAPT HPIR/QAFDEAISELDTLGEESYKDSTLIMQLLRD NLTLWTSDLTEDGGDEVKEASK KLDEYLLTR/LSGVTADGQGVKVESTAVPSASTPD VADAKAPAADDDDDDDVDLFGEETEEEKK/SSVLL DVKPWDDETDMTKLEEAVR/MEGLLWGASK

functional category Protein modification and chaperone

Secondary metabolite biosynthesis Antioxidant system Amino acid metabolite Defense-related protein

Transcriptional and translational proteins Transcriptional and translational protein

These proteins are indicated with small r (for root) throughout the text.

results show that, among these identified glycolysis proteins, root protein spots are decreased and shoot protein spots are mostly increased, which might account for their growth differences. In addition, increased spot s10 in the shoot was identical to putative oxalyl-CoA decarboxylase. The protein catalyzes oxalylCoA into formyl CoA and CO2 and has 62% positive homology with 2-hydroxyphytanoyl-Coenzyme A lyase participating in the breakdown of long chain 2-hydroxyfatty acids.67 Spot r50 decreased in root was identified as methylmalonate semialdehyde dehydrogenase (MMSA dehydrogenase) that catalyzes oxidative decarboxylation of malonate and MMSA into acetyland propinoyl-CoA, respectively. MMSA dehydrogenase, whose products (NADH, acetyl-CoA, and propinoyl-CoA) inhibit the β-oxidation of fatty acid, participates in catabolic pathway of branched-chain amino acids (valine, leucine, and isoleucine) for cellular energy production.68 Thus, although oxalyl-CoA decarboxylase and MMSA dehydrogenase are reversely regulated in different tissues, it appears that cellular energy in JAtreated rice seedlings may be generated preferentially from β-oxidation of fatty acid than carboxylation of amino acids. 3.2.2.2. Acetyl-CoA Oxidation. Three spots identified as putative aconitate hydratase were increased (s1 and r1) and decreased (spot r2) in shoots and roots. Aconitate hydratase catalyzes the interconversion of citrate into isocitrate in citric acid cycle or glyoxylate cycle. Cytoplasmic aconitate hydratase of Cucurbita maxima, which has 95% positive homology with spot s1, r1, and r2, was reported to participate in the glyoxylate cycle used to convert acetate to carbohydrate.69,70 In roots, increased spot R18 was identified as mitochondrial malate dehydrogenase that reversibly catalyzes malate and NAD into oxaloacetate and NADH in citric acid cycle. Decreased spot r32 was found to be identical to citrate synthase, which catalyzes acetyl-CoA and oxaloacetate into citrate and CoA. Citrate synthase and malate dehydrogenase in mitochondria of strawberry was reported to play potential roles as important determinants of ripening via regulation of the sugar/ acid balance.70,71 The result suggests that a combination of 3594


citrate synthase and malate dehydrogenase in roots of JAtreated rice seedlings may regulate root growth. Furthermore, cytoplasmic malate dehydrogenases (spots s31 and r17), which were decreased in both shoots and roots, may participate in gluconeogenesis in the cytosol. The enzyme in the cytosol mesophyll cell of C4 plants is used for the fixing of CO2 to pass bundle-sheath cells, where CO2 is released and is fixed by RuBisCO for photosynthetic carbon reduction.38 Cytoplasmic malate dehydrogenase has been shown to be involved in rice leaf sheath elongation process.72 Therefore, a decrease in spots s31and r17 may be an important aspect of the causes for growth inhibition after JA treatment. 3.2.3. Transcriptional and Translational Factors. JA treatment induced the differential expression of 4 translation-related spots (S10, s5, s23, and s48) in shoots. Spots s5 and S10 were increased and respectively identified as OSJNBa0020P07.3, which has 100% positive homology with transcription elongation factor (EF: NCBI accession number ABL74570), and putative plastid-specific ribosomal protein 2 precursor (S10), which has 79% positive homology to chloroplast plastid-specific 30S ribosomal protein 2 precursor (PSRP-2, Spinacia oleracea). PSRP-2 has an important role in translating chloroplast mRNAs for active protein synthesis.73,74 Spots s23 and s48 were decreased and identical to EF-Tu (s23) and putative ribosomal protein L12 (s48), respectively. Spot s23 has 99% positive homology with chloroplast EF-Tu, which plays an essential function in the elongation of chloroplastic mRNA translation and is highly expressed in the leaf.75 Ribosomal protein L12 (spot s48) also has 77% homology to chloroplast 50S ribosomal protein L12-2 precursor from Secale cereale.76 Moreover, decreased transcription-related protein spot s53 was identified as putative chloroplast 29 kDa ribonucleoprotein precursor, which has 76% positive homology with single-stranded DNA binding protein from Solanum tuberosum. The protein is homologous with silencing element binding factor that represses the potato PR-10a involved in response to pathogen infection or elicitor treatment.77 Thus, JA may affect the


research articles

Figure 5. Distribution of 98 identified nonredundant proteins in JA-treated shoots and roots. In total, 134 protein spots were excised from 2-D gels of shoot and root proteins, stained with colloidal CBB, and analyzed by nLC-IT-TOF-MS/MS or nESILC-MS/MS. One-hundred eight proteins were unambiguously identified, representing 98 nonredundant proteins; 10 proteins overlapped between shoots and roots.

