Kyoungwon Cho

Integrated transcriptomics, proteomics, and metabolomics analyses to survey ozone responses in the leaves of rice seedling.

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 metabolomic 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.

Rice OsACDR1 ( Oryza sativa accelerated cell death and resistance 1) is a potential positive regulator of fungal disease resistance

Rice Oryza sativa accelerated cell death and resistance 1 (OsACDR1) encodes a putative Raf-like mitogen-activated protein kinase kinase kinase (MAPKKK). We had previously reported upregulation of the OsACDR1 transcript by a range of environmental stimuli involved in eliciting defense-related pathways. Here we apply biochemical, gain and loss-of-function approaches to characterize OsACDR1 function in rice. The OsACDR1 protein showed autophosphorylation and possessed kinase activity. Rice plants overexpressing OsACDR1 exhibited spontaneous hypersensitive response (HR)-like lesions on leaves, upregulation of defense-related marker genes and accumulation of phenolic compounds and secondary metabolites (phytoalexins). These transgenic plants also acquired enhanced resistance to a fungal pathogen (Magnaporthe grisea) and showed inhibition of appressorial penetration on the leaf surface. In contrast, loss-offunction and RNA silenced OsACDR1 rice mutant plants showed downregulation of defense-related marker genes expressions and susceptibility to M. grisea. Furthermore, transient expression of an OsACDR1:GFP fusion protein in rice protoplast and onion epidermal cells revealed its localization to the nucleus. These results indicate that OsACDR1 plays an important role in the positive regulation of disease resistance in rice.

Kinetics of wound-induced activation of antioxidative enzymes in Oryza sativa: differential activation at different growth stages

Abstract Superoxide dismutase, peroxidase and catalase are major antioxidative enzymes that contribute to the oxidative stress response in plants. The activity of these enzymes was measured in wound-stressed rice plants that were wounded at seedling stage, maximum tillering stage or flowering stage. Kinetics of enzyme activity were examined in leaf extracts post-wounding. Superoxide dismutase activity increased rapidly until 6 h after wounding and was maintained at a high level throughout development. Wound stress-induced changes in manganese superoxide dismutase and copper–zinc superoxide dismutase activities were detected using a native activity gel. In contrast, catalase was rapidly deactivated and peroxidase was transiently activated and then deactivated after wounding. However, high peroxidase activity was maintained at maximum tillering stage of development. These observations suggest that rice antioxidant enzymes are differentially activated by wound stress depending on the plant growth stage, and that the antioxidative enzymes are activated by wounding in two phases. The first phase involves activation of superoxide dismutase and peroxidase and rapid deactivation of catalase. The second phase involves deactivation of peroxidase under conditions of severe stress. Deactivation of peroxidase and catalase may lead to accumulation of hydrogen peroxide, which can activate of a series of additional defense mechanisms.

Protein extraction/solubilization protocol for monocot and dicot plant gel-based proteomics

Sample preparation in plant proteomics is tedious, requiring modifications depending on the type of tissue involved. Here, we describe a protein extraction protocol for both monocotyledonous (monocot) and dicotyledonous (dicot) species, which significantly improves the solubilization of total proteins. For example, we used the primary leaf tissue and seeds from rice, a cereal crop and genome model system. Total protein was first precipitated with trichloroacetic acid/acetone extraction buffer (TCAAEB) and subsequently solubilized with a modified O’Farrell lysis buffer (LB) containing thiourea and tris (LB-TT). Separation of total leaf proteins by two-dimensional gel electrophoresis (2-DGE) revealed improved solubilization, as determined by an increased number of spots detected with Coomassie brilliant blue (CBB) staining. In addition, the resolution was better than when LB-TT was used alone for protein extraction. Seed proteins could be extracted in LB-TT itself without the need for TCAAEB, which resulted in a highly insoluble precipitate. Our CBB-stained 2-D gel protein profiles also demonstrated the efficacy of this protocol for total protein extraction/solubilization from the dicot genome model (Arabidopsis), a dicot disease model (cucumber), and two other important monocot cereal crop models (maize and wheat). Moreover, this is the first report on generating a 2-D gel proteome profile for wheat crown and cucumber leaf tissues. Finally, as examples of proteome reference maps, we obtained silver nitrate-stained, large-format 2-D gels for rice leaf and wheat crown LB-TT solubilized proteins.

