Characterization of Vacuolar Membrane Proteins Changed in Rice

Rice vacuolar membrane proteins changed by gibberellin (GA) were analyzed using a proteome approach. Vacuolar membrane fractions were isolated using a...
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Characterization of Vacuolar Membrane Proteins Changed in Rice Root Treated with Gibberellin Hirosato Konishi,† Masayoshi Maeshima,‡ and Setsuko Komatsu*,† Department of Molecular Genetics, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan, and Laboratory of Cell Dynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Aichi, Japan Received March 28, 2005

Rice vacuolar membrane proteins changed by gibberellin (GA) were analyzed using a proteome approach. Vacuolar membrane fractions were isolated using a discontinuous sucrose/sorbitol system and 10 proteins increased in vacuolar membrane of the root, treated with GA3 as compared with control. Fructose-1,6-bisphosphate aldolase C-1 and vacuolar H+-ATPase (V-ATPase) increased in root vacuolar membrane by GA3 interacted in rice roots. It suggests that aldolase C-1 regulates the V-ATPase mediated control of cell elongation that determines root growth. Keywords: rice • vacuolar membrane • gibberellin • root • aldolase • ATPase

Introduction The vacuole is the largest and most conspicuous compartment within plant cells and has numerous functions, which include the accumulation and storage of metabolites, the regulation of cytosolic homeostasis, the compartmentalization of toxic substances, and the hydrolysis and recycling of cellular components.1 Plant cells with defects in vacuole expansion do not expand2 and the rapid uptake of water by expanding vacuoles and quick osmoregulation between the cytosol and the vacuole are regulated by vacuolar membrane.3 The accumulation of osmotically active ions accompanies vacuole expansion, and vacuolar membrane proton pumps generate the trans-vacuolar membrane electrical and proton gradient that drives the uptake of many solutes.4 Blocking the expression of vacuolar H+-ATPase (V-ATPase) subunit A causes the inhibition of root elongation, confirming the importance of this proton pump in cell expansion.5 Vacuolar solute accumulators that are energized by the H+ electrochemical potential gradient also influence the expansion of root cells.6 Plant growth requires cell elongation including vacuole expansion. Plant hormones are the most important factors affecting plant cell division, growth, and differentiation that need to be precisely controlled during development to ensure the coordinated growth of tissues. Thus, the plant hormone gibberellin (GA) is an essential endogenous regulator of plant growth and developmental processes.7 Exogenous application of GA3 promotes the leaf sheath elongation in seedlings of rice.8 The leaf sheath is a functionally important part of the rice plant, where many critical metabolic and regulatory activities take place * To whom correspondence should be addressed. Department of Molecular Genetics, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan. Tel: 81-29-838-7446. Fax: 81-29-838-7408. E-mail: [email protected]. † National Institute of Agrobiological Sciences. ‡ Nagoya University. 10.1021/pr050079c CCC: $30.25

 2005 American Chemical Society

which eventually control the height and robustness of the plant. Furthermore, GA is a regulator of cell elongation in roots and plants with low levels of GA usually have short roots.4 Root elongation of rice seedling is accelerated by GA3 treatment.9 Since the architecture of the root system influences water and nutrient absorption, GA regulation of root growth is essential for plant survival. Vacuole expansion and cell elongation are dependent on several factors, such as rates of cell component biosyntheses, metabolite concentrations, and pH gradients across the vacuolar membrane. GA is highly likely to be connected with the vacuolar membrane function for plant growth. However, the mechanism for activation of vacuolar function triggered by GA is less well understood. High-resolution 2-DE is very useful for separating complex protein mixtures.10 Sequence analysis of protein separated by 2-DE is routine now using automated mass spectrometers. In rice, genome information provides a rich resource for understanding the biological process. Rice proteomics is becoming an increasingly powerful tool for the investigation of complex cellular process and turning out to be a major subject of research. Image analysis and 2-DE have contributed to the development of rice protein databases.11-13 Significant progress has been made toward identifying and cataloguing of the proteins from rice tissues and organelles. Proteomics of rice leaf sheath,14 root,15 and different subcellular compartments16 have opened an avenue to critically understand the functions of rice proteins. Furthermore, proteome analysis has been employed to study alterations in protein expression in response to GA3 in rice seedlings.17 However, there is no detailed report on proteins correlated with activation of vacuolar function triggered by GA. In this study, vacuolar membrane proteins regulated by GA in rice seedlings were investigated using proteome analysis techniques. Journal of Proteome Research 2005, 4, 1775-1780