expression of PR proteins in shoots. However, the regulation of several translation factors by JA is still poorly understood. In roots, spots r45 and r46 identical to a glycine-rich protein were increased and decreased, respectively. The glycine-rich protein has 98% positive homology with glycine-rich RNAbinding protein 1A (GRP1A) related with disease resistance,78 and 94% homology with GRP1A of Sinapis alba, which plays a general role in circadian phenomena associated with meristematic or growing tissues.79 Increased spot r54 (GF14-c protein) is a 14-3-3 homologue involved in growth factor signaling and interaction with MEK kinases. GF14-c protein has 100% positive homology with 14-3-3-like protein GF14-C from rice, which is associated with a DNA binding complex that binds to the G box (a well-characterized cis-acting DNA regulatory element). The GF14-C protein has been shown to be induced by various stresses such as wounding, drought and salt stresses, methyl JA, hydrogen peroxide (H2O2), abscisic acid (ABA), and rice blast fungus and bacterial blight.80,81 Spot r55 (translational elongation factor 1-beta) was increased in roots. The protein has 88% positive homology with translational elongation factor 1 subunit 1B beta from Pisum sativum that interacts with a cyclin-dependent kinase involved in regulating progression of meiotic and mitotic cell cycles.82,83 Furthermore, 2 decreased spots r19 and r28 were identified as guanine nucleotide-binding protein beta subunit-like protein (GPB-LR) and adenosine kinase-like protein, respectively. GPB-LR contains WD40 domain involved in adaptor/regulatory modules in signal transduction, pre-mRNA processing, and cytoskeleton assembly, and is expressed constitutively in all organs of rice plants.84 The adenosine kinase-like protein (r28) is associated with stress response and seed development.85 Interestingly, from these results, it can be suggested that JA regulates putative development- and stress response-related proteins in roots. 3.2.4. Protein Modification and Chaperone. The increased spots S12 and r8 identified as calreticulin precursor have 100% identical homology to each other. Calreticulin has been implicated in folding and oligomeric assembly of glycoproteins in endoplasmic reticulum (ER), and was suggested to be involved in developmental regulation of rice cultured suspension cells.86 Five protein spots (s11, s12, r4, r33, and r38) were identified as a DnaK-type molecular chaperone HSP70 (heat

Figure 6. Identified proteins in shoots and roots belonged to 10 functional categories. The pie chart shows the distribution of redundant proteins identified in shoots (66) and roots (68) into their functional classes in percentage.

shock protein), where spots s11 and r38 were increased and s12, r4, and r33 were decreased by JA treatment. The DnaKtype molecular chaperone HSP70 has 96% positive homology with glyoxysome-bound HSP from Cucumis sativus known to be involved in the interaction with cytosolic HSP70.87 Spots s6 and S9 found to be increased in shoot were identified as OSJNBa0039C07.4 and proteasome component C2, respectively. OSJNBa0039C07.4 (s6) has 94% positive homology with ATP-dependent protease from Solanum lycopersicum, which participates in energy-dependent proteolysis to regulate the availability of short-lived regulatory proteins, to ensure the proper stoichiometry for multi-protein complexes, and to rid the cell of abnormal proteins.88 Proteasome component C2 (S9) is a participant of the cell proliferation-related proteasome, a multicatalytic proteinase complex, which degrades ubiquitinconjugated proteins with an ATP-dependent proteolytic activity.89 Decreased spots s7, s13, and s14 were identified as HSP (s7), a putative RuBisCO subunit-binding protein subunit (CPN60 alpha; s13), and a putative CPN60 beta (s14). HSP (s7) is involved in stress response and seed development and has 86% positive homology with stromal 70 kDa HSP from P. sativum, which is implicated in protein transport and a variety of cellular processes such as DNA replication and protein folding.85,90 CPN60 is composed of alpha and beta subunits and is implicated in the folding and assembly of multimeric proteins in plastids. CPN60 beta has 91% positive homology with RuBisCO large subunit-binding protein subunit beta from A. Journal of Proteome Research • Vol. 6, No. 9, 2007 3595

