Rice Proteomics

Abstract

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 R. Komatsu S. of in the rice using proteome Scholar). major of the rice two-dimensional PAGE data in which known proteins are is the of proteins on which can be the and molecular In to the identification of known proteins, these sequences can be used to which are for cloning the The of this study was to proteins from rice, to determine their molecular and and to N-terminal and internal amino acid sequence analysis using a protein sequencer and Finally, an was also to study the physiological significance of some proteins thus identified from rice. Rice sativa L. were used for and and Rice were under leaf and were from rice (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 of the rice tissues was with a (2.O’Farrell P.H. High resolution two-dimensional electrophoresis of proteins.J. Biol. Chem. 1975; 250: 4007-4021Google Scholar), and The was to two-dimensional PAGE (2.O’Farrell P.H. High resolution two-dimensional electrophoresis of proteins.J. Biol. Chem. 1975; 250: 4007-4021Google Scholar). or was out in a with a of and a of in the was with The and molecular weight of protein were determined using the two-dimensional PAGE The of proteins on the gels were with by two-dimensional the proteins were electroblotted onto a polyvinylidene difluoride and by Coomassie Brilliant (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 from the polyvinylidene difluoride and applied to the of the in a gas-phase protein sequencer was to the program by The proteins were separated by two-dimensional PAGE and stained with Gel protein spots were and the protein was from the gel using an of power for the protein was for and The protein in the sample was applied to a sample well in The sample was with a Electrophoresis was the sample and were in the gel and for to the protein by in and analysis by gel.J. Biol. Chem. 1977; Scholar). Electrophoresis was and the separated were electroblotted onto the polyvinylidene difluoride and to gas-phase sequencing H. Komatsu S. A. Kajiwara H. S. homology between related proteins and seed protein in rice (Oryza sativa Appl. Genet. Scholar). 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H. K. sequences of cDNA encoding expressed in rice seed (Oryza sativa Biol. Scholar). MS, these proteins were identified with spots and were to and was to number and the N-terminal sequences and were to stress protein B. R. R. M. M. A. of a rice expression in response to stress and Scholar). In MS analysis, the proteins a mass and were to proteins. Two proteins and were to the rice number and was to a protein number of rice plant is important for and for the the leaf sheath is the for of rice the plant be if the growth is the of leaf sheath The calreticulin and protein are expressed in the leaf also protein and the of rice cultured suspension Z. Komatsu S. cloning and characterization of a calcium-binding protein involved in the of rice cultured suspension J. Scholar), that the signal is important for growth and The proteome data file proteins S. M. T. N. Komatsu S. 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K. of a protein is related to the of rice cultured suspension cells.Plant Cell Scholar). the proteome data file of rice cultured this protein was identified as a calcium-binding protein, which is to calreticulin of on the of the amino acid sequence of the protein, were used for A cDNA library was using the as a were and by A cDNA was Z. Komatsu S. cloning and characterization of a calcium-binding protein involved in the of rice cultured suspension J. Scholar). the function of calreticulin in rice the cDNA for calreticulin was into rice in the and In calreticulin the rice callus and growth S. T. K. of a protein is related to the of rice cultured suspension cells.Plant Cell Scholar). In the study, the of calreticulin plant from callus and growth that calreticulin is a in the differentiation and of rice are by plant a in plant signal are a of plant that growth and and seed The cellular responses to are to be by A proteins have been identified as for by using a of A protein in rice was identified using a S. T. Hirano H. Rice gibberellin-binding related to ribulose-1,5-bisphosphate carboxylase/oxygenase Scholar). The protein was and N-terminal and internal amino acid sequences were determined. the proteome data file of rice leaf sheath and leaf this protein was identified as activase in on the of the amino acid sequence of were used for A cDNA library was using the as a were and by Two cDNA and were Z. Komatsu S. cloning and characterization of encoding of ribulose-1,5-bisphosphate carboxylase/oxygenase activase in rice (Oryza sativa Scholar). the function of the activase in rice the cDNA for activase was into the rice in the and The of growth the of and rice. from callus and growth was in rice. is known that of is of the first by In the of activase is the The of ribulose-1,5-bisphosphate carboxylase/oxygenase activase in to 1997; Scholar). In physiological of the has and in comparison with the N. of of a specific for the activase by Natl. Acad. Sci. U. S. A. 1999; Scholar). the function of the activase has been the function of the of activase is The that the small have a in the of leaf sheath The that activase is a for the signal transduction The and leaf that the signal is for rice PAGE and analysis a to study the various proteins that are or in rice under growth normal and which proteins are being in specific tissues and of rice under a of physiological can to the for these proteins. The study of partial amino acid sequence analysis of plant proteins will greatly to the of molecular for the identification of proteins through homology The information thus obtained from the amino acid sequence of these proteins will be helpful in predicting the function of the proteins and will aid in their molecular cloning in