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First published online December 29, 2005; 10.1104/pp.105.068395 Plant Physiology 140:734-745 (2006) © 2006 American Society of Plant Biologists Proteomics of Rac GTPase Signaling Reveals Its Predominant Role in Elicitor-Induced Defense Response of Cultured Rice Cells1,[W]Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology, Ikoma 630–0101, Japan (M.F., T.K., K.S.); and Agricultural and Veterinary Research Labs, Meiji Seika Kaisha, Sakado, Saitama 350–0289, Japan (K.U.)
We have previously shown that a human small GTPase Rac homolog, OsRac1, from rice (Oryza sativa) induces cascades of defense responses in rice plants and cultured cells. Sphingolipid elicitors (SEs) have been similarly shown to activate defense responses in rice. Therefore, to systematically analyze proteins whose expression levels are altered by OsRac1 and/or SE treatment, we performed a differential display analysis of proteins by the use of two-dimensional gel electrophoresis and mass spectrometry. A total of 271 proteins whose expression levels were altered by constitutively active (CA)-OsRac1 or SE were identified. Interestingly, of 100 proteins that were up-regulated by a SE, 87 were also induced by CA-OsRac1, suggesting that OsRac1 plays a pivotal role in defense responses induced by SE in cultured rice cells. In addition, CA-OsRac1 induces the expression of 119 proteins. Many proteins, such as pathogenesis-related proteins, SGT1, and prohibitin, which are known to be involved in the defense response, were found among these proteins. Proteins involved in redox regulation, chaperones such as heat shock proteins, BiP, and chaperonin 60, proteases and protease inhibitors, cytoskeletal proteins, subunits of proteasomes, and enzymes involved in the phenylpropanoid and ethylene biosynthesis pathways were found to be induced by CA-OsRac1 or SE. Results of our proteomic analysis revealed that OsRac1 is able to induce many proteins in various signaling and metabolic pathways and plays a predominant role in the defense response in cultured rice cells.
Plant proteomics has rapidly advanced over the last several years, and a number of studies have been undertaken in various species (van Wijk, 2001  ; Rose et al., 2004). So far, the most successful studies are those of subcellular compartments, since they contain a limited number of proteins. Such studies include the proteomics of the chloroplast (Peltier et al., 2000; Friso et al., 2004; Kleffmann et al., 2004; Lonosky et al., 2004; Rose et al., 2004), mitochondria (Kruft et al., 2001; Millar et al., 2001; Bardel et al., 2002), endoplasmic reticulum (Maltman et al., 2002), peroxisome (Fukao et al., 2002), and vacuole (Carter et al., 2004). Proteomic studies of specific stages in development or physiological conditions have been also performed with various plants. They include the proteomics of seed maturation (Finnie et al., 2002; Hajduch et al., 2005), seed germination (Gallardo et al., 2002), senescence (Wilson et al., 2002), nitrogen mobilization (Schiltz et al., 2004), salinity tolerance (Liska et al., 2004), and somatic embryogenesis (Imin et al., 2005). These studies mainly employ two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometric analysis using matrix-assisted laser-desorption ionization time of flight (TOF) mass spectrometry (MS) or electrospray ionization-TOF to identify proteins.
One possible limitation in the application of proteomics in plant biology is the lack of complete genome data in most species (van Wijk, 2001
Although proteomics is a promising technique that can be used for the study of signal transduction in plants, so far this method has not been extensively used in such studies in plants. In rice, proteomics has been utilized for studies of defensive responses, such as the elicitor response (Rakwal and Komatsu, 2000
Rac GTPase belongs to a family of Rac/Rop GTPases in plants, which are closely related to the Rho-type GTPase of animals, and the Rac/Rop GTPases have been shown to play key roles in a number of cellular activities such as growth and differentiation, hormone signaling, defense signaling against pathogens, and responses to various stresses in plants (Valster et al., 2000
Sphingolipid elicitors (SEs) were isolated from membranes of the rice blast fungus and shown to cause the accumulation of phytoalexins, cell death, increased resistance to infection by compatible pathogens, and induction of PR (pathogenesis-related) gene expression (Koga et al., 1998 Therefore, to systematically analyze proteins whose expression levels are constitutively activated in rice cells expressing CA-OsRac1 or induced by a SE, we employed a proteomics approach by using 2-DE and mass spectrometric analysis. We identified 271 proteins whose expression levels were altered by elicitor or CA-OsRac1. The analysis of these proteins revealed that close to 90% of the proteins, which were induced by the SE, were constitutively activated in rice cells expressing CA-OsRac1. In addition to those proteins induced by elicitor and CA-OsRac1, CA-OsRac1 induces many other proteins that may be involved in defensive responses. These results strongly suggested that OsRac1 plays a pivotal role in the elicitor-induced defense response in cultured rice cells.