Unraveling Low-Level Gamma Radiation–Responsive Changes in Expression of Early and Late Genes in Leaves of Rice Seedlings at litate Village, Fukushima

In the summer of 2012, 1 year after the nuclear accident in March 2011 at the Fukushima Daiichi nuclear power plant, we examined the effects of gamma radiation on rice at a highly contaminated field of Iitate village in Fukushima, Japan. We investigated the morphological and molecular changes on healthy rice seedlings exposed to continuous low-dose gamma radiation up to 4 µSv h(-1), about 80 times higher than natural background level. After exposure to gamma rays, expression profiles of selected genes involved in DNA replication/repair, oxidative stress, photosynthesis, and defense/stress functions were examined by RT-PCR, which revealed their differential expression in leaves in a time-dependent manner over 3 days (6, 12, 24, 48, and 72 h). For example, OsPCNA mRNA rapidly increased at 6, 12, and 24 h, suggesting that rice cells responded to radiation stress by activating a gene involved in DNA repair mechanisms. At 72 h, genes related to the phenylpropanoid pathway (OsPAL2) and cell death (OsPR1oa) were strongly induced, indicating activation of defense/stress responses. We next profiled the transcriptome using a customized rice whole-genome 4×44K DNA microarray at early (6h) and late (72 h) time periods. Low-level gamma radiation differentially regulated rice leaf gene expression (induced 4481 and suppressed 3740 at 6 h and induced 2291 and suppressed 1474 genes at 72 h) by at least 2-fold. Using the highly upregulated and downregulated gene list, MapMan bioinformatics tool generated diagrams of early and late pathways operating in cells responding to gamma ray exposure. An inventory of a large number of gamma radiation-responsive genes provides new information on novel regulatory processes in rice.

Cellular localization of dual positional specific maize lipoxygenase-1 in transgenic rice and calcium-mediated membrane association

The dual positional maize lipoxygenase-1 was introduced into rice and T2 transgenic plants were produced. Cellular location of maize lipoxygenase-1 in transgenic rice and effects of calcium ion on membrane association in vitro were analyzed. Localization study by confocal microscopic analysis indicated that the maize lipoxygenase-1 was localized in cytoplasm. Sucrose-density fractionation experiment and in vitro protein transport to chloroplast showed that the maize lipoxygenase-1 can be associated with chloroplast. Secondary structure alignment revealed putative calcium binding sites in the PLAT domain of maize lipoxygenase-1 and the association of the maize lipoxygenase-1 with membranes was mediated by calcium ion in vitro. Our results provide evidences for calcium-mediated translocation of dual positional LOX without chloroplast targeting sequence from cytoplasm to chloroplast in plants for the first time.

Transgenic expression of dual positional maize lipoxygenase-1 leads to the regulation of defense-related signaling molecules and activation of the antioxidative enzyme system in rice

Effects of transgenic expression of dual positional maize lipoxygenase-1 on the defense system were analyzed in rice. The activities of hydroperoxidelyase and antioxidative enzymes (superoxide dismutase, catalase, peroxidase) were increased and high levels of aldehydes including malondialdehyde were produced. The constitutive level of jasmonic was slightly increased and the constitutive salicylic acid level was decreased. Kinetic analysis of wound response indicated that the levels of jasmonic acid and salicylic acid are inversely correlated in nully transgenic rice plants, suggesting that there is an antagonistic interaction between jasmonic acid and salicylic acid. Microarray analysis indicated that several defense-related genes encoding antioxidative enzymes and pathogen-related proteins were up-regulated, and the resistance to rice blast fungus was enhanced in transgenic rice. Taken together, our results suggest that maize lipoxygenase-1 expressed in the cytoplasm plays an important role for the regulation of defense system including the antioxidative enzymes in transgenic rice, and that these effects may be mediated by reactive oxygen species generated through the enzyme-initiated catalytic peroxidation mechanism of maize lipoxygenase-1.

