Project description: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
Project description:Arabidopsis thaliana and Eutrema salsugineum show the ability to cold acclimate. However, the degree of freezing tolerance depends in both cases on the accession. To elucidate the transcriptional basis of this differencial freezing tolerance, we performed where we grew plants under control conditions (20°C/18°C day/night) or under cold conditions (additional 4°C for 2 weeks). Rosettes were harvested from non-acclimated and cold acclimated plants for RNA isolation. Expression patterns were compared between treatments, accessions and species.
Project description: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 Experiment Overall Design: 6 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 capture 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. Keywords: strain_or_line_design
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 ÃÂcaptureÃÂ 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.
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 Âcapture 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
Project description:Brassinosteroids (BRs) are growth-promoting plant hormones that play a role in abiotic stress responses, but molecular modes that enable this activity remain largely unknown. Here we show that BRs participate in the regulation of freezing tolerance. BR signaling-defective mutants of Arabidopsis thaliana were hypersensitive to freezing before and after cold acclimation. The constitutive activation of BR signaling, in contrast, enhanced freezing resistance. Evidence is provided that the BR-controlled basic helix–loop–helix transcription factor CESTA (CES) can contribute to the constitutive expression of the C-REPEAT/DEHYDRATION-RESPONSIVE ELEMENT BINDING FACTOR (CBF) transcriptional regulators that control cold responsive (COR) gene expression. In addition, CBF-independent classes of BR-regulated COR genes are identified that are regulated in a BR- and CES-dependent manner during cold acclimation. A model is presented in which BRs govern different cold-responsive transcriptional cascades through the posttranslational modification of CES and redundantly acting factors. This contributes to the basal resistance against freezing stress, but also to the further improvement of this resistance through cold acclimation. We used microarray data to investigate the contribution of different pathways to cold tolerance of Arabidopsis thaliana .
Project description:Plants in temperate regions have evolved mechanisms to survive sudden temperature drops. Previous reports have indicated that the cold acclimation mechanism is light-dependent and does not fully operate under a low light intensity. In these studies, plants were grown under a long-day photoperiod and were more sensitive to freezing stress. However, winter annuals like Arabidopsis thaliana Col-0 germinate in the fall, overwinter as rosettes, and therefore must acclimate under short photoperiods and low irradiance. The role of light intensity was analysed in plants grown under a short-day photoperiod at the growth stage 1.14. Plants were acclimated at 4 °C for seven days under 100 and 20 μmol m-2s-1 PPFD for control and limited-light conditions, respectively. All cold acclimated plants accumulated molecular markers reportedly associated with acquired freezing tolerance, including proline, sucrose, CBFs, and COR gene protein products dehydrins and low-temperature-responsive proteins LTIs. Observed changes indicated that low PPFD did not inhibit the cold acclimation process, and the freezing stress experiment confirmed similar survival rates. The molecular analysis found distinct PPFD-specific adaptation mechanisms that were manifested in contrasting content of anthocyanins, cytokinin conjugates, abundances of proteins forming photosystems, and enzymes of protein, energy, and ROS metabolism pathways. Finally, this study led to the identification of putative proteins and metabolite markers correlating with susceptibility to freezing stress of non-acclimated plants grown under low PPFD. Our data show that Arabidopsis plants grown under short-day photoperiod can be fully cold-acclimated under limited light conditions, employing standard and PPFD-specific pathways.
Project description:St (common potato) is a freezing sensitive species unable to cold acclimate. The close wild relative Sc is freezing tolerant and able to cold acclimate. Here we compare the cold transcriptome of these two species with different levels of freezing tolerance. We also identify the putative CBF regulons by comparing the transcriptomes of wild type plants with that of 35S::AtCBF3 transgenic lines in both species.
Project description:During cold acclimation plants increase their freezing tolerance in response to low non-freezing temperatures. This is accompanied by many physiological, biochemical and molecular changes that have been extensively investigated. In addition, many cold acclimated plants become more freezing tolerant during exposure to mild, non-damaging sub-zero temperatures. There is hardly any information available about the molecular basis of this adaptation. However, Arabidopsis thaliana is among the species that acclimate to sub-zero temperatures. This makes it possible to use the molecular and genetic tools available in this species to identify components of sub-zero signal transduction and acclimation. Here, we have used microarrays and a qRT-PCR primer platform covering 1880 genes encoding transcription factors to monitor changes in gene expression in the accessions Columbia-0, Rschew and Tenela during the first three days of sub-zero acclimation at -3°C. The results indicate that gene expression during sub-zero acclimation follows a tighly controlled time-course. Especially AP2/EREBP and WRKY transcription factors may be important regulators of sub-zero acclimation, although the CBF signal transduction pathway seems to be less important during sub-zero than during cold acclimation. Globally, we estimate that approximately 5% of all Arabidopsis genes are regulated during sub-zero acclimation. Particularly photosynthesis-related genes were down-regulated and genes belonging to the functional classes of cell wall biosynthesis, hormone metabolism and RNA regulation of transcription were up-regulated. Collectively, these data provide the first global analysis of gene expression during sub-zero acclimation and allow the identification of candidate genes for forward and reverse genetic studies into the molecular mechanisms of sub-zero acclimation. We used whole genome microarrays to monitor changes in gene expression in the Arabidopsis thaliana accessions Columbia-0, Rschew and Tenela during three days of acclimation to sub-zero temperature at -3°C after cold acclimation