research articles thaliana. These results suggest that CPN60 alpha (s13) and CPN60 beta (s14) may be involved in assembly of RuBisCO subunits.91,92 If so, the decrease of these proteins may result in the inactivity of RuBisCO, leading to a decrease in the efficiency of photosynthesis and finally affecting the growth of rice plant. A putative disulfide-isomerase precursor (r39) and trypsin (R15) increased in the root. Protein disulfide-isomerase from Z. mays, which has 93% positive homology with spot r39, is one of the molecular chaperones that assist formation of proper disulfide bonds during protein folding.93 Trypsin catalyzes inactive precursor zymogens into active forms by proteolysis.94 Furthermore, a mitochondrial CPN60 (r10) and a putative leucine aminopeptidase (r49), were found to be decreased in roots. CPN60-1 from Z. mays, which has 98% positive homology with spot r10, is implicated in import and correct assembly of mitochondrial protein under seed germination and heat stress.95 Putative leucine aminopeptidase having 100% identical homology with chloroplastic leucine aminopeptidase 2 is involved in the processing and regular turnover of intracellular proteins.18 3.2.5. Cell Wall and Membrane Biogenesis. Spots S11 and r7 that were increased and decreased, respectively, were identified as beta-5 tubulin and beta-tubulin R2242, respectively. β-Tubulin is the basic component of microtubulines, which play important roles in cell division and elongation in plants. The inhibition of polymerization of β-tubulin by the toxin of rice seedling blight reduces root growth and finally causes plant death.96,97 These results may provide a crucial clue for understanding why root growth is more inhibited than shoot growth. In roots, 2 spots were identified as putative actin depolymerizing factor (r35) and actin (r41). Decreased putative actin depolymerizing factor (r35) has 100% identical homology with actin-depolymerizing factor 4 (ADF-4). ADF-4, one of the small actin-binding proteins that regulates actin dynamics in cell, enhances the turnover rate of actin and interacts with actin monomers as well as actin filaments.98 Increased spot r41, which has 100% identical homology with actin-2, is an important determinant in cytoplasmic streaming, cell shape determination, cell division, organelle movement, and extension growth.99 Actin-7 from Arabidopsis with 99% identical homology to spot r41 is involved in the regulation of hormone-induced plant cell proliferation and callus formation.100,101 Moreover, cell wall synthesis-related proteins were identified in roots. Two increased spots (r29 and r31) were identical to putative beta1,3-glucanase related to plant growth.102 Decreased spot r18 was identical to a reversibly glycoylated polypeptide and has 96% positive homology with alpha-1,4-gulcan-protein synthase from Z. mays involved in the synthesis of cell wall polysaccharides.103,104 In addition, 2 decreased spots were identified as UDP-glucose pyrophosphorylase (r12) and UDP-glucose dehydrogenase (r48). UDP-glucose pyrophosphorylase catalyzes UTP and glucose 1-phosphate into diphosphate and UDPglucose, which is subsequently oxidized into UDP-glucuronic acid by UDP-glucose dehydrogenase. UDP-glucuronic acid is a precursor for sugar nucleotides, which are needed for the biosynthesis of many components of hemicellulose, a major component of primary plant cell walls. UTP-glucose-1-phosphate uridylyltransferase from H. vulgare, which has 96% positive homology with spot r12, was reported to join reactions of sucrose metabolism and starch synthesis in this tissue.105 Moreover, putative UDP-glucose dehydrogenase 1 from Nicotiana tabacum and UDP-glucose 6-dehydrogenase from Gly3596


Cho et al.

cine max, which have, respectively, 96 and 95% positive homology with spot r48, showed high expression in tissues undergoing secondary wall synthesis.106,107 3.2.6. Amino Acid Metabolism. Spot S3 identified as methionine synthase was found to be increased in shoots. Methionine synthase catalyzes the formation of methionine by the transfer of a methyl group from 5-methyltetrahydrofolate to homocysteine at the last step in methionine biosynthesis. Nutritional starvation has been shown to induce a strong, transient transcriptional activation of methionine synthase in the Catharanthus roseus cell culture.108 Two increased spots were found to be identical to S-adenosylmethionine synthetase 1 (SAM synthetase 1, s20) and OSJNBa0011P19.5 (s21) that has 100% identity with SAM synthetase 2. SAM synthetase catalyzes the formation of SAM, a universal methyl group donor, from methionine and ATP. Methionine synthase and SAM synthetase are participants in the biosynthetic pathway of Asp-derived amino acids such as ethylene and polyamine, which are, respectively, a curial phytohormone for plant germination and an important component in the regulation of cell proliferation and differentiation. Indeed, inhibition of methionine biosynthesis during seed germination in Arabidopsis was shown to inhibit the seedling growth.109 Therefore, these results indicate that, due to an increase of methionine synthase and SAM synthetase in shoot, the growth of JA-treated shoot may be less inhibited than that of JA-treated root. In roots, decreased spot r3 was identified as putative aspartate-tRNA ligase involved in aspartate synthesis. Cytosolic glutamine synthetase isozyme 1-1 (r15) and glutamine synthetase root isozyme (r52), which catalyze glutamate using ATP and NH3 into glutamine, were also decreased. In cytosolic glutamine synthetase-deficient plants, the reduction of leaf blades elongation, plant height, panicle size, and grain filling has been reported.110 This study supports our finding that JA treatment caused reduction in seedling growth, where aspartate-tRNA ligase and glutamine synthetase proteins were decreased and might play in role in the observed growth reduction in roots. 3.2.7. Secondary Metabolism. Among the enzymes involved in secondary metabolism, we identified several proteins involved in the biosynthesis of chlorophyll and compounds of the phenylpropanoid pathway. A putative chloroplast glutamate1-semialdehyde 2,1-aminomutase (spot s22) was decreased in shoots. Glutamate-1-semialdehyde 2,1-aminomutase, which catalyzes (S)-4-amino-5-oxopentanoate into 5-aminolevulinate, has 94% positive homology with glutamate-1-semialdehyde 2,1aminomutase from H. vulgare. Aminolevulinate is the universal precursor for the biosynthesis of tetrapyrroles involved in the biosynthesis of chlorophyll and heme.111 Thus, the decrease of spot s22 in shoots may induce the reduction of chlorophyll biosynthesis thereby decreasing the photosynthetic efficiency. Furthermore, 3 putative phenylalanine ammonia-lyases (PAL; s9, R16, and R17) were increased in shoots and roots. The PAL catalyzes deamination of phenylalanine for production of cinnamate, the first reaction in the biosynthesis of phenylpropanoid, an important secondary metabolite involved in response to various stresses.112,113 Spot r16 was decreased in roots and identified as putative caffeic acid 3-O-methyltransferase, which has 100% identical homology with quercetin 3-Omethyltransferase 1 that methylates OH residues of flavonoid compounds in phenylpropanoid pathway.114 It should be noted that JA was reported to induce the accumulation of sakuranetin, a major phytoalexin in JA-treated rice, which is produced from


