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Rice Proteomics

2003; Elsevier BV; Volume: 2; Issue: 1 Linguagem: Inglês

10.1074/mcp.r200008-mcp200

1535-9484

Setsuko Komatsu, Hirosato Konishi, Shihua Shen, Guangxiao Yang,

Microbial Metabolic Engineering and Bioproduction

The technique of proteome analysis with two-dimensional PAGE has the power to monitor global changes that occur in the protein expression of tissues and organisms and/or expression that occurs under stresses. In this study, the catalogues of the rice proteome were constructed, and a functional characterization of some of these proteins was examined. Proteins extracted from tissues of rice and proteins extracted from rice under various kinds of stress were separated by two-dimensional PAGE. An image analyzer was used to reveal a total of 10,589 protein spots on 10 kinds of two-dimensional PAGE gels stained by Coomassie Brilliant Blue. The separated proteins were electroblotted onto a polyvinylidene difluoride membrane, and the N-terminal amino acid sequences of 272 of 905 proteins were determined. The internal amino acid sequences of 633 proteins were determined using a protein sequencer or mass spectrometry after enzyme digestion of the proteins. Finally, a data file of rice proteins that included information on amino acid sequences and sequence homologies was constructed. The major proteins involved in the growth and development of rice can be identified using the proteome approach. Some of these proteins, including a calcium-binding protein that turned out to be calreticulin and a gibberellin-binding protein, which is ribulose-1,5-bisphosphate carboxylase/oxygenase activase in rice, have functions in the signal transduction pathway. The information thus obtained from the rice proteome will be helpful in predicting the function of the unknown proteins and will aid in their molecular cloning. The technique of proteome analysis with two-dimensional PAGE has the power to monitor global changes that occur in the protein expression of tissues and organisms and/or expression that occurs under stresses. In this study, the catalogues of the rice proteome were constructed, and a functional characterization of some of these proteins was examined. Proteins extracted from tissues of rice and proteins extracted from rice under various kinds of stress were separated by two-dimensional PAGE. An image analyzer was used to reveal a total of 10,589 protein spots on 10 kinds of two-dimensional PAGE gels stained by Coomassie Brilliant Blue. The separated proteins were electroblotted onto a polyvinylidene difluoride membrane, and the N-terminal amino acid sequences of 272 of 905 proteins were determined. The internal amino acid sequences of 633 proteins were determined using a protein sequencer or mass spectrometry after enzyme digestion of the proteins. Finally, a data file of rice proteins that included information on amino acid sequences and sequence homologies was constructed. The major proteins involved in the growth and development of rice can be identified using the proteome approach. Some of these proteins, including a calcium-binding protein that turned out to be calreticulin and a gibberellin-binding protein, which is ribulose-1,5-bisphosphate carboxylase/oxygenase activase in rice, have functions in the signal transduction pathway. The information thus obtained from the rice proteome will be helpful in predicting the function of the unknown proteins and will aid in their molecular cloning. Rice (Oryza sativa L.) is an important crop in eastern Asia. A vast number of rice cultivars as well as wild species of rice are widely grown, and their genetic and molecular makeup is being actively investigated. Rice is also considered a model plant in monocots because of the relatively small size of its genome. The rice genome conceivably consists of about 430 million base pairs (1.Sasaki T. The rice genome project in Japan.Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2027-2028Google Scholar), and about 30,000 genes can be expressed in rice plant tissue. High resolution two-dimensional PAGE is useful for separating complex protein mixtures (2.O'Farrell P.H. High resolution two-dimensional electrophoresis of proteins.J. Biol. Chem. 1975; 250: 4007-4021Google Scholar). Due to its high resolving power, the technique has been used to study alterations in cellular protein expression in response to various stimuli or as a result of differentiation and development (3.Celis J.E. Bravo R. Two-Dimensional Gel Electrophoresis of Proteins Methods and Applications. Academic Press, New York1984: 1-487Google Scholar). The latter approach further allows cDNA cloning from the resultant sequence(s). The Rice Genome Research Project is a joint project of the National Institute of Agrobiological Sciences and the Institute of the Society for Techno-innovation of Agriculture, Forestry, and Fisheries. In addition, partial support comes from the Genome Research Program of the Japanese Ministry of Agriculture, Forestry, and Fisheries. The program started in October 1991, and the first phase continued through 1997, resulting in the establishment of some of the basic tools of rice genome analysis. The Rice Genome Research Project was reorganized into a national project in 1998; objectives of the organization are to completely sequence the entire rice genome and to pursue integrated goals in functional genomics, genome informatics, and applied genomics. Two important objectives in rice proteome research are 1) to determine whether the cDNA encoding particular proteins from the cDNA library constructed from rice can be identified by a computer search of an amino acid sequence homology and 2) to predict the function of the proteins and study the physiological significance of functional proteins in rice. Sequencing of a protein separated by two-dimensional PAGE became possible with the introduction of protein electroblotting methods that allow the efficient transfer of a sample from the gel matrix onto a support that is suitable for gas-phase sequencing or related techniques (4.Mastsudaria P.J. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes.J. Biol. Chem. 1987; 262: 10035-10038Google Scholar). Proteins can also be recognized by their amino acid composition, their exact molecular weight as determined by mass spectrometry (MS), 1The abbreviations used are: MS, mass spectrometry; GA, gibberellin; BR, brassinosteroid; BL, brassinolide; CBB, Coomassie Brilliant Blue; MALDI, matrix-assisted laser desorption ionization; TOF, time-of-flight; SOD, superoxide dismutase; RuBisCO, ribulose-1,5-bisphosphate carboxylase/oxygenase. or their partial amino acid sequence. In conjunction with automated gel scanning and computer-assisted analysis, two-dimensional PAGE has contributed greatly to the development of a protein data base (5.Anderson L. Anderson N.G. High resolution two-dimensional electrophoresis of human plasma proteins.Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5421-5425Google Scholar, 6.Celis J.E. Ratz G.P. Madsen P. Gasser B. Lauridsen J.B. Hansen K.P.B. Kwee S. Rasmussem H.H. Nielsen H.V. Cruger D. Basse B. Honore H. Moller O. Celis A. Computerized, comprehensive databases of cellular and secreted proteins from normal human embryonic lung MRC-5 fibroblasts: identification of transformation and/or proliferation sensitive proteins.Electrophoresis. 1989; 10: 76-115Google Scholar, 7.Garrels J.I. Franz B.R. Transformation-sensitive and growth related changes of protein synthesis in REF52 cells.J. Biol. Chem. 1989; 264: 5229-5321Google Scholar, 8.Hirano H. Microsequence analysis of winged bean seed proteins electroblotted from two-dimensional gel.J. Protein Chem. 1989; 8: 115-130Google Scholar). Gel-separated proteins can be identified rapidly by MS, and if genomic information is also available, such analyses permit the systematic identification of the protein complement of a genome (9.Yetes III, J.R. J. Mass Spectrom. 