The Expression Levels of 258 Proteins Are Altered by CA-OsRac1
To investigate alterations in protein expression induced by CA-OsRac1 expression, we used a combination of 2-DE and MS. For the analysis of cultured rice cells transformed with CA-OsRac1 against the wild type (Kinmaze), we extracted total proteins from rice cells with a urea/thio-urea buffer. The total proteins from cultured rice cells were resolved into approximately 1,500 spots in reproducible SDS-polyacrylamide gels (isoelectric focusing pH range; 4–7 size; 24 cm, SDS-PAGE gel size; 26 x 20 cm). Protein spots were visualized by staining with colloidal Coomassie Brilliant Blue. Figure 1 shows the entire image of the Coomassie Brilliant Blue-stained 2-D gels of total extracted proteins from wild-type and CA-OsRac1 cultured rice cells. The image analysis of these gels was carried out with PDQuest. Overall, the protein levels of 258 spots were found to be altered by CA-OsRac1. Of 258 proteins, 206 were up-regulated, while 52 were down-regulated. Representative samples of proteins that were up-regulated by CA-OsRac1 are shown in Figure 2. They are RGP2 (reverse-glycosylating protein 2), putative NADPH-dependent oxidoreductase, naringenin-2-oxoglutarate 3-dioxygenase, and putative 5'-phosphoribosyl-5-aminoimidazole synthetase, and their protein levels were increased 17-fold, 3-fold, 4-fold, and 7.5-fold, respectively (Table I; Supplemental Table I). Their levels in DN-OsRac1-expressing cells were decreased as shown in Figure 2. Those proteins that showed more than 3-fold changes in all of three or more replicate gels from the independent protein extractions were chosen to be differentially regulated proteins (see "Materials and Methods"). These spots were excised from the gels, digested with trypsin, and analyzed by Q-TOF mass spectrometer. Protein identification was performed with the nonredundant database National Center for Biotechnology Information using the MASCOT search program (Perkins et al., 1999
A Total of 100 Proteins Induced by SE Are Identified To analyze proteins whose expression levels are altered by SE, alterations in protein levels were examined at 4 and 8 h after initiation of the SE treatment (Fig. 3A). For instance, the tau class glutathione S-transferase (GST) protein 4 was not visible in the wild-type cells, while it was clearly induced at 4 and 8 h after elicitor treatment and in CA-OsRac1-expressing cells (Fig. 3B). A putative nucleic acid-binding protein was induced only at 8 h after the elicitor treatment and expressed in CA-OsRac1-expressing cells (Fig. 3C). Putative methylenetetrahydrofolate reductase (MTHFR) was induced at 4 h after elicitor treatment but disappeared at 8 h after treatment. This protein was not expressed in CA-OsRac1-expressing cells (Fig. 3D). We have identified a total of 100 proteins that were induced by SE in cultured rice cells (Fig. 4). Those proteins that were found only at either 4 h or 8 h after elicitor treatment were included in this category. We did not find any protein spots that were clearly down-regulated by SE. This is in contrast to CA-OsRac1-expressing cells, in which a number of proteins were down-regulated as shown below.
Proteins Whose Abundance Is Altered by CA-OsRac1 or SE Are Grouped into Four Categories Proteins whose expression levels were altered by CA-OsRac1 or SE were grouped into four categories (Fig. 4). Proteins grouped in class I (87 proteins; Table I) were those whose abundance was increased by both CA-OsRac1 and SE treatment. Those in class II (119 proteins; Supplemental Table I) were proteins that were up-regulated by CA-OsRac1 but not increased by SE treatment. Class III (13 proteins; Supplemental Table II) included those that were induced by SE treatment but not by CA-OsRac1. Class IV (52 proteins; Supplemental Table III) included those whose expression levels were decreased by CA-OsRac1. For each protein listed in the tables an average fold induction or fold repression is shown at the right end of the column. For SE-induced proteins fold increase was calculated based on the higher values obtained at 4 or 8 h treatment.