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Table 1. Effects of Specific Inhibitors on the Activity of H+-ATPase in the Vacuolar membranea H+-ATPase activity treatment

µmol Pi/mg protein h

% control

control + KNO3 + Na3VO4 + NaN3

42.3 17.1 38.2 41.3

100 40 90 98

a The sensitivity of H+-ATPase activity to nitrate, vanadate and azide was used to distinguish between vacuolar membrane, plasma membrane, and mitochondrial enzymes, respectively.

Materials and Methods Plant Materials. Rice (Oryza sativa L. cv. Nipponbare) seedlings were grown in plastic seedling pots (280 × 160 × 90 mm) in a greenhouse. At 12 days after sowing, seedlings were treated with 0.1 and 5 µM GA3 (Wako, Osaka, Japan) or 100 µM glucose (Wako) for 24, 48, and 72 h. At the end of these treatments, leaf sheaths, and roots were utilized for isolation of vacuolar membrane. Isolation of Vacuolar Membrane. All procedures described below were carried out at 4 °C. Roots of rice seedlings were chopped and ground with homogenization medium consisting of 0.25 M sorbitol, 50 mM Tris-acetate (pH 7.5), 1 mM EGTA, 1% PVP, 10 µM PMSF and 2 mM DTT using a mortar and pestle. The homogenate was filtered through a Miracloth (Calbiochem, La Jolla, CA). The extract was centrifuged at 3600 × g for 10 min. The supernatant was collected and centrifuged at 120 000 × g for 25 min. The precipitate was suspended in Tris-sucrose buffer consisting of 0.5 M sucrose, 20 mM Tris-acetate (pH 7.5), 1 mM EGTA, 2 mM MgCl2 and 2 mM DTT, and the suspension was overlaid with equal volume of Tris-sorbitol buffer consisting of 0.25 M sorbitol, 20 mM Tris-acetate (pH 7.5), 1 mM EGTA, 2 mM MgCl2, and 2 mM DTT. After centrifugation at 120 000 × g for 45 min, vacuolar membranes that formed a band at the interface between the two solutions were collected, diluted with the Tris-sorbitol buffer, and centrifuged at 130 000 × g for 25 min. The resulting pellet was suspended in the Trissorbitol buffer and used as the vacuolar membrane fraction. KNO3, Na3VO4, and NaN3 are specific inhibitors of V-, P-, and F-type H+-ATPases, which are specifically associated with the vacuolar membrane, the plasma membrane and the mitochondrial membrane, respectively.18 The quality of isolated vacuolar membranes was determined by assaying these specific ATPase activities. Gel Electrophoresis. Samples (50 µg, 100 µL) solubilized with lysis buffer10 were separated in the first dimension by IEF or IPG tube gel (Daiichi Pure Chemicals, Tokyo, Japan) and in the second dimension by SDS-PAGE. IEF tube gel solution consisted of 8 M urea, 3.5% acrylamide, 2% NP-40, 2% Ampholines (pH 3.5-10.0 and pH 5.0-8.0), ammonium persulfate, and TEMED. Electrophoresis was carried out at 200 V for 30 min, followed by 400 V for 16 h and 600 V for 1 h. For IPG electrophoresis, samples were applied to acidic side of gels and electrophoresis using IPG tube gels (pH 6.0-10.0) was carried out at 400 V for 1 h, followed by 1000 V for 16 h and 2000 V for 1 h. After IEF or IPG, SDS-PAGE in the second dimension was performed using 15% polyacrylamide gel. The gels were stained with silver, and the image analysis was performed. Images of 2-DE were synthesized and the positions of individual proteins on the gels were evaluated automatically 1776

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Figure 1. Changes in the protein patterns of vacuolar membrane of the rice root treated with GA3. Proteins were extracted from vacuolar membrane of the root treated without or with 0.1 µM GA3 for 48 h, separated by 2-DE with IEF and IPG in the first dimension and SDS-PAGE in the second dimension, and detected by silver staining. The isoelectric point and relative molecular mass of each protein were determined using 2-D markers (BioRad). Circles show the positions of 10 proteins increased in vacuolar membrane of the root treated with GA3 as compared with control. Table 2. Specific Proteins Increased by GA3 at the Vacuolar Membrane of Roots spot no.

homologous protein

accession no.