1998; 33: 1-19Google Scholar). In addition, MS is a powerful tool for the analysis of isoforms, secondary modifications of proteins such as glycosylation and phosphorylation, and proteolysis, which only require low amounts (picomoles to attomoles) of proteins (10.Wilkins M.R. Gasteiger E. Gooley A.A. Herbert B.R. Molloy M.P. Binz P.A. Ou K. Sanchez J.C. Bairoch A. Willams K.L. Hochstrasser D.F.J. High-throughput mass spectrometric discovery of protein post-translational modifications.Mol. Biol. 1999; 289: 645-657Google Scholar). Such systematic analyses of protein populations are summarized by the term proteomics. Thus, proteomics bridges the gap between genomic sequence information and the actual protein population in a specific tissue, cell, or cellular compartment. Concerning the rice plant, some well known studies have dealt with the construction of proteomes from complex origins, such as the leaf, embryo, endosperm, root, stem, shoot, and callus proteome (11.Komatsu S. Kajiwara H. Hirano H. A rice protein library: a data-file of rice proteins separated by two-dimensional electrophoresis.Theor. Appl. Genet. 1993; 86: 935-942Google Scholar, 12.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 two-dimensional electrophoresis.Breed. Sci. 1997; 47: 245-251Google Scholar, 13.Komatsu S. Muhammad A. Rakwal R. Separation and characterization of proteins from green and etiolated shoots of rice: towards a rice proteome.Electrophoresis. 1999; 20: 630-636Google Scholar, 14.Komatsu S. Rakwal R. Li Z. Separation and characterization of proteins in rice suspension cultured cells.Plant Cell Tissue Organ Cult. 1999; 55: 183-192Google Scholar, 15.Tsugita A. Kawakami T. Uchiyama Y. Kamo M. Miyatake N. Nozu Y. Separation and characterization of rice proteins.Electrophoresis. 1994; 15: 708-720Google Scholar). Proteomic studies to date have mainly focused on those changes in genome expression that are triggered by environmental factors. Examples of descriptive proteomes include the global comparison of green and etiolated rice shoots (13.Komatsu S. Muhammad A. Rakwal R. Separation and characterization of proteins from green and etiolated shoots of rice: towards a rice proteome.Electrophoresis. 1999; 20: 630-636Google Scholar) and an analysis of defense-associated responses in the rice leaf and leaf sheath following a jasmonic acid treatment (16.Rakwal R. Komatsu S. Role of jasmonate in the rice self-defense mechanism using proteome analysis.Electrophoresis. 2000; 21: 2492-2500Google Scholar). One major advantage of the rice two-dimensional PAGE data base, in which most known proteins are recorded, is the wealth of new proteins on which experiments can be conducted at the biochemical and molecular levels. In addition to facilitating the identification of known proteins, these sequences can be used to prepare oligodeoxyribonucleotides, which are essential for cloning the corresponding cDNA. The aim of this study was to separate proteins from rice, to determine their relative molecular weights and isoelectric points, and to perform N-terminal and internal amino acid sequence analysis using a protein sequencer and MS. Finally, an attempt was also made to study the physiological significance of some proteins thus identified from rice. Rice (O. sativa L. cv. Nipponbare) seeds were used for collecting embryos and endosperms and preparing suspension-cultured cells. Rice seedlings were grown under white fluorescent light (6,000 lux, 12-h light period/day) at 25 °C. Leaf sheaths, leaf blades, roots, and lamina joints were collected from 2-week-old rice seedlings (11.Komatsu S. Kajiwara H. Hirano H. A rice protein library: a data-file of rice proteins separated by two-dimensional electrophoresis.Theor. Appl. Genet. 1993; 86: 935-942Google Scholar, 12.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 two-dimensional electrophoresis.Breed. Sci. 1997; 47: 245-251Google Scholar, 13.Komatsu S. Muhammad A. Rakwal R. Separation and characterization of proteins from green and etiolated shoots of rice: towards a rice proteome.Electrophoresis. 1999; 20: 630-636Google Scholar, 14.Komatsu S. Rakwal R. Li Z. Separation and characterization of proteins in rice suspension cultured cells.Plant Cell Tissue Organ Cult. 1999; 55: 183-192Google Scholar). A portion of the rice tissues was removed, homogenized with a lysis buffer (2.O'Farrell P.H. High resolution two-dimensional electrophoresis of proteins.J. Biol. Chem. 1975; 250: 4007-4021Google Scholar), and centrifuged. The supernatant was subjected to two-dimensional PAGE (2.O'Farrell P.H. High resolution two-dimensional electrophoresis of proteins.J. Biol. Chem. 1975; 250: 4007-4021Google Scholar). Isoelectric focusing or immobilized