Surprisingly, 87 of 100 SE-inducible proteins (87%) were constitutively expressed in the cultured rice cells expressing CA-OsRac1 (class I). A total of 206 proteins that were up-regulated by CA-OsRac1 (classes I and II) were identified. Therefore, 42% of CA-OsRac1-induced proteins were similarly up-regulated by SE. These results suggest that OsRac1 could induce most SE-inducible proteins and that OsRac1 is likely to be placed very close to SE reception in the same signaling pathway. This conclusion is consistent with the previous findings that CA-OsRac1 could induce a variety of responses in rice cells and plants and leads to resistance against pathogens (Kawasaki et al., 1999
A large number of proteins were identified in the differential display analysis, and they could be classified into several distinct categories based on their functions. They will be discussed separately below.
Proteins that are known to be induced during defense responses were identified in SE-treated cells (Table I) and CA-OsRac1-expressing cells (Supplemental Table I). We identified PR proteins, such as
Prohibitin, a mitochondrial protein, was previously shown to be phosphorylated in rice lesion-mimic mutants (Takahashi et al., 2003
ROS have signaling roles in the induction of changes in gene expression associated with a number of physiological and developmental processes, including defense responses (Lamb and Dixon, 1997
Ferredoxin-NADP(H) reductase, putative NADPH-dependent oxidoreductase, Gln synthetase, putative quinine oxidoreductase, putative quinine oxidoreductase QR2, and putative NADPH-thioredoxin reductase were constitutively up-regulated in CA-OsRac1-expressing cells. Putative MTHFR was induced by SE in the wild-type cells. GSTs can detoxify lipid peroxidation products to prevent ROS toxicity (Gronwald and Plaisance, 1998
Heat-shock proteins (Hsps) and chaperonin were identified in CA-OsRac1-expressing cells. We have previously identified four molecular chaperones that were up-regulated during the appearance of cell death in the rice lesion-mimic mutant, cdr2 (Tsunezuka et al., 2005
We found that a Cys protease was down-regulated, whereas a Cys protease inhibitor was increased by CA-OsRac1. Both proteins are shown to be important for the regulation of programmed cell death. The Cys protease inhibitor exhibits high homology to cyctatin, which is probably involved in the regulation of protein turnover, and plays an important role in resistance against insects and pathogens (Danon et al., 2004
We identified several proteins associated with the phenylpropanoid pathway in CA-OsRac1-induced proteins (Table I). They include the NADP-dependent malic enzyme and pyruvate decarboxylase (PDC), caffeic acid 3-o-methyltransferase, and putative isoflavone reductase. These proteins are enzymes involved in the lignin and phytoalexin biosynthesis of the phenylpropanoid pathway and known to be highly induced after infection of pathogens (Dumas et al., 1988
We identified Met synthase and S-adenosyl-Met (SAM) synthase as CA-OsRac1-inducible proteins. SAM is involved in the biosynthesis of ethylene and polyamine (Even-Chen et al., 1982
Many enzymes in the polyamine biosynthetic pathway were found to be induced by CA-OsRac1. Decarboxylated SAM, which is a SAM decarboxylation form, was catalyzed by SAM decarboxylase. Thus, this compound links the ethylene biosynthesis pathway with the polyamine biosynthesis pathway. Decarboxylated SAM is used as a substrate for spermidine synthase and spermine synthase, and these enzymes convert from putrescine to spermidine and from spermidine to spermine, respectively. Arginase is an enzyme in the polyamine biosynthesis pathway and is shown to be induced by wounding, jasmonic acid treatment, and bacterial pathogen attack (Chen et al., 2004
We identified several proteins associated with alcoholic fermentation, PDC, alcohol dehydrogenase, and aldehyde dehydrogenase (ALDH), among proteins that were induced by CA-OsRac1. These enzymes are necessary for energy production of plants in anaerobic environments (Nakazono et al., 2000
Rho GTPase is known to regulate cytoskeleton organization in animal cells (Etienne-Manneville and Hall, 2002
We identified 52 proteins that were down-regulated by CA-OsRac1. Although proteins grouped into this category do not clearly show a particular tendency, they might have negative regulators of defense responses (Fig. 5; Supplemental Table III). Calreticulin and the calreticulin precursor were down-regulated in CA-OsRac1. Calreticulin, a Ca2+-binding protein associated with several functions, including Ca2+ homeostasis and molecular chaperoning, is required for the stress response (Simpson et al., 1997