methylation of naringenin by naringenin 7-O-methyltransferase (NOMT) with substrate specificity.115,116 3.2.8. Defense-Related Proteins. Plant responses to abiotic and biotic stresses involve the expression of a large number of proteins, many of which are believed to be crucial components of the plant’s self-defense mechanism and used widely as marker(s) to study the defense mechanism.25 In the shoot, spot s46 was increased, whereas spots s43 and s47 were decreased. Spots s46 and s47 are, respectively, identical to putative r40c1 protein and r40g2 protein, previously reported to be involved in ABA- and salt stress-response in rice.117 Spot s43 was identified as OSJNBb0048E02.12, which has 94%, 89%, and 89% positive homology with salt tolerant protein (NCBI accession number AAY26389) from Triticum aestivum, PR protein 2 from Z. mays,118 and Bet vI allergen (NCBI accession number AAV28626) from Z. mays, respectively. In roots, 3 spots (r21, r23, and r30) were decreased, and 7 spots (R13, R20, R21, R23, r37, r44, and r53) were increased. Spots r21 and r23 in root are identical to s46 (putative r40c1 protein) and s47 (r40g2 protein) in shoots, respectively. Spot r30 was identified as glyoxalase I, which has 100% positive homology with lactoylglutathione lyase. Lactoylglutathione lyase catalyzes glutathione and methylglyoxal into S-lactoylglutathione, causes an allergic reaction in human, and induces salt tolerance.119 Interestingly, the results showed that the upregulated proteins by salt stress are down-regulated by JA except for r40c1, which is root-specifically up-regulated by salt stress in rice. These results provide cues for further study on the reverse relationship between salt stress pathway and JA downstream pathway, and the effect of different regulation of r40c1 in shoot and root on rice development. Spot R13 is identical to lipoxygenase 2. Lipoxygenase 2 catalyzes the hydroperoxidation of fatty acid containing a cis,cis-1,4-pentadiene motif and initiates biosynthesis of oxylipin, which is involved in a number of diverse aspects of plant physiology including growth and development, pest resistance, and senescence or responses to wounding.120 Spot R20 was identified as putative Chitinase with 76% positive homology. Chitinase able to digest chitin, the main component of cell wall, is induced by phytohormones such as dichlorophenoxyacetic acid and benzyl adenine.121 Spot R21 was identified as Prb1, which has 72% positive homology with type-1 PR protein from H. vulgare, whose transcription level is increased after pathogen inoculation.122 Probenazole-induced protein (PBZ; spot R23) was identified. PBZ1, which has 100% identity with spot R23, is markedly transcripted not by wounding but by inoculation with rice blast fungus, and plays an important role during the disease resistance response in rice.123 Spot r37 is identical to probable germin protein 4, which has 82% positive homology with germin-like protein 6a from H. vulgare ssp. vulgare. Germin-like protein 6a, which has a role of defense against pathogen attack, is expressed highly in very young seedlings.124 Spot r44 was identified as translationally controlled tumor protein homologue involved in calcium binding, microtubule stabilization, and disease resistance.78 Spot r53 has 46% positive homology with polyketide synthase type I from Streptomyces aizunensis that has potent antifungal activity.125 The abovememtioned JA up-regulated proteins are involved in activation of immune system of plants as well as inducers of programmed cell death, the composition change of membrane and cell wall, or limitation of plant growth. Therefore, the results suggest that root growth might be strategically limited to activate JAinducible defense mechanism.