pH gradient was carried out in a glass tube with a length of 13 cm and a diameter of 3 mm. SDS-PAGE in the second dimension was performed with 15% separation gels. The isoelectric point and relative molecular weight of each protein were determined using the two-dimensional PAGE standard (Bio-Rad). The localization sites of individual proteins on the gels were evaluated automatically with Image Master 2D Elite software (Amersham Biosciences). Following separation by two-dimensional PAGE, the proteins were electroblotted onto a polyvinylidene difluoride membrane (ProBlott, Applied Biosystems, Foster City, CA) and detected by Coomassie Brilliant Blue (CBB) (11.Komatsu S. Kajiwara H. Hirano H. A rice protein library: a data-file of rice proteins separated by two-dimensional electrophoresis.Theor. Appl. Genet. 1993; 86: 935-942Google Scholar). The spots were excised from the polyvinylidene difluoride membrane and applied to the upper glass block of the reaction chamber in a gas-phase protein sequencer (Procise, Applied Biosystems, Foster City, CA). Edman degradation was performed according to the standard program supplied by Applied Biosystems (Foster City, CA). The proteins were separated by two-dimensional PAGE and stained with CBB. 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 watts of constant power for 2 h. After electroelution, the protein solution was dialyzed against deionized water for 2 days and lyophilized. The protein dissolved in the SDS sample buffer (pH 6.8) was applied to a sample well in SDS-PAGE. The sample solution was overlaid with a solution containing Staphylococcus aureus V8 protease (Pierce, Rockford, IL). Electrophoresis was performed until the sample and protease were stacked in the upper gel and interrupted for 30 min to digest the protein (17.Cleveland D.W. Fischer S.G. Kirschner M.W. Laemmli U.K. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel.J. Biol. Chem. 1977; 252: 1102-1106Google Scholar). Electrophoresis was then continued, and the separated digests were electroblotted onto the polyvinylidene difluoride membrane and subjected to gas-phase sequencing (18.Hirano H. Komatsu S. Nakamura A. Kikuchi F. Kajiwara H. Tsunasawa S. Sakiyama F. Structural homology between semidwarfism related proteins and glutelin seed protein in rice (Oryza sativa L.).Theor. Appl. Genet. 1991; 83: 153-158Google Scholar). The amino acid sequences obtained were compared with those of proteins in the amino acid sequence data base (MPSRCH-pp protein-protein data base, University of Edinburgh, Edinburgh, Scotland, UK). The CBB-stained protein spots were excised from a gel, washed with 25% (v/v) methanol and 7% (v/v) acetic acid for 12 h at room temperature, and destained with 50 mm NH4HCO3 in 50% (v/v) methanol for 1 h at 40°C. The proteins were reduced with 10 mm dithiothreitol in 100 mm NH4HCO3 for 1 h at 60°C and incubated with 40 mm iodoacetamide in 100 mm NH4HCO3 for 30 min at room temperature. The gel pieces were minced, allowed to dry, and then rehydrated in 100 mm NH4HCO3 with 1 pmol of trypsin at 37°C overnight. The digested peptides were extracted from the gel slices with 0.1% trifluoroacetic acid in 50% (v/v) acetonitrile/water three times. The peptide solution thus obtained was dried, reconstituted with 30 μl of 0.1% trifluoroacetic acid in 5% acetonitrile/water, and then desalted by Zip Tip C18™ pipette tips (Millipore, Bedford, MA). Matrix-assisted laser desorption ionization (MALDI) MS was performed using a Voyager Elite XL time-of-flight mass spectrometer (Applied Biosystems, Framingham, MA). The above peptide solution was mixed with the matrix solution, the supernatant of a 50% acetonitrile solution saturated with α-cyano-4-hydroxycinnamic acid, and then air-dried on the flat surface of a stainless steel plate. Calibrations were carried out using a standard peptide mixture (19.Jensen O.N. Wilm M. Shevchenko A. Mann M. 2-D Proteome Analysis Protocols. Humana Press Inc., Totowa, NJ1999: 513-530Google Scholar). The mass spectra were subjected to a sequence data base search with Mascot software (Matrix Science Ltd., London, UK). A cDNA microarray containing independent rice cDNA clones of 9,600 expressed sequence tags was used. One microgram of