Differential Expression of Defense/Stress-Related Marker Proteins in Leaves of a Unique Rice Blast Lesion Mimic Mutant (blm) Young-Ho Jung,† Randeep Rakwal,*,‡,§ Ganesh Kumar Agrawal,§ Junko Shibato,‡ Jung-A Kim,† Mi Ok Lee,† Pil-Kyu Choi,† Seung-Hee Jung,† So Hee Kim,† Hee-Jong Koh,| Masami Yonekura,⊥ Hitoshi Iwahashi,‡ and Nam-Soo Jwa*,† Department of Molecular Biology, College of Natural Science, Sejong University, Seoul 143-747, Korea, Human Stress Signal Research Center (HSS), National Institute of Advanced Industrial Science and Technology (AIST) WEST, Onogawa 16-1, Tsukuba, Ibaraki 305-8569, Japan, Research Laboratory for Agricultural Biotechnology and Biochemistry (RLABB), G.P.O. Box 8207, Kathmandu, Nepal, School of Agricultural Biotechnology, Seoul National University, Seoul 151-742, Korea, and Food Function Laboratory, School of Agriculture, Ibaraki University, Ami, Ibaraki 300-0393, Japan Received March 14, 2006
We analyzed a unique rice (Oryza sativa L.) blast lesion mimic (blm) mutant for differentially expressed proteins in leaves of one- and two-week-old seedlings manifesting the lesion mimic phenotype. Gelbased one- and two-dimensional electrophoresis (1- and 2-DGE) was performed using leaves (blm and wild-type, WT) before (stage 1, S1) and after (stage 2, S2) lesion formation. 1-DGE immunoblotting revealed potent increase in the expression of a key pathogenesis-related (PR) marker biosynthetic enzyme, naringenin 7-O-methyltransferase, involved in rice phytoalexin sakuranetin biosynthesis, and three oxidative-stress-related marker proteins, catalase, ascorbate peroxidase (APX), and superoxide dismutase (SOD) in leaves of the blm mutant. 2-D gel immunoblotting analysis with anti-APX and antiSOD antibodies revealed newly appearing cross-reacting protein spots in blm. 2-DGE analysis detected 50 Coomassie brilliant blue-stained protein spots differentially expressed in blm. A total of 23 and 44 protein spots was excised for analysis by N-terminal amino acid sequencing and nano-electrospray ionization liquid chromatography mass spectrometry, respectively; 26 nonredundant proteins were identified. The pathogenesis-related class 5 and 10 proteins, including a new OsPR10d protein, were significantly induced in blm. The OsPR5 protein spot was stained with Pro-Q Diamond phosphoprotein gel stain suggesting OsPR5 to be a putative phosphoprotein. Surprisingly, protein spot 20, a leaf OsPR10b, showed identity to a rice root-specific PR-10 (RSOsPR10). To resolve this discrepancy, we checked its expression in leaves of blm and WT (S1 and S2), respectively, using gene-specific primers and reverse transcriptase-polymerase chain reaction; RSOsPR10 mRNA was found to express in the leaves. Keywords: Biomarkers • lesion mimic mutant • Oryza sativa • oxidative stress • proteomics
1. Introduction Rice (Oryza sativa L.) is a staple food crop for more than one-third of the world’s population accounting for 20% of total calories consumed globally in 2000 (Word rice statistics from International Rice Research Institute, http://www.irri.org/science/ricestat/index.asp). The importance of rice in the socio* Authors for correspondence: Dr. Randeep Rakwal, HSS, AIST-WEST, Onogawa 16-1, Tsukuba, Ibaraki 305-8569, Japan. E-mail: rakwal-68@ aist.go.jp. Tel./fax: +81-29-861-8508. Dr. Nam-Soo Jwa, Department of Molecular Biology, College of Natural Science, Sejong University, Seoul 143747, Korea. E-mail:
[email protected]. Tel./fax: +82-31-378-0486/0485. † Sejong University. ‡ National Institute of Advanced Industrial Science and Technology (AIST) WEST. § Research Laboratory for Agricultural Biotechnology and Biochemistry (RLABB). | Seoul National University. ⊥ Ibaraki University.
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Journal of Proteome Research 2006, 5, 2586-2598
Published on Web 08/29/2006
economic fabric of our society makes it one of the most important crop species in South Asia to study, especially on its response to stress and disease, the main research focus of our group. With tremendous advances in new technologies, it is now possible to get more precise and broad information on plant response to a wide variety of biotic and/or abiotic stresses. Rice is a reference plant for the monocots,1 especially cereal crops, and therefore, it was termed a cornerstone for functional genomics of crop plants (for review, see ref 2). Among the “omic pillars” that constitute functional genomics, proteomics is a rapidly expanding field today. Proteomics is one of the high-throughput approaches currently being used to address biological function of plant by studying globally expressed proteins in a given tissue.2-10 Two-dimensional gel electrophoresis (2-DGE) is the most commonly used proteomics technology for monitoring changes 10.1021/pr060092c CCC: $33.50
2006 American Chemical Society
Differential Expression of Defense/Stress-Related Marker Protein
in the expression levels of complex protein mixtures, and is also the most widely utilized.2,8-10 2-DGE can simultaneously separate thousands of proteins (and their modified forms) to homogeneity, deliver high-quality protein resolution and dynamic range, and generate reference maps in a short period of time, and at a cost “affordable” to proteomics researcher. Moreover, 2-DGE, in conjunction with immunological detection techniques, is a very powerful tool to identify a range of proteins for which suitable antibodies are available. Thus, we have every reason to believe that 2-DGE remains one of the most suitable approaches toward the systematic characterization of the plant proteome. Notably, recent use of immobilized pH gradient (IPG) technology for development of high-quality 2-D gel reference maps with greater spot reproducibility is the next target in rice proteomics. Embracing IPG technology for the gel-based approach will make 2-DGE even more attractive soon. It is also emphasized that 2-DGE is not suitable for highand low-masses (>150 and