research articles 3.2.9. Antioxidant System. To reduce oxidative injuries induced from reactive oxygen species (ROS) such as superoxide anion (O2-), hydrogen peroxide (H2O2), and hydroxyl radical (OH-), plants have developed enzymatic systems for scavenging the ROS.126,127 These include superoxide dismutase (SOD, EC 1.15.1.1), catalyzing the conversion of O2- to oxygen (O2) and H2O2, catalase (EC 1.11.1.6), peroxidase (EC 1.11.1.7), and ascorbate peroxidase (APX, EC 1.11.1.11) which convert H2O2 to water and O2, and serve together as frontline enzymatic antioxidant defenses from yeast to complex organisms like humans and plants. Three spots (s49, s51, and s52) identified as catalase were decreased in shoots. These were identified as putative catalase (s49), putative catalase A (s51), and catalase A (s52). Catalase is involved in the protection of cells from the toxic effects of peroxides. Catalase isozyme A, which has 100% identity with spots s51 and s52, is expressed at high levels in seeds during early development and also in young seedlings.128,129 Increased spot S6 and decreased spot s54, which were identified as dehydroascorbate reductase (DHAR), have 100% identity to each other. DHAR catalyzes regeneration of ascorbic acid from its oxidized form. Ascorbate is essential for the detoxification of environmental toxins and products of oxidative stress.130 Five decreased spots (R14, r24, r42, r47, and r51) related to isoforms of glutathione S-transferase (GST), which catalyzes conjugation of reduced glutathione to hydrophobic electrophiles, were identified. Among these proteins, decreased spot R14 was identified as probable GSTF2, which has 100% identical homology with spot r24 (GST II) that is constitutively expressed in roots, anthers, callus, panicles, sheaths, and stems and induced by the herbicide safener fenclorim and drought stress.131,132 Three spots were identified as putative GST (r42), tau class GST protein 4 (r47), and putative GST (r51). Spot r42 has 100% identity with probable GSTU6 (28 kDa cold-induced protein) involved in chilling tolerance.133 Tau class GST protein 4 (r47) and tau class GST protein 3, which have 100% positive homology with spot r51, are induced in roots by hypoxic stress and regulated by redox perturbations and ROS.134 Moreover, 4 spots were identified as isoforms of peroxidase such as cationic peroxidase (R19), L-APX (r25, r26), and APX (r34). Peroxidase participates in a multistep oxidative reaction involving H2O2 as the electron acceptor. Cationic peroxidase (R19), which was increased in root, has 96% positive homology with class III peroxidase precursor involved in the mechanism of cell elongation, cell wall construction and differentiation, and defense against pathogens.135 Decreased L-APX (r25 and r26) and increased APX (r34) have, respectively, 100% identical homologies with cytosolic L-APX1 and cytosolic L-APX2 that are induced by stress, hormones, and infection with rice blast fungus and expressed by circadian-regulation.136-138 Furthermore, decreased spots r36 and r43 were identified as cytosol SOD (Cu-Zn SOD1) and mitochondrial SOD2, respectively. The SOD destroys radicals that are normally produced within cells.139,140 Decreased spot r9 was identified as gammaglutamylcysteine synthetase. Putative gamma-glutamylcysteine synthetase from Z. mays, which has 97% positive homology with spot r9, is imposed in leaf redox state in response to shortterm chilling stress by control of glutathione synthesis.141 Differential regulation of these antioxidative systems suggest that JA is intimately involved in their regulation in rice. We could observe decrease not increase in these protein spots, suggesting that the regulation of ROS-inducible mechanism is involved in not only regulation of photosynthesis, pathogen Journal of Proteome Research • Vol. 6, No. 9, 2007 3597

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Figure 7. The effect of JA on genes expressed in roots is more prominent than genes expressed in shoots. In total, 107 and 325 genes were induced in shoots and roots, respectively, and to our surprise, only 22 genes overlapped. In total, 34 and 213 genes were suppressed in shoots and roots, respectively, and to our surprise, only 5 genes overlapped.

response, and hormonal action, but also plant growth and development.127 3.3. Expression Profiling of JA-Influenced Genes. In the second part of our study, we checked the transcriptional levels of approximately 22 000 rice genes (cDNA clones) using DNA microarray chip, in order to analyze the genes related in the response to JA in rice seedlings. After normalization (Supplementary Figure 2 in Supporting Information), we summarized the genes whose transcriptional levels were altered more than 2-fold in shoot and root of JA (5 µM)-treated rice seedlings over the control (the gene lists are presented in the microarray data list of Supplementary Tables 2-5 in Supporting Information). The results showed that 107 and 325 genes are, respectively, induced in shoots and roots, and 34 and 213 genes are reduced (Figure 7). By the use of the COG database (section 2.6), these genes were categorized according to their function (Figure 8). Finally, the analysis by RT-PCR of some JA-induced and JAsuppressed genes reconfirmed the reliability of microarray data (Figure 9). 3.3.1. Overview of JA-Regulated Genes in Shoots and Roots. Functional categorization of genes using the COG database divided the identified genes into 3 groups such as information storage and processing, cellular process and signaling, and metabolism groups (Figure 8). It is clear that among 107 and 325 induced, and 34 and 213 suppressed genes in shoots and roots, respectively, 62 and 215, and 22 and 147 genes are unidentified or poorly characterized. Interestingly, the induced genes in shoots of JA-treated rice seedlings are mainly divided into secondary metabolites biosynthesis, transport and catabolism, lipid transport and metabolism, carbohydrate transport and metabolism, and amino acid transport and metabolism groups. The suppressed genes are mostly classified into carbohydrate transport and metabolism group. In roots, the upand down-regulated genes are commonly categorized into posttranslational modification, protein turnover and chaperones, energy production and conversion, carbohydrate transport and metabolism, lipid transport and metabolism, and secondary metabolites biosynthesis, transport, and catabolism groups. It was also seen that induced but not suppressed genes were highly represented in the signal transduction mechanism- and amino acid transport and metabolism-related gene categories. We discuss below both the novel genes/categories regulated by JA, and the genes that are matched with the proteomics data. 3.3.1.1. Novel Regulation of 14 Transporter-Related Genes by JA. Transcript profiling showed that the expression of various transporter-related genes, which could not be provided by 2-DGE, are regulated in JA-treated rice seedlings. This is a novel finding of the present study. Among transporter-related genes, AK102404 (putative oligopeptide transporter, isp4-like protein) and AK105635 (putative nitrate transporter) were up3598