mRNA sample prepared from lamina treated with 1 μg of BL for 48 h or water as a control was reverse-transcribed in a 20-μl volume containing 1 mm Cy5 dCTP (Amersham Biosciences), anchored oligo(dT)25, random nonamer, dithiothreitol, dNTP, and SuperScript II (Invitrogen). After incubation at 42 °C for 2 h, the reaction was stopped, and RNA was degraded by first heating at 94 °C for 3 min and then treating with NaOH at 37 °C for 15 min. Fluorescently labeled probes were purified using a QIAquick PCR purification kit (Qiagen, Hilden, Germany). Probe hybridization and scanning of the hybridized microarray slide were conducted. Data were analyzed using Array Vision (Imaging Research Inc., Ontario, Canada) (20.Yazaki J. Kishimoto N. Nakamura K. Fuji F. Wu J. Yamamoto K. Sakata K. Sasaki T. Kikuchi S. Embarking on rice functional genomics via cDNA microarray: use of 3′ UTR probes for specific gene expression analysis.DNA Res. 2000; 7: 367-370Google Scholar). Agrobacterium tumefaciens strain EHA101 (a gift from Dr. E. Hood) has been described previously (21.Hiei Y. Ohta S. Komari T. Kumashiro T. Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis.Plant J. 1994; 6: 271-282Google Scholar). Plasmids were introduced into this strain by electroporation. A. tumefaciens was grown on AB medium (1 g/liter NH4NO3, 296 mg/liter MgSO4, 10 mg/liter CaCl2, 1.3 g/ml NaH2PO4, 3 g/liter K2HPO4, 150 mg/liter KCl, 2.5 mg/liter FeSO4, 5 g/liter glucose, 15 g/liter bacto agar) at 28 °C. The rice gene was connected to 35S promoter in pIG121-Hm (a gift from Dr. K. Nakamura), a binary vector that contains a kanamycin resistance gene (npt) and a hygromycin resistance gene (hpt), and the intron-gus in the T-DNA region. The transformation was done as reported previously (22.Komatsu S. Masuda T. Abe K. Phosphorylation of a protein (pp56) is related to the regeneration of rice cultured suspension cells.Plant Cell Physiol. 1996; 37: 748-753Google Scholar). The regenerated rice was eventually transferred to soil in pots and grown to maturity in a greenhouse. The technique of proteome analysis with two-dimensional PAGE has the power to monitor global changes that occur in the protein expression of tissues and organisms whether or not they are under stress. In this study, proteins extracted from endosperm, embryo, root, callus, green shoot, etiolated shoot, leaf sheath, leaf blade, lamina joint, and panicle were separated by two-dimensional PAGE (Fig. 1). Using an image analyzer, it was revealed that a total of 10,589 protein spots could be detected on 10 kinds of two-dimensional PAGE gels stained by CBB. The separated proteins were electroblotted onto a polyvinylidene difluoride membrane, and the N-terminal amino acid sequences of 272 of 905 proteins were determined. The N-terminal regions of the remaining proteins could not be sequenced, and it was concluded that they had a blocking group at the N terminus. The internal amino acid sequences of 633 proteins were determined using the gas-phase protein sequencer or MS after an enzyme digestion of proteins. Finally, a data file of rice proteins was constructed that included information on amino acid sequences and sequence homologies (Table I). The rice cDNA catalogue contains about 39.6% of the genes in the entire genome. The cDNA encoding particular proteins could be screened with a 40% probability using a computer search for sequence homology.Table INumber of proteins of which cDNAs were identified in the cDNA catalogueOrganNumber of proteins analyzedNumber of proteins sequencedaThe partial amino acid sequences of 905 proteins of 1,220 proteins were determined by protein sequencer or MS.Number of proteins identifiedbThe sequences of 358 proteins of 905 proteins were matched to those of a cDNA.