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Cold induced changes: differences between the Arabidopsis thaliana wild type and freezing sensitive mutants.

ABSTRACT: Arabidopsis sfr mutants are deficient in cold acclimation during exposure to coolnon-freezing temperatures. Although not visibly affected by the cold they have lost the ability to survive subsequent freezing. We plan to investigate how the sfr2 and sfr6 mutants respond to low temperature on the gene expression level. Wild type plants that have undergone identical treatments in parallel are necessary controls. The cold treatment of plants in the rosette stage (soil grown in a 8/16 hours day/night cycle) will be carried out in a cooled growth chamber at 4 degrees for 24 hours (same light regimetreatment starting/ending at the 4th hour of light). The aerial parts of the treated and untreated plants will be collected and frozen immediately in liquid nitrogen for RNA extraction. Comparison of the cold response of thousands of Arabidopsis genes in the wild type to the situation in our freezing sensitive mutants will enhance our understanding of the cold response itself and illuminate the effect of the mutations on the cold acclimation process. Experimenter name = Irene Bramke Experimenter phone = 01784 44 3770 Experimenter fax = 01784 43 4326 Experimenter address = Royal Holloway Experimenter address = University of London Experimenter address = School of Biological Sciences Experimenter address = Bourne Building Experimenter address = Laboratory 406 Experimenter zip/postal_code = TW20 OEX Experimenter country = UK Keywords: growth_condition_design; genetic_modification_design

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE5711 | GEO | 2007/01/08

SECONDARY ACCESSION(S): PRJNA96999

REPOSITORIES: GEO

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Project description:The sfr6 mutant was identified on the basis of its failure to cold acclimate, and exhibits a marked deficiency in cold-and osmotic stress-inducible gene expression (Knight et al., 1999). We have demonstrated that genes of this type (so-called COR genes) are misregulated if they contain the DRE (drought-responsive element, or CRT; C-repeat) cis acting element in their promoter (Boyce et al., 2003). Micro-array analysis has allowed us to identify a number of COR genes misregulated in sfr6, all of which contain the DRE element. However, these experiments have indicated that other genes, not of the COR group, are also misregulated in the mutant and these do not contain the DRE element. We chose the three non-COR genes that were most clearly down-regulated in sfr6 on our previous micro-array, and identified each as of these as dark-inducible. We now seek to address two questions: (1) Can we discover more dark-regulated genes that are down-regulated in the mutant, and thus identify a second cis-acting element with which SFR6 may be interacting? (2) Do all of the non-COR genes that are misregulated in sfr6 fall into the category of dark-inducible, or is SFR6 likely to be interacting with three or more regulons? For this micro-array experiment, we will subject sfr6 and wild type Arabidopsis, grown in a 16/8-h light/dark cycle, to darkness for 3h or 6h at ambient growth temperature. Under these conditions we see strong up-regulation of the three genes we have identified as dark-inducible, and we see clear down-regulation of expression in sfr6. References. Knight H, Veale E, Warren GJ, Knight MR (1999) The sfr6 mutation in Arabidopsis suppresses low-temperature induction of genes dependent on the CRT/DRE sequence motif. Plant Cell 11: 875-886. Boyce JM, Knight H, Deyholos M, Openshaw MR, Galbraith DW, Warren G, Knight MR (2003). The sfr6 mutant of Arabidopsis is defective in transcriptional activation via CBF/DREB1 and DREB2 and shows sensitivity to osmotic stress. Plant Journal 34: 395-406. Experimenter name = Heather Knight Experimenter phone = 01865 275023 Experimenter fax = 01865 275074 Experimenter institute = University of Oxford Experimenter address = Department of Plant Sciences Experimenter address = University of Oxford Experimenter address = South Parks Road Experimenter address = Oxford Experimenter zip/postal_code = OX1 3RB Experimenter country = UK Keywords: growth_condition_design; genetic_modification_design