Genome-wide mapping of the ozone-responsive transcriptomes in rice panicle and seed tissues reveals novel insight into their regulatory events

The ‘ozone (O3)-responsive transcriptome’ behavior in the panicles and grains of rice plant was studied individually through high-throughput oligo-DNA microarray technique. O3 differentially and separately regulated 620 and 130 genes in the panicles and grains. Among the O3-responsive genes, 176 and 444 genes were up- and down-regulated in panicle compared to 24 and 106 genes in grain, respectively. Further mapping revealed that the majority of differentially expressed genes were mainly involved in signaling, hormonal, cell wall, transcription, proteolysis, and defense events. Many previously unknown O3-responsive novel genes were identified. Inventory of 745 O3-responsive genes and their mapping will expand our knowledge on novel regulatory processes in both panicles and grains of rice; and, serve as a resource towards the designing of rice crops for future high-O3world.Purpose of workTropospheric ozone (O3) severely affects agricultural production worldwide. Present study aims to reveal a detailed O3 responsive gene network in panicle and grains of rice plants through transcriptomics approach. Our results provide an insight into the basis of O3-response in rice plants, and will help to develop suitable rice genotype for future high O3- world.

Changes in metabolic markers in insulin-producing β-cells during hypoxia-induced cell death as studied by NMR metabolomics.

This study was designed to investigate changes in the metabolites in the intracellular fluid of the pancreatic β-cell line INS-1 to identify potential early and late biomarkers for predicting hypoxia-induced cell death. INS-1 cells were incubated under normoxic conditions (95% air, 5% CO₂) or hypoxic conditions (1% O₂, 5% CO₂, 95% N₂) for 2, 4, 6, 12, or 24 h. The biological changes indicating the process of cell death were analyzed using the MTT assay, flow cytometry, Western blotting, and immunostaining. Changes in the metabolic profiles from cell lysates were identified using ¹H nuclear magnetic resonance (¹H NMR) spectroscopy, and the spectra were analyzed by the multivariate model Orthogonal Projections to Latent Structure-Discriminant Analysis. Cell viability decreased approximately 40% after 12-24 h of hypoxia, coincident with a high level of cleaved caspase-3. A high level of HIF-1α was detected in the 12-24 h hypoxic conditions. The metabolite profiles were altered according to the degree of exposure to hypoxia. A spectral analysis showed significant differences in creatine-containing compounds at the early stage (2-6 h) and taurine-containing compounds at the late stage (12-24 h), with the detection of HIF-1α and cleaved caspase-3 in cells exposed to hypoxia compared to normoxia. Glycerophosphocholine decreased during the early stage hypoxia. The change in taurine- and creatine-containing compounds and choline species could be involved in the β-cell death process as inhibitors or activators of cell death. Our results imply that assessment by ¹H NMR spectroscopy would be a useful tool to predict the cell death process and to identify molecules regulating hypoxia-induced cell death mechanisms.

Rice Proteomic Analysis: Sample Preparation for Protein Identification

Rice is one of the most important food and cereal crop plants in the world. Rice proteomics began in the 1990s. Since then, considerable progress has been made in establishing protocols from isolation of rice proteins from different tissues, organs, and organelles, to separation of complex proteins and to their identification by mass spectrometry. Since the year 2000, global proteomics studies have been performed during growth and development under numerous biotic and abiotic environmental conditions. Two-dimensional (2-D) gel-based proteomics platform coupled with mass spectrometry has been retained as the workhorse for proteomics of a variety of rice samples. In this chapter, we describe in detail the different protocols used for isolation of rice proteins, their separation, detection, and identification using gel-based proteomics and mass spectrometry approaches.