% (b/a)Endosperm2010880.0Embryo10024833.3Root100762836.4Callus1501033836.9Green shoot100502958.0Etiolated shoot10030930.0Panicle50291034.5Others60058322839.1Total1,220cDetected proteins = 10,589.905dBlocked proteins = 633.35839.6a The partial amino acid sequences of 905 proteins of 1,220 proteins were determined by protein sequencer or MS.b The sequences of 358 proteins of 905 proteins were matched to those of a cDNA.c Detected proteins = 10,589.d Blocked proteins = 633. Open table in a new tab Brassinosteroids (BRs) are a group of naturally occurring plant steroids with structural similarities to insect and animal steroid hormones (23.Mandava N.B. Plant growth-promoting brassinosteroids.Annu. Rev. Plant Physiol. Plant Mol. Biol. 1988; 39: 23-52Google Scholar). Exogenous application of BRs to plant tissues evokes various growth responses such as cell elongation, proliferation, differentiation, organ bending, and a number of other physiological processes (24.Sasse J.M. Recent progress in brassinosteroid research.Physiol. Plant. 1997; 100: 696-701Google Scholar). It is believed that BR affects plant growth through the regulation of gene expression. However, only a few BR-regulated genes have been identified. The changes of gene expression caused by BR were systematically analyzed in rice seedlings using a combination of proteome and cDNA microarray approaches. The bending of the second leaf and its leaf sheath in rice seedlings is very sensitive to BRs, and this unique characteristic of rice leaves has been used as a quantitative bioassay for BRs (25.Wada K. Marumo S. Ikekawa N. Morisaki M. Mori K. Brassinolide and homobrassinolide promotion of lamina inclination of rice seedlings.Plant Cell Physiol. 1981; 22: 323-325Google Scholar). We adopted this model system and found that 1 μm BL treatment caused the maximum bending under these experimental conditions (Fig. 2). First, proteins were extracted from lamina joints treated with 1 μm BL for 48 h or water as a control and analyzed by two-dimensional PAGE. A systematic comparison of protein patterns showed that six proteins were increased, when compared with the water control, in the lamina joint treated with BL (Fig. 2). Sequence analysis revealed that two protein spots (LJ258 and LJ262) were homologous to the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit, and one protein (LJ133) showed homology to glyceraldehyde-3-phosphate dehydrogenase, which is a key enzyme in glycolysis. The other three protein spots (LJa, LJb, and LJ195) did not display any significant homology to proteins in the data bases researched. Second, a cDNA microarray containing 1,265 independent rice genes randomly selected from 9,600 expressed sequence tags was used to analyze differential gene expression caused by BL. The arrays were hybridized with Cy5 fluorescently labeled probes of lamina joint sample treated with 1 μm BL or water as a control (Fig. 2). Fluorescent signal differences greater than 2-fold between the control and BL-treated samples were considered to be significant. Data analysis showed that the expression of 12 genes was enhanced by the BL treatment (Fig. 2). Among them, five genes had homologies based on a search in the GenBank™ data base using the BLAST program. A vacuolar H+-transporting ATPase homologue (Element 0145) showed higher expression in the BL-treated lamina joint. Element 0550 was homologous to Bromheadia finlaysonia mRNA for extension, suggesting a role in BR-mediated cell expansion. Elements 0250 and 1029 showed homologies to the Arabidopsis ubiquitin-conjugating enzyme and Helianthus annuus mRNA for the 1-aminocyclopropane-1-carboxylic acid oxidase-related protein, respectively. Element 0654 was a rice photosystem II oxygen-evolving complex protein I. The other seven had no significant homologies in the data base. By using proteome analysis of differential protein expression and cDNA microarray analysis of differential gene expression, we identified some changes at the transcription and translation levels caused by the BL in the lamina joint. However, we did not find any overlaps in the results of the two approaches in the present study. This can be explained in the following manner. 1) The amount of some proteins is far beyond the detection sensitivity of CBB staining on two-dimensional PAGE. 2) The cDNA microarray that was used in the present study contains 1,265 genes and accounts for about 4% of the total number of genes predicted in rice, and those changed proteins detected in the proteome are not contained in the cDNA microarray used. Fifty-four proteins of leaf sheath from rice seedlings were analyzed by Edman sequencing and MS. For Edman sequencing, most of the proteins were N-terminally blocked. Using MS, all proteins were identified by matching the protein from rice and other species (Table II). The similar proteins are spot LS083, homologous to calreticulin, a calcium-binding protein located in the endoplasmic reticulum (26.Li Z. Komatsu S. Molecular cloning and characterization of calreticulin, a calcium-binding protein involved in the regeneration of rice cultured suspension cells.Eur. J. Biochem. 