Project description:The sfr6 mutant was identified on the basis of its failure to cold acclimate, and exhibits a marked deficiency in cold-and osmotic stress-inducible gene expression (Knight et al., 1999). We have demonstrated that genes of this type (so-called COR genes) are misregulated if they contain the DRE (drought-responsive element, or CRT; C-repeat) cis acting element in their promoter (Boyce et al., 2003). Micro-array analysis has allowed us to identify a number of COR genes misregulated in sfr6, all of which contain the DRE element. However, these experiments have indicated that other genes, not of the COR group, are also misregulated in the mutant and these do not contain the DRE element. We chose the three non-COR genes that were most clearly down-regulated in sfr6 on our previous micro-array, and identified each as of these as dark-inducible. We now seek to address two questions: (1) Can we discover more dark-regulated genes that are down-regulated in the mutant, and thus identify a second cis-acting element with which SFR6 may be interacting? (2) Do all of the non-COR genes that are misregulated in sfr6 fall into the category of dark-inducible, or is SFR6 likely to be interacting with three or more regulons?; For this micro-array experiment, we will subject sfr6 and wild type Arabidopsis, grown in a 16/8-h light/dark cycle, to darkness for 3h or 6h at ambient growth temperature. Under these conditions we see strong up-regulation of the three genes we have identified as dark-inducible, and we see clear down-regulation of expression in sfr6. References. Knight H, Veale E, Warren GJ, Knight MR (1999) The sfr6 mutation in Arabidopsis suppresses low-temperature induction of genes dependent on the CRT/DRE sequence motif. Plant Cell 11: 875-886. Boyce JM, Knight H, Deyholos M, Openshaw MR, Galbraith DW, Warren G, Knight MR (2003). The sfr6 mutant of Arabidopsis is defective in transcriptional activation via CBF/DREB1 and DREB2 and shows sensitivity to osmotic stress. Plant Journal 34: 395-406. Experimenter name = Heather Knight; Experimenter phone = 01865 275023; Experimenter fax = 01865 275074; Experimenter institute = University of Oxford; Experimenter address = Department of Plant Sciences; Experimenter address = University of Oxford; Experimenter address = South Parks Road; Experimenter address = Oxford; Experimenter zip/postal_code = OX1 3RB; Experimenter country = UK Experiment Overall Design: 4 samples were used in this experiment

Project description:Our analysis of the sfr6 freezing-sensitive mutant (Knight, H., Veale, E., Warren, G. J. and Knight, M. R. (1999). Plant Cell 11, 875-886.) and cls8 (unpublished) chilling-sensitive mutant of Arabidopsis, has revealed that the expression of certain cold-regulated genes is aberrant in both these mutants. In order to understand the molecular basis of chilling and freezing stress in Arabidopsis and also to determine commonalities and differences between these 2 different physiological stress-tolerance processes, we request transcriptome analysis for both of these mutants compared to wild type in one experiment, upon cold treatment and at ambient conditions. The sfr6 mutant shows the most severe phenotype with respect to cold gene expression, but is tolerant to chilling (Knight, H., Veale, E., Warren, G. J. and Knight, M. R. (1999). Plant Cell 11, 875-886.). However, it is unable to cold acclimate and hence is sensitive to freezing. The cls8 mutant, on the other hand, has a relatively mild phenotype relative to the cold-regulated genes we have examined, but is very sensitive to chilling temperatures (15 to 10 degree centigrade). It is thus likely that in cls8 we have not yet identified the genes which are most affected, and which account for the physiological phenotype. Both sfr6 and cls8 have been fine-mapped and are close to being cloned. The cls8 mutant has an altered calcium signature in response to cold which means it is likely to be affected in early signalling, e.g. cold perception itself.We will compare the expression profiles of genes in sfr6, cls8 and Columbia (parental line for both mutants), both at ambient, and after treatment with cold (5 degrees) for 3 hours. This timepoint is designed to ÂcaptureÂ both rapidly responding genes e.g. CBF/DREB1 transcription factors, and also more slow genes e.g. COR genes (KIN1/2 and LTI78). Pilot northerns confirm that this time point is suitable.This analysis will provide new insight into 2 novel genes required for tolerance to low temperature in Arabidopsis, and additionally will determine the nature of overlap between the separate processes of chilling and freezing tolerance. Experiment Overall Design: Number of plants pooled:40-60

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Cold induced changes: differences between the Arabidopsis thaliana wild type and freezing sensitive mutants.

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