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Comparative Analysis of Seed Transcriptomes of Ambient Ozone-Fumigated Two Different Rice Cultivars

ABSTRACT: High ozone (O3) concentration causes serious damages in plant productivity. Climate models forecast that ground O3 level in the future will reach phytotoxic range, resulting in crop yield losses. With an ultimate goal to screen molecular factors to minimize losses of crop production by the rise of O3 level, we have started an investigation on effects of O3 on rice using rice DNA chip. Herein, we have utilized the samples of dry mature rice seeds harvested in an ozone-sensitive rice cultivar (Oryza sativa L. indica cv. Takanari) and a tolerant cultivar (Oryza sativa L. japonica cv. Koshihikari) which were fumigated with ambient air (mean O3: 32.7 ppb) in small open-top chambers (OTCs). First, we extracted total RNA from dry mature rice seeds of Takanari and Koshihikari using a modified protocol based on cethyltrimethylammonium bromide extraction buffer and phenol-chloroform-isoamylalcohol treatment. Furthermore, to perform microarray analysis using the Agilent 4x44 rice DNA Chip and the dye-swap method, we designed a balanced block design comparing seeds in an ambient air-fumigated rice cultivar and those in a filtered air-fumigated rice cultivar. Direct comparison of Koshihikari and Takanari O3 transcriptomes in seeds of rice plants fumigated with ambient O3 in OTCs successfully showed that genes encoding proteins involved in jasmonic acid, GABA biosynthesis, cell wall and membrane modification, starch mobilization, and secondary metabolite biosynthesis are differently regulated in an O3-sensitive cv. Takanari and a tolerant cv. Koshihikari.

ORGANISM(S): Oryza sativa

PROVIDER: GSE49963 | GEO | 2013/08/20

SECONDARY ACCESSION(S): PRJNA215708

REPOSITORIES: GEO

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Project description:In this study, integrated transcriptomics, proteomics and metabolomics approaches were applied to investigate the molecular responses of O3 in the leaves of two-weeks old rice (cv. Nipponbare) seedlings exposed to 0.2 ppm O3 for a period of 24 h. Based on the morphological alteration of O3-exposed rice leaves, transcript profiling of rice genes was performed in leaves exposed for 1, 12 and 24 h using rice DNA microarray chip, proteomics and metabolomics. This systematic survey showed that O3 triggers a chain reaction of altered gene, protein and metabolite expressions involved in multiple cellular processes in rice. Also investigated were the molecular responses in the leaves of two-weeks old rice (cv. Nipponbare) seedlings under continuous light and pure air (as a positive control for ozone exposure experiments) for a period of 24 h. Transcript profiling of rice genes was performed in leaves exposed for 1, 12 and 24 h using rice DNA microarray chip, proteomics and metabolomics. This systematic survey showed that continuous light and growth for 24 h also triggers a chain reaction of altered gene expressions involved in multiple cellular processes in rice, but different from those against ozone, in general. Experiment Overall Design: Ozone-treated Samples: An in vivo 14-days-old rice seedling model system was used, where whole seedlings were used for exposure/fumigation to the ozone (0.2 ppm). Dye-swap or reverse labeling with Cy3 and Cy5 dyes procedure was applied followed by hybridization and wash processes, and the hybridized microarrays (G4138A) were scanned using a GenePix microarray scanner followed by the Gene Pix 4000 analysis application program for image analysis and data extraction processes. The GeneSpring Ver. 4 software was used for normalization. Experiment Overall Design: Control Samples: An in vivo 14-days-old rice seedling model system was used, where whole seedlings were used for exposure to continuous light and pure air. Dye-swap or reverse labeling with Cy3 and Cy5 dyes procedure was applied followed by hybridization and wash processes, and the hybridized microarrays (G4138A) were scanned using a Standard protocol of Agilent DNA Microarray Scanner and data processing was done by Feature Extraction software Version 8.1 from Agilent Technologies.

Project description:A combined approach of evaluating ozone (O3)-caused foliar injury symptom and global gene expression profiling was used to identify potential genes associated with severity of injury on leaves of O3 (200 ppb)-fumigated (1, 12, 24, 48, and 72 h) two-week-old rice (cv. Nipponbare) seedling along with appropriate control. Foliar injuries were evaluated up to 72 h using both qualitative visual scale and quantitative RGB (red-green-blue) image analysis methods. The (R-G)/(R+G) was found as the optimal quantitative RGB parameter to assess the foliar injury. Large-scale transcript profiling of leaves identified 270 genes linked with foliar injury. Of these genes, the expression levels of 139 genes showed significant differences (P < 0.05) between leaves without and with injury symptoms. When a rigorous correlation test was applied on these genes for their expression changes and relative (R-G)/(R+G) parameters, the expression of 93 genes were found to increase with increased foliar severity up to 72 h, showing a positive, tight correlation between a subset of gene expression and commonly observed O3-triggerred symptom of foliar injury with correlation coefficient below -0.80. Of 93 genes, genes involved in metabolism (29%) formed a major functional category. Reconstruction of the metabolic networks with identified metabolic genes provided insight into the cellular responses such as photorespiration, biosynthesis of secondary metabolites, and detoxification. The O3 effect on these cellular responses has been previously reported based on physiological and biochemical studies, validating our approach used in this study to globally identify O3-responsive biomarkers tightly linked with foliar injury symptom. This study provides evidence for the presence of large number of genes associated with the foliar injury symptom than thought before, and could serve as a resource of potential biomarkers to study mechanisms of visible injury development by O3. Comparison between healthy rice seedling leaves and ozone treated (for 48 h and 72 h) rice seedling leaves was performed. Three biological replicates (5 leaves in each replicate; pooled) were used, and dye-swaped.

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Comparative Analysis of Seed Transcriptomes of Ambient Ozone-Fumigated Two Different Rice Cultivars

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