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regulated, and AK071310 (putative oligopeptide transporter, isp4-like protein) was down-regulated in shoots. In roots, genes encoded by AK065895 and AK072119 (peptide transporters), AK072531 (tonoplast membrane integral protein), and AK107681 (iron regulated metal transporter) were up-regulated, whereas AK102404 (isp4-like protein), AK068409 (nitrate transporter), AK107204 (putative ammonium transporter), AK107848 (copper transporter), AK065426 (ABC-type transport protein-like protein), AK067556 (putative ABC transporter), and AK073531 (delta-type tonoplast intrinsic protein) were down-regulated. These genes seem to be mainly involved in amino acid or peptide transport. Although the correlation between plant response to JA and these transporters is poorly reported, our results imply that JA is a regulator of transporters in plants. 3.3.1.2. Cytochrome P450s. Cytochrome P450 catalyzes the biosynthesis of epoxy fatty acid. The main product of linoleic acid epoxidation with cytochrome P450 is (9Z)-12,13-epoxy9-octadecenoic (vernolic) acid.142 The epoxy fatty acids are converted into diols or triols by epoxide hydrolase.143 Epoxy fatty acids and products of epoxide hydrolase are known as plant defensive compounds against pathogenic fungi.143-145 Epoxides like 9,10-epoxystearate as well as the corresponding vicinal diols and their ω-hydroxyderivatives are cutin monomers which are components of cuticle, the first barrier between the plant and its environment, and plays a key role in providing protection against mechanical damage, ultraviolet radiation, or penetration of xenobiotics and pathogens.6,146 Among the cytochrome P450-related genes, AK060486 and AK100163 (cytochrome P450s), AK103336, AK065238, and AK065302 (putative cytochrome P450), AK109775 (cytochrome P450-like protein), and AK109504 (cytochrome P450-dependent fatty acid hydroxylase) were up-regulated in shoots. In roots, AK060563 (epoxide hydrolase) was up-regulated, whereas AK109676 and AK099924 (cytochrome P450s) and AK110852 (putative cytochrome P450) were down-regulated. This result implies that JA is involved in mediating external signals to trigger a defensive response in plants.10 3.3.2. Comparative Analyses of Proteomics and Transcriptomics Data Sets. First, in the cellular respiration group for energy production, microarray data showed the up-regulation of pyruvate decarboxylase 1 (AK100678) and isocitrate lyase (AK063353) in roots. Pyruvate decarboxylase catalyzes pyruvate into actyl-CoA, which participates in citric acid cycle or glyoxylate cycle, and isocitrate lyase catalyzes isocitrate into glyoxylate and succinate in glyoxylate pathway.70 2-DGE analysis showed that putative aconitate hydratase (spots r1 and r2), which catalyzes the interconversion of citrate into isocitrate, was regulated in roots. Thus, results indicate that the glyoxylate cycle may participate in the JA response to regulate plant growth. Furthermore, the transcription of GAP dehydrogenase subunit (AK071685) is down-regulated. However, 2-DGE analysis indicated that cytosolic GAP dehydrogenases (spots r20 and r40) were increased in roots. To clarify this discrepancy, we checked amino acid sequence of the product encoded by the cDNA clone AK071685. It was found that the amino acid residues of AK071685 have 98% positive homology with chloroplastic GAP dehydrogenase, which participates in photosynthesis. The result shows inhibition of photosynthesis in JAtreated rice seedlings, which correlates well with the finding of large number of photosynthesis-related protein changes by proteome analysis. In addition, various glycosyl hydrolases (AK066710, AK065130, AK072245, and AK070962 in shoots; AK072245, AK068499, AK100820, AK101744, and AK065793 in


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Figure 8. Functional categorization of induced (up-regulated) and suppressed (down-regulated) genes in rice seedling’s shoots and roots. The functional categorization was performed using the COG database as described in section 2.6. The functional categories are indicated on the left-hand side in line with each bar of the histogram representing each named category.

roots) are up-regulated to produce glucose, which may be used for energy production and carbohydrate conversion. Microarray data showed that none of the differentially expressed transcriptional- and translational-related genes matched with the proteins identified from 2-DGE analysis. However, the result indicated that 5 genes (AK109011, AK112056, AK069082, AK108452, and AK111859) encoded MYB transcription factors that were up-regulated in roots. Two genes (AK067269 and AK064143) in shoots and 1 gene (AK067269) in roots containing the pfam00651 motif were down-regulated. The pfam00651 motif is present in zinc finger proteins and is thought to mediate transcriptional repression and to interact with components of histone deacetylase co-repressor complexes.147 Moreover, the self-incompatibilty RNases (S-RNases) were up-regulated in both shoots (AK061438 and AK059757) and roots (AK059757). The S-RNases in plant are expressed to scavenge phosphate from ribonucleotides during leaf senescence and are involved in response to wounding or pathogen invasion.147 2-DGE analysis had revealed the induction of two proteaserelated proteins (spots s6 and S9) in shoots. Microarray data also showed the changed expression of many protease-related genes. Cysteine protease were up- and down-regulated in shoots (AK110974 and AK070448) and roots (AK060534, AK072235, and AK071948), respectively. In roots, down-regulated AK061045 (putativechloroplastDNA-bindingprotein)containspfam00026.13 motif involved in activity of aspartyl protease. Moreover, genes related to serine carboxypeptidases (AK067912 and AK068732) and aminopeptidase M (AK068165) were up-regulated in roots. Aminopeptidase M has 61% positive homology with puromy-