Are the Changes at the Protein Level Correlated with Those at the mRNA Level? One important question originating from our proteomic study was whether the changes at the protein level caused by CA-OsRac1 expression or SE treatment were similar at the mRNA level. We have performed a preliminary analysis of global mRNA expression by the use of a rice microarray carrying 22,000 genes (T. Togashi, T. Kawasaki, and K. Shimamoto, unpublished data). Results showed that, for 87 class I proteins, which were up-regulated by both CA-OsRac1 and SE, 69 (79.3%) did not show a clear increase at the mRNA level. For 119 class II proteins, which were up-regulated by CA-OsRac1 but not by SE treatment, 52 (43.7%) exhibited no increase at the mRNA level, whereas 44 (37.0%) showed a similar increase at the mRNA level. Therefore, in these two classes, 121 of 206 proteins (58.7%) showed no clear increase at the mRNA level. These results indicate that almost 60% of the proteins whose levels were increased by either CA-OsRac1 or SE treatment did not show a similar increase at the mRNA level, suggesting that changes at the mRNA level do not necessarily show changes at the protein level under the conditions used in this study. It is possible that various modifications of proteins or changes in protein stability cause increased protein levels without a concomitant increase in mRNA levels. Results of reverse transcription-PCR analysis for some examples are shown in Figure 6. Six proteins whose mRNA levels were analyzed were class I proteins whose protein levels were increased by CA-OsRac1 and the SE treatment (Fig. 6, A and B). Three class I proteins whose abundance was essentially parallel with that of RNA are shown in Figure 6A while three other class I proteins whose mRNA levels did not greatly change by CA-OsRac1 or SE treatment are shown in Figure 6B. Figure 6C shows an example whose protein level was increased by CA-OsRac1 but not changed by SE. However, its RNA level was increased by CA-OsRac1 and SE. This kind of information can be obtained only after having conducted extensive studies on both functional proteomics and transcriptome analysis. Although this is preliminary information at the moment, our results on the functional proteomics of defense signaling in cultured rice cells suggest the importance of proteomics in plant cell and molecular biology.
The Role of OsRac1 in Signaling in Cultured Rice Cells
From our proteomic study of OsRac1 it is clear that it plays an important role in various signaling pathways in rice cells as shown in Figure 7. Current study seems to indicate that OsRac1 regulates signaling pathways in various stress responses including defense. Increased levels of proteins involved in redox regulation by CA-OsRac1 may be the results of increased ROS production (Kawasaki et al., 1999
A systematic analysis of proteins whose expression was altered by CA-OsRac1 and/or a SE by 2-DE and mass spectrometric analysis identified 271 proteins. Although a large-scale proteome analysis of various tissues in rice identified more than 2,500 proteins (Koller et al., 2002 ), extensive studies on the proteome analysis of functionally separated proteins involved in particular signal transduction pathways have not been performed. Therefore, this study may represent one of the most extensive analyses of proteins involved in the defense signaling pathway, at least in rice. The most interesting observation in this study is that the majority of SE-induced proteins were also activated by CA-OsRac1. Considering the previous findings that a SE is able to elicit almost all the cellular responses induced by the rice blast fungus, these results might indicate that OsRac1 is indeed a molecular switch of defense in rice. Furthermore, the observation that CA-OsRac1 is able to induce many other proteins, which were not induced by a SE, indicates that OsRac1 is also involved in other signaling pathways. They may be other branches in the defense pathway and others in various signaling pathways. Our preliminary comparison of microarray data on global mRNA expression with the protein data shown here indicates that, for a large fraction of proteins whose expression levels were increased by CA-OsRac1 or SE treatment in cultured rice cells, their mRNA levels did not appreciably change. These results suggest the importance of proteomics in the analysis of actual modifications in protein expression that occur during responses to various internal and external stimuli.