2000; 267: 737-745Google Scholar); spot LS261, matched to Bowman Birk trypsin inhibitor; spot LS317, identified as a Cu,Zn-SOD 1, a cytoplasmic protein that destroys radicals that are normally produced within the cells and are toxic to biological systems (27.Sakamoto A. Okumura T. Kaminaka H. Tanaka K. Molecular cloning of the gene (SodCc1) that encodes a cytosolic copper/zinc-superoxide dismutase from rice (Oryza sativa L.).Plant Physiol. 1995; 107: 651-652Google Scholar); spot LS322, found to be a C97454 rice callus cDNA clone; spot LS332, identified as a chloroplast Cu,Zn-SOD (28.Kaminaka H. Morita S. Yokoi H. Masumura T. Tanaka K. Molecular cloning and characterization of a cDNA for plastidic copper/zinc-superoxide dismutase in rice (Oryza sativa L.).Plant Cell Physiol. 1997; 38: 65-69Google Scholar); spot LS346, matched to RuBisCO small subunit from chloroplast protein, which catalyzes the first reaction in the Calvin cycle (29.Matuoka M. Kano-Murakami Y. Tanaka Y. Ozeki Y. Yamamoto N. Classification and nucleotide sequence of cDNA encoding the small subunit of ribulose-1,5-bisphosphate carboxylase from rice.Plant Cell Physiol. 1988; 29: 1015-1022Google Scholar).Table IIRice leaf sheath proteins identified using protein sequencer and mass spectrometrySpot no.MasspIHomologous protein (% of identified)Accession numberkDaLS08356.04.0CalreticulinaThe values indicate the homology for the identity protein sequences obtained from Edman sequence. (100) (40)BAA88900aThe values indicate the homology for the identity protein sequences obtained from Edman sequence.BAA88900bThe values indicate percentage of predicted protein sequence obtained from mass spectrometry covered by matched peptides.LS26131.05.8Bowman Birk protein inhibitoraThe values indicate the homology for the identity protein sequences obtained from Edman sequence. (100) (43)CBA88208aThe values indicate the homology for the identity protein sequences obtained from Edman sequence.CBA88208bThe values indicate percentage of predicted protein sequence obtained from mass spectrometry covered by matched peptides.LS28628.34.4UnknownLS29028.04.5UnknownLS29127.84.6UnknownLS30925.05.6G-box binding factor 4aThe values indicate the homology for the identity protein sequences obtained from Edman sequence. (66.7)P42777aThe values indicate the homology for the identity protein sequences obtained from Edman sequence.Glutathione-dependent dehydroascorbate reductase 1 (72.8)BAA90672bThe values indicate percentage of predicted protein sequence obtained from mass spectrometry covered by matched peptides.LS31024.85.5PET122 protein precursoraThe values indicate the homology for the identity protein sequences obtained from Edman sequence.(75)O13374aThe values indicate the homology for the identity protein sequences obtained from Edman sequence.Probable superoxide dismutase (45)TU4312bThe values indicate percentage of predicted protein sequence obtained from mass spectrometry covered by matched peptides.LS31723.15.6Cu,Zn-SOD 1aThe values indicate the homology for the identity protein sequences obtained from Edman sequence. (100) (64)p28756aThe values indicate the homology for the identity protein sequences obtained from Edman sequence.P28756bThe values indicate percentage of predicted protein sequence obtained from mass spectrometry covered by matched peptides.LS31922.65.7ATP synthase subunits region ORF 5aThe values indicate the homology for the identity protein sequences obtained from Edman sequence.(87.5)P05448aThe values indicate the homology for the identity protein sequences obtained from Edman sequence.605 ribosomal protein 1.5 (84)CAA79041bThe values indicate percentage of predicted protein sequence obtained from mass spectrometry covered by matched peptides.LS32022.55.8UnknownLS32122.35.7Major capsid