cin-sensitive aminopeptidase (Homo sapiens) that is involved in proteolytic events essential for cell growth and viability.148 Induced gene AK062091 (low molecular HSP) in roots contains the cd00298.2 motif, which is thought to be an ATP-independent chaperone that prevents aggregation and to be involved in refolding in combination with other HSPs.147 Among the cell wall and membrane biogenesis-related genes, actin (AK063572) was down-regulated in shoots; the amino acid sequence of actin has 95% positive homology with spot r41 induced in roots. Cinnamoyl-CoA reductase (AK072872), which participates in lignin biosynthesis, was up-regulated in shoots. Lignin is a component of cell wall and plays a crucial role in plant defense responses as a barrier against pathogens.149 In secondary metabolism, out of 3 genes encoding UDPglucosyltransferases, AK106312 was up-regulated in shoots and roots, while AK104985 was down-regulated in roots. UDPglucosyltransferase (AK106312) has 70% positive homology with anthocyanidin 3-O-glucosyltransferase (Z. mays), which catalyzes the glucosylation of anthocyanidin involved in antioxidation and pigment biosynthesis.150 UDP-glucosyltransferase (AK104985) has 73% positive homology with zeatin O-glucosyltransferase 2 (Arabidopsis) which glucosylates cytokinins.151 Moreover, 3 genes (AK061247 in shoots; AK069721 and AK069308 in roots) encoding O-methyltransferase were up-regulated. The genes AK061247, AK069721, and AK069308 have 21%, 31%, and 22% positive homology, respectively, with spot r16 protein. Among the defense-related genes, AK059449 (HSR203J), which may be involved in disease resistance,78 was up-regulated in shoots. A Chitinase (AK065866) whose amino acid sequence was identical to an induced spot R20 in roots was also Journal of Proteome Research • Vol. 6, No. 9, 2007 3599

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Figure 9. Validation of some differentially expressed genes by RT-PCR. Eight induced and 5 suppressed genes from the shoots and roots, respectively, were randomly selected from among the identified differentially expressed genes. The values besides each gel image represent relative intensity of mRNA level of control (C) and treatment (JA). Actin serves as a control. The AK numbers represent the gene array numbers and correspond to the cDNA clones identified in the rice full-length cDNA cloning project (http:// cdna01.dna.affrc.go.jp/cDNA/).

respectively, 57% and 65% positive homologies with decreased spot r47, AK103316 and AK064612 had 41% homology with decreased spot r42, and AK062937 and AK065921 have, respectively, 60% and 23% homology with decreased spot r24. A down-regulated catalase (AK066378) in roots was found to be identical to spot s49 in shoots.

4. Conclusions

Figure 10. Toward unraveling the role of JA in rice. Applications of proteomics and transcriptomics approaches on 7-day-old rice seedling’s shoots and roots has resulted in large inventory of differentially regulated proteins and genes by JA. This data set provides a foundation for discovery-based research for dissecting the function of these genes using plants carrying genes of interest in an overexpressed, silenced, or completely knocked-out form.

up-regulated in the shoot. In roots, another Chitinase (AK102369) and 2 class III Chitinase homologues (AK073267 and AK064356) were also up-regulated. However, amino acid sequences of AK102369, AK073267, and AK064356 have 5, 38, and 50% positive homologies with spot R20 protein. Moreover, AK111610 (Col-0 resistance to Pseudomonas syringae), AK071926 (stripe rust resistance protein Yr10), AK108620 (xyloglucan-specific fungal endoglucanase inhibitor protein), and AK067214 (pollen allergen) were up-regulated in roots. Finally, an antioxidant system-related gene, AK070895 (DHAR), whose amino acid sequence is identical to induced spot S6 and suppressed spot s54, was up-regulated in shoots. In roots, 6 genes (AK063773, AK105926, AK103316, AK064612, AK062937, and AK065921) encoding GSTs were repressed. In particular, amino acid sequences of AK063773 and AK105926 have, 3600


It can be stated that among the phytohormones, JA is the only hormone that plays a prominent role in controlling various aspects of plant growth and development, including their responses to the abiotic and biotic stresses. Inspite of its importance, the role of JA in monocot is poorly characterized at a global level on proteins and genes. This systematic study provides new and detailed information on the effect of JA on both the morphological responses in rice and molecular changes at protein and transcript level therein, by employing an elegant rice seedling culture study. Application of 2-DGE approach in combination with LC-MS/MS resulted in the identification of numerous JA-responsive proteins in shoots and roots. In shoots, photosynthesis-related proteins were largely down-regulated and represented 44% of the total identified proteins indicating that photosynthetic apparatus is the primary target for JA. Contrastingly, in the root, antioxidant systems, cellular respiration-, and defense-related proteins comprised 50% of the total identified proteins whose expressions were altered by JA. A parallel study of transcript profiling on shoot and root revealed 107 and 34 (in shoot) and 325 and 213 (in root) differently expressed genes with respect to their controls, by JA treatment. Several novel genes were identified by JA treatment; one of these was the transporter-related genes. This systematic study has extended our knowledge on how JA globally affects the expression of proteins and genes in rice. As shown in Figure 10, this inventory of differentially expressed proteins and genes can be further exploited to dissect the function of proteins or genes of interest using a combination


of genetic (loss-of-function) and transgenic (overexpression and silencing) approaches.