Rice Cell Cultures and Elicitor Treatment
Rice (Oryza sativa) suspension cultured cells expressing the CA and DN-OsRac1 have been described previously (Kawasaki et al., 1999
Rice cells in suspension cultures were harvested either before or after SE treatment and ground in a protein extraction buffer (7 M urea/2 M thio-urea, 4% w/v CHAPS, 20 mM dithiothreitol [DTT], 2% v/v, pharmalyte [Amersham Biosciences]; pH 3–10). Crude homogenates were sonicated and centrifuged at 4°C (9,000g for 30 min) to remove cellular debris. The supernatants were centrifuged again (20,000g for 30 min) and kept frozen at –80°C. The protein contents were determined by the Bradford (1976)
Isoelectric focusing was performed for 90 kVh at 20°C with a Multiphor II unit (Amersham Biosciences). For analytical separations, precast 24-cm immobilized pH gradient strips (pH 4–7; Immobiline DryStrip; Amersham Biosciences) were rehydrated overnight with 500 µg of sample proteins/450 µL of a rehydration buffer (7 M urea/2 M thio-urea, 4% w/v CHAPS, 20 mM DTT, 2% v/v, IPG buffer [Amersham Biosciences]; pH 4–7, bromphenol blue). The strips were placed in a reduction buffer (1% DTT, 6 M urea, 2% SDS, 30% glycerol, 50 mM Tris-HCl, pH 8.8) for 20 min with gentle shaking. The strips were then transferred to an alkylation buffer (2.5% iodoacetamide, 6 M Urea, 2% SDS, 30% glycerol, 50 mM Tris-HCl, pH 8.8) and shaken for 20 min. SDS-PAGE was carried out using an Ettan DALT twelve 2-D electrophoresis unit (Amersham Biosciences). The SDS polyacrylamide gels used contained 12.5% polyacrylamide gel (260 x 200 x 1.5 mm) and run in a Tris-Gly buffer (25 mM Tris, 192 mM Gly, 0.1% SDS) at a current setting of 10 mA/gel at 25°C. The 2-DE gels were stained with Coomassie Brilliant Blue R-350 (Amersham Biosciences) or SYPRO Ruby protein gel stain (Invitrogen). Gel images were scanned using the GS-800 Calibrated Imaging Densitometer (Bio-Rad Laboratories). Noise reduction, background subtraction, spot detection, quantification, gel-to-gel matching, and differential protein display analysis were carried out using the PDQuest software (Bio-Rad Laboratories). After normalization of the spot intensities on each gel, the spot intensities of each protein were quantified using at least three gels from the independent extractions. High reproducibility of spot intensities was obtained among the replicates. The average of spot intensities of each protein was calculated and used to compare the protein levels among the samples. Those proteins exhibiting 3-fold changes in all of replicate gels were chosen to be differentially regulated proteins. Selected spots were excised and stored at 4°C in deionized water.
Protein spots excised from the Coomassie Brilliant Blue-stained gels were washed twice with HPLC grade water containing 30% acetonitrile (Wako), washed with 100% of acetonitrile, and dried in a vacuum concentrator. The dried gel pieces were absorbed with 2 µL of a 0.5 µg/µL trypsin (Promega)/50 mM ammonium bicarbonate (Shevchenko and Shevchenko, 2001
Total RNA was extracted using EASYPrep RNA (TaKaRa) according to the manufacturer's protocol. One microgram of total RNA was reverse transcribed into first-strand cDNA with an oligo poly-T primer (21-mer) and SuperScript II reverse transcriptase (Invitrogen) in 20 µL total volume. One microliter of the synthesized first-strand cDNA was used for PCR analysis with different sets of gene-specific primers. The primer's sequences, annealing temperatures, and number of cycles are shown in Supplemental Table IV.
We thank the members of the Laboratory of Plant Molecular Genetics at Nara Institute of Science and Technology for technical assistance, comments, and participation in discussions. Received July 17, 2005; returned for revision November 9, 2005; accepted November 9, 2005.
1 This work was supported by the Research for the Future Program of the Japan Society for Promotion of Science (JSPS; grant no. JSPS–RFTF 00L01604) and the Ministry of Agriculture, Forestry, and Fisheries of Japan, Rice Genome Project.  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Ko Shimamoto (simamoto{at}bs.naist.jp).
[W] The online version of this article contains Web-only data. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.068395. * Corresponding author; e-mail simamoto{at}bs.naist.jp; fax 81–743–72–5509.
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ABSTRACT
RESULTS AND DISCUSSION
-subunit has been shown to act upstream of OsRac1 and regulate ROS production and defense gene activation (Suharsono et al., 2002
-1, 3-glucanase and root-specific PR-10, and stress-induced proteins, such as salt tolerance protein 5, putative glucosyltransferase IS5a (salicylate induced), and salt stress-induced protein. In addition, SGT1, which has been shown to be a key regulator of R-gene-mediated disease resistance, was found to be induced by CA-OsRac1. SGT1 mRNA was previously shown to be up-regulated by infection by rice blast fungus in rice (Cooper et al., 2003
