Integrated Transcriptomics, Proteomics, and Metabolomics Analyses To Survey Ozone Responses in the Leaves of Rice Seedling Kyoungwon Cho, † Junko Shibato, ‡ Ganesh Kumar Agrawal, §,¶ Young-Ho Jung, | Akihiro Kubo, † Nam-Soo Jwa, | Shigeru Tamogami, ⊥ Kouji Satoh, ∇ Shoshi Kikuchi, ∇ Tetsuji Higashi, O Shinzo Kimura, # Hikaru Saji, † Yoshihide Tanaka, O Hitoshi Iwahashi, O Yoshinori Masuo, ‡ and Randeep Rakwal* ,‡,§ Environmental Biology Division, National Institute for Environmental Studies, Tsukuba, Ibaraki 305-8506, Japan, Health Technology Research Center (HTRC), National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba West, 16-1 Onogawa, Tsukuba 305-8569, Japan, Research Laboratory for Biotechnology and Biochemistry (RLABB), Kathmandu, Nepal, Department of Molecular Biology, College of Natural Science, Sejong University, Seoul 143-747, Korea, Laboratory of Growth Regulation Chemistry, Department of Biological Production, Akita Prefectural University, Akita 010-0195, Japan, Plant Genome Research Unit, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba 305-8602, Ibaraki, Japan, HTRC, AIST Kansai Center, 1-8-31 Midorigaoka, Ikeda 563-8577, Japan, and Hazard Assessment and Epidemiology Research Group, Japan National Institute of Occupational Safety and Health (Japan NIOSH), Kawasaki 214-8585, Japan Received February 15, 2008 Ozone (O 3 ), a serious air pollutant, is known to significantly reduce photosynthesis, growth, and yield and to cause foliar injury and senescence. Here, integrated transcriptomics, proteomics, and metabolomics approaches were applied to investigate the molecular responses of O 3 in the leaves of 2-week-old rice (cv. Nipponbare) seedlings exposed to 0.2 ppm O 3 for a period of 24 h. On the basis of the morphological alteration of O 3 -exposed rice leaves, transcript profiling of rice genes was performed in leaves exposed for 1, 12, and 24 h using rice DNA microarray chip. A total of 1535 nonredundant genes showed altered expression of more than 5-fold over the control, representing 8 main functional categories. Genes involved in information storage and processing (10%) and cellular processing and signaling categories (24%) were highly represented within 1 h of O 3 treatment; transcriptional factor and signal transduction, respectively, were the main subcategories. Genes categorized into information storage and processing (17, 23%), cellular processing and signaling (20, 16%) and metabolism (18, 19%) were mainly regulated at 12 and 24 h; their main subcategories were ribosomal protein, post- translational modification, and signal transduction and secondary metabolites biosynthesis, respectively. Two- dimensional gel electrophoresis-based proteomics analyses in combination with tandem mass spectrometer identified 23 differentially expressed protein spots (21 nonredundant proteins) in leaves exposed to O 3 for 24 h compared to respective control. Identified proteins were found to be involved in cellular processing and signaling (32%), photosynthesis (19%), and defense (14%). Capillary electrophoresis-mass spectrometry-based metabo- lomic profiling revealed accumulation of amino acids, gamma-aminobutyric acid, and glutathione in O 3 exposed leaves until 24 h over control. This systematic survey showed that O 3 triggers a chain reaction of altered gene, protein and metabolite expressions involved in multiple cellular processes in rice. Keywords: ozone • oxidative stress • jasmonic acid • ethylene • gel-based approach • mass spectrometry • DNA microarray 1. Introduction Ozone (O 3 ) is produced by the photochemical reactions of volatile organic compounds with nitrogen oxides in the tro- posphere. Annually, used fossil fuels have further increased the ground-level of O 3 causing serious biological and environmen- tal problems. In plants, gaseous O 3 enters a leaf through the stomata, reacts with water, spontaneously generates the reac- tive oxygen species (ROS) leading to multiple and complex oxidative injuries. 1–6 Plants exposed to O 3 over long period manifest early senescence (a chronic symptom) and reduction of photosynthesis, growth rate and crop yields. But a high-dose of O 3 to plants even for short period causes visible lesions on leaves (an acute symptom) similar to hypersensitive cell death. 2,6,7 O 3 -triggered molecular responses in plants have been investigated mainly in Arabidopsis, a dicot model (Arabidopsis thaliana) plant. 2,6,7 However, considering the importance of rice as a model plant for socio-economic crop transcriptomes and proteomes, 8,9 O 3 responses in rice (Oryza sativa L.), a monocot genome model plant, 10 remain largely unknown. * To whom correspondence should be addressed. Dr. Randeep Rakwal, HTRC, AIST, Tsukuba West, 16-1 Onogawa, Tsukuba 305-8569, Japan. E-mail, rakwal-68@aist.go.jp; fax, +81-29-861-8508. † Environmental Biology Division, National Institute for Environmental Studies. ‡ Health Technology Research Center (HTRC), National Institute of Advanced Industrial Science and Technology (AIST). ¶ Present address: University of Missouri-Columbia, Biochemistry Depart- ment, 204 Life Sciences Center, Columbia, MO 65211, USA. § Research Laboratory for Biotechnology and Biochemistry (RLABB). | Department of Molecular Biology, Sejong University. ⊥ Department of Biological Production, Akita Prefectural University. ∇ Plant Genome Research Unit, National Institute of Agrobiological Sciences. O HTRC, AIST Kansai Center. # Hazard Assessment and Epidemiology Research Group, Japan National Institute of Occupational Safety and Health (Japan NIOSH). 2980 Journal of Proteome Research 2008, 7, 2980–2998 10.1021/pr800128q CCC: $40.75  2008 American Chemical Society Published on Web 06/03/2008