Acknowledgment. Authors appreciate the institutional support from HSS, and to O.H. from ARPC and BK21. The study was in part funded by a grant from the Plant Signaling Network Research Center, Korea Science and Engineering Foundation to N.-S.J. The study was also carried out with the support of “On-SiteCooperativeAgricultureResearchProject(No.20070301080003), RDA, Republic of Korea (N.-S.J.). Supporting Information Available: Figures showing the microarray protocol with pooled biological samples and dye-swap, scatter plots, and differentially expressed protein profiles in JA-treated shoots and roots by 2-DGE; tables listing the primer combinations used for confirmatory RT-PCR, the common induced and repressed genes in shoot in Rice 923 and 924, and the common induced and repressed genes in root in Rice 906 and 907. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Vick, B. A.; Zimmermann, D. C. Biochem. Biophys. Res. Commun. 1983, 111, 470-477. (2) Hamberg, M.; Gardner, H. W. Biochim. Biophys. Acta 1992, 1165, 1-18. (3) Farmer, E. E.; Ryan, C. A. Plant Cell 1992, 4, 129-134. (4) Beale, M. H.; Ward, J. L. Nat. Prod. Rep. 1998, 15, 533-548. (5) Conconi, A.; Smerdon, M. J.; Howe, G. A.; Ryan, C. A. Nature 1996, 383, 826-829. (6) Feussner, I.; Wasternack, C. Annu. Rev. Plant Biol. 2002, 53, 275297. (7) Wasternack, C.; Hause, B. Prog. Nucleic Acid Res. 2002, 72, 165221. (8) Browse, J. Vitam. Horm. 2005, 72, 431-56. (9) Mueller, M. J. Physiol. Plant 1997, 100, 653-663. (10) Agrawal, G. K.; Tamogami, S.; Han, O.; Iwahashi, H.; Rakwal, R. Biochem. Biophys. Res. Commun. 2004, 317, 1-15. (11) Agrawal, G. K.; Yonekura, M.; Iwahashi, Y.; Iwahashi, H.; Rakwal, R. J. Chromatogr., B 2005, 815, 109-123. (12) Agrawal, G. K.; Yonekura, M.; Iwahashi, Y.; Iwahashi, H.; Rakwal, R. J. Chromatogr., B 2005, 815, 125-136. (13) Agrawal, G. K.; Yonekura, M.; Iwahashi, Y.; Iwahashi, H.; Rakwal, R. J. Chromatogr., B. 2005, 815, 137-145. (14) Agrawal, G. K.; Rakwal, R. Mass Spectrom. Rev. 2006, 25, 1-53. (15) Agrawal, G. K.; Jwa, N. S.; Iwahashi, Y.; Yonekura, M.; Iwahashi, H.; Rakwal, R. Proteomics 2006, 6, 5549-5576. (16) Baginsky, S.; Gruissem, W. J. Exp. Bot. 2006, 57, 1485-1491. (17) DeRisi, J. L.; Iyer, V. R., et al. Science 1997, 278, 680-686. (18) Kikuchi, S.; Satoh, K.; Nagata, T.; Kawagashira, N.; Doi, K.; Kishimoto, N.; Yazaki, J.; Ishikawa, M.; Yamada, H.; Ooka, H.; Hotta, I.; Kojima, K.; Namiki, T.; Ohneda, E.; Yahagi, W.; Suzuki, K.; Li, C. J.; Ohtsuki, K.; Shishiki, T.; Otomo, Y.; Murakami, K.; Iida, Y.; Sugano, S.; Fujimura, T.; Suzuki, Y.; Tsunoda, Y.; Kurosaki, T.; Kodama, T.; Masuda, H.; Kobayashi, M.; Xie, Q.; Lu, M.; Narikawa, R.; Sugiyama, A.; Mizuno, K.; Yokomizo, S.; Niikura, J.; Ikeda, R.; Ishibiki, J.; Kawamata, M.; Yoshimura, A.; Miura, J.; Kusumegi, T.; Oka, M.; Ryu, R.; Ueda, M.; Matsubara, K.; Kawai, J.; Carninci, P.; Adachi, J.; Aizawa, K.; Arakawa, T.; Fukuda, S.; Hara, A.; Hashizume, W.; Hayatsu, N.; Imotani, K.; Ishii, Y.; Itoh, M.; Kagawa, I.; Kondo, S.; Konno, H.; Miyazaki, A.; Osato, N.; Ota, Y.; Saito, R.; Sasaki, D.; Sato, K.; Shibata, K.; Shinagawa, A.; Shiraki, T.; Yoshino, M.; Hayashizaki, Y.; Yasunishi, A. Science 2003, 301, 376-379. (19) Narusaka, Y.; Narusaka, M.; Seki, M.; Ishida, J.; Nakashima, M.; Kamiya, A.; Enju, A.; Sakurai, T.; Satoh, M.; Kobayashi, M.; Tosa, Y.; Park, P.; Shinozaki, K. Plant Cell Physiol. 2003, 44, 377-387. (20) Sasaki-Sekimoto, Y.; Taki, N.; Obayashi, T.; Aono, M.; Matsumoto, F.; Sakurai, N.; Suzuki, H.; Hirai, M. Y.; Noji, M.; Saito, K.; Masuda, T.; Takamiya, K.; Shibata, D.; Ohta, H. Plant J. 2005, 44, 653668. (21) Strassner, J.; Schaller, F.; Frick, U. B.; Howe, G. A.; Weiler, E. W.; Amrhein, N.; Macheroux, P.; Schaller, A. Plant J. 2002, 32, 585601. (22) Heidel, A. J.; Baldwin, I. T. Plant Cell Environ. 2004, 27, 13621373.

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Survey of Differentially Expressed Proteins and Genes in Jasmonic

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