Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of Arabidopsis thaliana during UV-induced programmed cell death

ABSTRACT: We have published results demonstrating that UV-C induces apoptotic-like changes in Arabidopsis (Danon and Gallois FEBS lett (1998) 437: 131-136). The Programmed Cell Death "phenotype" of nucleus shape, DNA laddering caspase-like activity is similar to what has been described in other plant PCD. This demonstrates that UV-C is an appropriate and controllable trigger to study PCD in plants. Using selected mutants and a set of chosen conditions we are aiming at identifying genes that are part of the PCD pathways in plants and filter out the general stress response. We are asking for a medium size experiment knowing that additional microarray analysis are likely to be required to refine the results obtained. We are currently awaiting the results of Affymetrix RNAs hybridisation of PCD-induced wild type that will define the appropriate single time point of the proposed analysis. The rationale is to vary treatments in order to distinguish changes in gene transcription arising from general cellular stress responses to the trigger used from those specific to PCD: 1. We will use wild-type Arabidopsis seedlings as a negative control to provide the basal gene expression pattern. 2. A dose of UV-C radiation of 1KJ/m2. We will irradiate wild type with this non PCD-inducing dose of UV-C radiation to identify sets of genes that respond to UV-induced damage but whose expression is not linked to cell death. 3. A dose of UV-C radiation of 50 KJ/m2. We will irradiate wild type with a PCD-inducing dose of UV-C radiation to identify sets of genes that respond to UV-induced damage and cell death. 4. We have shown that the cell death induced by UV is light dependant, the control, sub-inducing dose and inducing dose treatments will be repeated and the plants kept in the dark until RNA sampling. In those conditions there is no PCD.

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: unknown unknown

PROVIDER: E-NASC-27 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:PCD is a highly organised process that is involved in development and in an organisms response to biotic stresses (toxins and avirulent pathogens) and abiotic stresses (such as temperature, water availability, etc.). It is a genetically regulated form of cellular suicide, however in plants the underlying process is poorly understood. Although PCD may occur in response to different stimuli; we believe once it is triggered, one core mechanism is responsible for the cellular demise. It is our aim to identify the elements of this mechanism. We will do this by expanding on the work of a previous user of GARNet's GeneChip microarray facility, Dr. Jodi Swidzinski. She utilised an Arabidopsis cell suspension system; performing microarray analysis on both senescing, and heat shock induced PCD samples. This resulted in data showing that a large number of genes were upregulated (or downregulated) in response to both treatments. We are working under the premise that some of the genes that were similarly regulated under both treatments must be core PCD genes. However because of the large amount of data generated it is difficult to choose appropriate candidate genes (with any degree of confidence that they are core genes) for further study. We propose to use a third PCD-inducing treatment, involving a mycotoxin, to generate another population of microarray results. The mycotoxin we intend to use is Fumonisin B1 (FB1). This is an extremely potent compound that induces PCD by disrupting ceramide synthesis. We have found Arabidopsis protoplasts to be much more sensitive than cells to the toxin at low concentrations. Protoplasts are treated with 20mM FB1 and RNA is extracted at time points when 0%, 20% and 40% of protoplasts have died. This RNA is then pooled. RNA from methanol treated protoplasts is used as a control. We intend for these RNA samples to be subjected to GeneChip microarray analysis. This would identify genes differentially regulated due to FB1 treatment, which would be interesting in itself. However, by combining this data with that from previous work by Swidzinski (2002) we will be able to decrease the pool of possible core genes further, and increase the chances of selecting an appropriate candidate gene. This will both improve upon existing work and add value to an existing GARNet data set.

Project description:UV-B (280-320 nm) exposure causes serious damage in plants, limiting their growth and survival, effects that are partly counteracted by repair mechanisms active in plants receiving accompanying visible radiation. Though no particular UV-B receptor has been identified to date, there is strong evidence to indicate that certain aspects of UV-B perception are receptor-mediated. Investigations of down-stream signalling events have thus far indicated broad similarities to pathogen-induced defence responses in plants. In order to identify genes in Arabidopsis that may be up- or down- regulated specifically in response to UV-B exposure and compare them to genes whose expression is altered in plants challenged by an avirulent isolate of Peronospora parasitica (downy mildew), we propose to analyse the transcriptional profiles for the following treatments: 1. UV-B Responses "A-1" Columbia (Col-0) exposed to supplementary UV-B/UV-A* with a background of low photosynthetically active radiation (PAR of 20 micromol m-2 s-1) for 1.5 photoperiods (photoperiod = 12h). [UV-B treatment] "A-2" Col-0 exposed to supplementary UV-A and low PAR for 1.5 photoperiods [control for UV-B treatment] "A-3" Col-0 exposed to visible light only (low PAR) (no UV) for 1.5 photoperiods [control for UV effects in general].* There are no pure sources of UV-B light available. 2. Pathogen Responses "A-4" Col-0 spray-inoculated with P. parasitica isolate HIKS-1 (recognised by the R-gene RPP7). After spraying, plants were kept covered in plant propagators and transferred to an 18 degreeC growth chamber. Samples for RNA extraction were taken 72h after inoculation. "A-5" The viability of spores was also checked by parallel spraying of the susceptible mutant, Col-rpp7. [pathogen treatment] "A-6" Col-0 mock treated with water, covered and transferred to an 18 degree C growth chamber, 72h prior to sampling. [control for pathogen treatment] In all experiments, we are using RNA from leaves taken at the same time of day (6 h into the 12 h photoperiod) from 4.5-week old plants grown under 12h photoperiod. All treatments were normalised against PR-1 expression levels to ensure comparability between UV-B and pathogen treatments. Due to the difficulty in distinguishing between local and systemic induced responses in UV-B treated plants, we are using RNA from whole rosettes for both the UV-B and pathogen treatment for better comparability among treatments. The degree of similarity between these two sets of transcriptional changes will complement and help interpret our experimental data on changes in resistance to pathogens in plants pre-treated with UV-B. Moreover, the data set obtained would allow for identification of UV-B specific changes in gene expression including cis-acting UV-B-responsive promoter elements.

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Transcription profiling of Arabidopsis thaliana during UV-induced programmed cell death

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