01 02 03 04 05 06 07 08 09 10

V-ATPase subunit B Ubiquitin RiP-20 V-ATPase subunit A functional unknowna Ferredoxin functional unknowna Proteinase 2 precursor functional unknowna Aldolase C-1 functional unknowna

AF375052 AF216530 P31450

a

AF010320 S53952 D50301

Increase rate

2.19 2.27 2.06 3.22 2.11 3.10 2.62 2.72 2.89 2.83

Functional unknown: protein existing in rice genome with no name.

using ImageMaster 2D Elite software (Amersham Biosciences, Piscataway, NJ). The pI and Mr of each protein were determined using 2-D markers (Bio-Rad, Hercules, CA). MALDI-TOF MS. The stained protein spots were excised from gels, washed with 25% methanol and 7% acetic acid for 12 h, and destained with 50 mM NH4HCO3 in 50% methanol for 1 h at 40 °C. Proteins were reduced with 10 mM DTT in 100 mM NH4HCO3 for 1 h at 60 °C and incubated with 40 mM iodoacetamide in 100 mM NH4HCO3 for 30 min. The gel pieces were minced and allowed to dry and then rehydrated in 100 mM NH4HCO3 with 1 pmol trypsin (Sigma, St. Louis, MO) at

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Figure 2. Comparison of amino acid sequences of rice aldolases. Amino acid sequences analyzed with Cleveland peptide mapping are boxed. Asterisks indicate specific amino acid residues of aldolase C-1 (accession number D50301) in boxed sequences as compared with those of aldolase C-2 (accession number X53130) and C-a (accession number D13512). Aldolase C, cytoplasmic fructose-1,6bisphosphate aldolase; Aldolase P (accession number D13513), chloroplastic fructose-1,6-bisphosphate aldolase.

37 °C overnight. The digested peptides were extracted from the gel slices with 0.1% TFA in 50% ACN/water 3 times. The peptide solution thus obtained was dried and reconstituted with 30 µL of 0.1% TFA in 5% ACN/water and then desalted with ZipTip C18 pipet tips (Millipore, Bedford, MA). The above peptide solution was mixed with R-cyano-4-hydroxycinnamic acid. MALDI-TOF MS was performed using a Voyager TOF mass spectrometer (Applied Biosystems, Framingham, MA). The mass spectra were subjected to sequence database search using Mascot software (Matrix Science Ltd, London, UK). Cleveland Peptide Mapping. For internal amino acid sequence analysis, Cleveland peptide mapping was carried out.19 Following separation by 2-DE, gel pieces containing protein spots were removed and the protein was electroeluted from the gel pieces using an electrophoretic concentrator (ISCO, Lincoln, CA) at 2 W constant power for 2 h. After electroelution, the protein solution was dialyzed against deionized water for 2 days and lyophilized. The protein was dissolved in 20 µL of SDS sample buffer consisting of 0.5 M Tris-HCl (pH 6.8), 10% glycerol, 2.5% SDS and 5% 2-mercaptoethanol, and applied to a sample well in an SDS-PAGE gel and the sample solution was overlaid with 20 µL of 1/2 SDS sample buffer containing 50 ng/µL Staphylococcus aureus V8 protease (Pierce, Rockford, IL). Electrophoresis was performed until the sample and protease

were stacked in the stacking gel, interrupted for 30 min to digest the protein, and then continued. Amino Acid Sequence Analysis. Following separation by Cleveland method, the peptides were electroblotted onto a PVDF membrane (Pall Bio Support Division, Port Washington, NY) using a semidry transfer blotter (Nippon Eido, Tokyo, Japan), and detected by CBB staining.20 The stained protein bands were excised from the PVDF membrane and applied to a gas-phase protein sequencer Procise 494 (Applied Biosystems, Foster City, CA). The amino acid sequences obtained were compared with those of known proteins in the Swiss-Prot, PIR, Genpept and PDB databases with Web-accessible search program FastA. Immunoprecipitation and Western Blot Analysis. Proteins were extracted from vacuolar membranes of roots treated with or without GA3 with extraction buffer consisting of 50 mM TrisHCl (pH 7.4), 150 mM NaCl, 1 mM EGTA, 5 µM Na3VO4, 1% sodium deoxycholate, 1 mM PMSF and 1% Triton X-100. After 100 µL of the extract was incubated with shaking for 2 h at 4 °C with 10 µL of anti-V-ATPase subunit A or B antibodies, 7.5 mg Protein A-Sepharose (Sigma) in 30 µL of immunoprecipitation buffer consisting of 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 1 mM PMSF, and 0.5% Triton X-100 was added and incubated with continuJournal of Proteome Research • Vol. 4, No. 5, 2005 1777

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ous shaking 4 °C overnight. Sepharose beads with antigen proteins were precipitated by centrifugation at 20 600 × g for 5 min. The pellet was washed twice with 500 µL of washing buffer consisting of 50 mM Tris-HCl (pH 7.8), 250 mM sucrose and 0.1% Triton X-100, and the suspended in SDS sample buffer. After boiling for 5 min, the suspension was centrifuged at 20 600 × g for 5 min and the supernatant was subjected to SDS-PAGE and followed by Western blot analysis with an antialdolase antibody or CBB staining. For separation of proteins by SDS-PAGE, 15% polyacrylamide gel was used. The proteins were electroblotted onto a PVDF membrane. The PVDF membranes were treated with antibodies and antigen-antibody complexes were detected with enhanced chemiluminescence using ECL-Plus kit (Amersham Biosciences).

Results and Discussion Plant cells with defects in vacuole expansion do not expand2 and vacuolar membrane regulates the rapid uptake of water by expanding vacuoles and quick osmoregulation between the cytosol and the vacuole.3 To analyze proteins that affect vacuolar function, the vacuolar membrane fraction was isolated using a discontinuous sucrose/sorbitol system. The purity of the fraction was examined using assay for H+-ATPase activity. The sensitivity of H+-ATPase activity to nitrate, vanadate and azide was used to distinguish between vacuolar membrane, plasma membrane and mitochondrial enzymes, respectively.18 The proportions of the total activity that were sensitive to nitrate, vanadate and azide amounted to 60%, 10% and 2%, respectively (Table 1). The nitrate-sensitive fraction was thus enriched in the vacuolar membrane fraction, but it is possible that the preparation contained traces of plasma membrane and mitochondrial contaminants. To examine changes of vacuolar membrane proteins by GA3 in the root, proteins were extracted from vacuolar membrane fractions of the rice root treated with or without 0.1 µM GA3 for 48 h, and separated by 2-DE. Ten proteins increased in vacuolar membrane of root by GA3 treatment (Figure 1). These proteins were identified by MALDI-TOF MS analysis (Table 2). Some of these proteins were relatively homologous to those from plants, such as V-ATPase subunit B (AF375052), ubiquitin RiP-20 (AF216530), V-ATPase subunit A (P31450), ferredoxin (AF010320), proteinase 2 precursor (S53952) and fructose-1,6bisphosphate aldolase C-1 (D50301). Aldolase C-1 increased in GA3-treated roots is at low levels in Tan-ginbozu roots, and levels further diminished in roots treated with uniconazole-P or ABA as compared with the control,9 therefore aldolase C-1 may act as a mediator between GA signaling and root growth. In rice, 4 aldolase isozymes that are 3 cytoplasmic aldolases (aldolase C-1, C-2, and C-a) and 1 chloroplastic aldolase (aldolase P) are already reported.21-23 Internal amino acid sequences of the protein 09 of root vacuolar membrane (aldolase C-1, Table 2) were determined by sequence analysis of peptides obtained using the Cleveland peptide mapping method. Although aldolase C-1 specific amino acid residues that were N (115), S (191), E (195), S (222), E (234), S (235), Q (241), and L (242) were detected (Figure 3), it became clear that the protein 09 of root vacuolar membrane was not aldolase C-2, aldolase C-a, and aldolase P. In the rice root, aldolase C-1 is specifically likely to influence the vacuolar function. To clarify how aldolase C-1 interacts the vacuolar membrane, immunoprecipitation analysis was performed to examine whether aldolase C-1 interacts with V-ATPase in rice roots. V-ATPase is a multi-subunit complex and two antibodies were 1778

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Figure 3. Co-immunoprecipitation of aldolase with V-ATPase of vacuolar membrane in the rice root. Root were treated with 0.1 µM GA3, respectively, for 48 h and vacuolar membranes were purified. Anti-V ATPase subunit A and B antibodies were added to the protein samples extracted from vacuolar membranes. The IgG-antigen complexes were precipitated by using Protein ASepharose in immunoprecipitation buffer and then subjected to Western blot analysis with anti-aldolase antibody. CBB stained gel was used as loading control.

prepared that are specific to the subunit A and B of V-ATPase. The antibodies were separately added to 1% TritonX-100extracted proteins from vacuolar membrane of roots. The immunoprecipitated proteins were isolated using Protein ASepharose and then analyzed by immunoblot analysis with an anti-aldolase C-1 antibody. Aldolase C-1 was detected in samples co-immunoprecipitated with anti-V ATPase subunit A and B antibodies (Figure 3). The association of aldolase C-1 with the V-ATPase complex in rice roots was indicated. In yeast cells, deficient in aldolase, the peripheral V1 domain of VATPase dissociates from the integral membrane V0 domain, indicating the coupling of glycolysis to the proton pump.24 Since the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase is physically associated with the aldolase-V ATPase complex and the glycolytic enzyme complex is directly coupled to the V-ATPase proton pump,25 association of aldolase with V-ATPase may result in the location of the ATP-generating glycolytic enzyme P-glycerate kinase close to the ATP-utilizing ATPase. Aldolase deletion mutant cells display a growth phenotype similar to that observed in V-ATPase subunit deletion mutants and the V-ATPase abnormalities shown in aldolase deletion mutant cells can be restored to normal levels by aldolase complementation.25 The binding of aldolase to V-ATPase should provide the cells with a means for localized ATP generation by glycolysis. Furthermore, aldolase C-1 is identified in the vacuolar membrane fruction isolated from roots (Figure 1 and Table 2), indicating that aldolase C-1 may associate with the vacuolar membrane protein in rice. In rice roots, aldolase C-1 was co-immunoprecipitated with both antibodies raised against V-ATPase subunit A and B (Figure 3), suggesting that aldolase physically associates with V-ATPase

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Rice Vacuolar Membrane Proteins Changed by Gibberellin

Figure 4. Time-course analyses of V-ATPase subunit A and B of vacuolar membrane in the rice roots treated with GA3 or glucose. Proteins extracted from vacuolar membrane in the roots treated with 0.1 µM GA3 or 100 µM glucose for 0, 24, 48, and 72 h were subjected to Western blot analysis with anti-V ATPase subunit A and B antibodies. Silver stained gel was used as a loading control.

in the root and is involved in the functional regulation of V-ATPase for cell elongation that results in root elongation. GA3 induces a sustained increase in cytosolic Ca2+ concentration26 and HvCDPK1 that encodes a Ca2+-dependent kinase regulates vacuolar function during the GA3 response in barley aleurone.27 Root growth requires cell elongation including vacuole expansion and is highly likely to be connected with the glycolytic enzyme aldolase C-1. To examine how GA3 and glucose change the level of V-ATPase subunit A and B in rice roots, proteins were extracted from the vacuolar membrane of rice root treated with 0.1 µM GA3 or 100 µM glucose 0 to 72 h and subjected to Western blot analysis with an anti-V ATPase subunit A and B antibodies. Measurable increases in V-ATPase subunit A and B accumulations were noted after 48 and 24 h, respectively, exposure to GA3 and glucose (Figure 4). By 48 h there was a pronounced increase in V-ATPase subunit A and B that weakened by 72 h exposure to GA3 and that continued until 72 h exposure to glucose (Figure 4). Exogenous glucose was able to inhibit root elongation at high concentrations (10 and 100 mM), and promote root elongation at 100 µM glucose.9 Aldolase C-1 might interact with V-ATPase by the treatment with 100 µM glucose affecting root elongation. Growth inhibition may be due to harmful osmotic influences on root growth. Glucose is a universal nutrient preferred by most organisms, and plants use hexokinase, an enzyme in glycolysis, as a glucose sensor to interrelate hormone signaling networks for controlling growth

Figure 5. Proposed model for promotion of rice root elongation by GA-induced aldolase C-1. The activation of glycolytic pathway function accelerates root growth and that GA-induced root aldolase C-1 may be modulated through OsCDPK13. Aldolase C-1 physically associates with V-ATPase in roots and may regulate the V-ATPase mediated control of cell elongation that determines root length.

and development.28 Aldolase C-1 and V-ATPase are involved in GA-stimulated root growth through activation of the glycolytic pathway.

Conclusions The present results suggest that GA3-induced aldolase C-1 enhances the metabolic rate of glycolysis in rice roots. It is suggested that aldolase C-1 activates the V-ATPase through physical interaction. As a result, the rate of cell growth of seedling roots may be efficiently enhanced. GA3 signaling for promotion of the root growth is mediated by CDPK13.29 The activation of glycolytic pathway function accelerates root growth and that GA3-induced root aldolase C-1 may be modulated through OsCDPK13. Aldolase C-1 physically associates with V-ATPase in roots and may regulate the V-ATPase mediated control of cell elongation that determines root length (Figure 5). Because target molecules of CDPK13 affecting the expression of aldolase C-1 are unclear, investigations using CDPK13-transgenic rice would be necessary.

Acknowledgment. We thank Dr. M. K. Khan for 2-DE and Dr. N. Tanaka for isolation of vacuolar membrane. We are also grateful to Dr. H. Yamane, University of Tokyo, for helpful discussion. This work was supported by a grant from the Program for Promotion of Basic Research Activities for Innovative Biosciences and MAFF Rice Genome Project Grant No. PR1201. References (1) Maeshima, M.; Nakanishi, Y.; Matsuura-Endo, C.; Tanaka, Y. Proton Pumps of the Vacuolar Membrane in Growing Plant Cells. J. Plant Res. 1996, 109, 119-125.

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research articles (2) Schumacher, K.; Vafeados, D.; McCarthy, M.; Sze, H.; Wilkins, T.; Chory, J. The Arabidopsis det3 mutant reveals a central role for the vacuolar H+-ATPase in plant growth and development. Genes. Dev. 1999, 13, 3259-3270. (3) Chaumont, F.; Barrieu, F.; Herman, E. M.; Chrispeels, M. J. Characterization of a maize tonoplast aquaporin expressed in zones of cell division and elongation. Plant Physiol. 1998, 117, 1143-1152. (4) Dolan, L.; Davies, J. Cell expansion in roots. Curr. Opin. Plant Biol. 2004, 7, 33-39. (5) Gogarten, J. P.; Fichmann, J.; Braun, Y.; Morgan, L.; Styles, P.; Taiz, S. L.; de Lapp, K.; Taiz, L. The use of antisense mRNA to inhibit the tonoplast H+-ATPase in carrot. Plant Cell 1992, 4, 851864. (6) Cheng, N. H.; Pitman, J. K.; Barkla, B. J.; Shigaki, T.; Hirchi, K. D. The Arabidopsis cax1 mutant exhibits impaired ion homeostasis, development and hormonal responses and reveals interplay among vacuolar transporters. Plant Cell 2003, 15, 347-364. (7) Kende, H.; Zeevaart, J. A. D. The five “classical” plant hormones. Plant Cell 1997, 9, 1197-1210. (8) Shen, S.; Sharma, A.; Komatsu, S. Characterization of proteins responsive to gibberellin in the leaf-sheath of rice (Oryza sativa L.) seedling using proteome analysis. Biol. Pharm. Bull. 2003, 26, 129-136. (9) Konishi, H.; Kitano, H.; Komatsu, S. Identification of rice root proteins regulated by gibberellin using proteome analysis. Plant Cell Environ. 2005, 28, 328-339. (10) O’Farrell, P. H. High-resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 1975, 250, 4007-4021. (11) Tsugita, A.; Kamo, M.; Kawakami, T.; Ohki, Y. Separation and characterization of rice proteins. Electrophoresis 1994, 17, 855865. (12) Koller, A.; Washburn, M. P.; Lange, B. M.; Andon, N. L.; Deciu, C.; Haynes, P. A.; Hays, L.; Schieltz, D.; Ulaszek, R.; Wei, J.; Wolters, D.; Yates, J. R. 3rd Proteomic survey of metabolic pathways in rice. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 1196911974. (13) Komatsu, S.; Kojima, K.; Suzuki, K.; Ozaki, K.; Higo, K. Rice Proteome Database based on two-dimensional polyacrylamide gel electrophoresis: its status in 2003. Nucleic Acids Res. 2004, 32, D388-D392. (14) Shen, S.; Matsubae, M.; Takao, T.; Tanaka, N.; Komatsu, S. A proteomic analysis of leaf sheaths from rice. J. Biochem. 2002, 132, 613-620. (15) Zhong, B.; Karibe, H.; Komatsu, S.; Ichimura, H.; Nagamura, Y.; Sasaki, T.; Hirano, H. Screening of rice genes from a cDNA catalog based on the sequence data-file of proteins separated by twodimensional electrophoresis. Breeding Sci. 1997, 47, 245-251. (16) Tanaka, N.; Fujita, M.; Handa, H.; Murayama, S.; Uemura, M.; Kawamura, Y.; Mitsui, T.; Mikami, S.; Tozawa, Y.; Yoshinaga, T.; Komatsu, S. Proteomics of the rice cell: systematic identification of the protein populations in subcellular compartments. Mol. Gen. Genomics 2004, 271, 566-576.

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Konishi et al. (17) Tanaka, N.; Konishi, H.; Khan, M. M. K.; Komatsu, S. Proteome analysis of rice tissues by two-dimensional electrophoresis: an approach to the investigation of gibberellin regulated proteins. Mol. Gen. Genomics 2004, 270, 485-496. (18) Sze, H. H+-translocating ATPases: advances using membrane vesicles. Annu. Rev. Plant Physiol. 1985, 36, 175-208. (19) Cleveland, D. W.; Fischer, S. G.; Kirschner, M. W.; Laemmli, U. K. Peptide mapping by limited proteolysis in sodium dodecyl sulphate and analysis by gel electrophoresis. J. Biol. Chem. 1977, 252, 1102-1106. (20) Komatsu, S.; Muhammad, A.; Rakwal, R. Separation and characterization of proteins from green and etiolated shoots of rice (Oryza sativa L.): Towards a rice proteome. Electrophoresis 1999, 20, 630-636. (21) Hidaka, S.; Kadowaki, K.; Tsutsumi, K.; Ishikawa, K. Nucleotide sequence of the rice cytoplasmic aldolase cDNA. Nucleic Acids Res. 1990, 18, 3991. (22) Tsutsumi, K.; Kagaya, Y.; Hidaka, S.; Suzuki, J.; Tokairin, Y.; Hirai, T.; Hu, D.; Ishikawa, K.; Ejiri, S. Structural analysis of the chloroplastic and cytoplasmic aldolase-encoding genes implicated the occurrence of multiple loci in rice. Gene 1994, 141, 215220. (23) Nakamura, H.; Satoh, W.; Hidaka, S.; Kagaya, Y.; Ejiri, S.; Tsutsumi, K. Genomic structure of the rice aldolase isozyme C-1 gene and its regulation through a Ca2+-mediated protein kinase-phosphatase pathway. Plant Mol. Biol. 1996, 30, 381-385. (24) Lu, M.; Holliday, L. S.; Zhang, L.; Dunn, W. A.; Gluck, S. L. Interaction between aldolase and vacuolar H+- ATPase. J. Biol. Chem. 2001, 276, 30407-30413. (25) Lu, M.; Sautin, Y. Y.; Holliday, L. S.; Gluck, S. L. The glycolytic enzyme aldolase mediates assembly, expression, and activity of vacuolar H+- ATPase. J. Biol. Chem. 2004, 279, 8732-8739. (26) Gilroy, S.; Jones, R. L. Gibberellic acid and abscisic acid coordinately regulate cytoplasmic calcium and secretory activity in barley aleurone protoplasts. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 3591-3595. (27) McCubbin, A. G.; Ritchie, S. M.; Swanson, S. J.; Gilroy, S. The calcium-dependent protein kinase HvCDPK1 mediates the gibberellic acid response of the barley aleurone through regulation of vacuolar function. Plant J. 2004, 39, 206-218. (28) Moore, B.; Zhou, L.; Rolland, F.; Hall, Q.; Cheng, W. H.; Liu, Y. X.; Hwang, I.; Jones, T.; Sheen, J. Role of the Arabidopsis glucose sensor HXK1 in nutrient, light, and hormonal signaling. Science 2003, 300, 332-336. (29) Konishi, H.; Yamane, H.; Maeshima, M.; Komatsu, S. Characterization of fructose-bisphosphate aldolase regulated by gibberellin in roots of rice seedling. Plant Mol. Biol. 2004, 56, 839-848.

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