Project description:Reactive oxygen species (ROS) are key signalling molecules that regulate growth and development and coordinate responses to biotic and abiotic stresses. ROS homeostasis is controlled through a complex network of ROS production and scavenging enzymes. Recently, the first genes involved in ROS perception and signal transduction have been identified and, currently, we are facing the challenge to uncover the other players within the ROS regulatory gene network. The specificity of ensuing cellular responses depends on the type of ROS and their subcellular production sites. Various experimental systems, including catalase-deficient plants, in combination with genome-wide expression studies demonstrated that increased hydrogen peroxide (H2O2) levels significantly affect the transcriptome of plants and are capable of launching both defence responses and cell death events. We used microarrays to assess differential gene expression provoked by H2O2 from plastid or peroxisomal origin, respectively.
Project description:In plants, reactive oxygen species and, more particularly, hydrogen peroxide (H2O2) play a dual role as toxic by-products of normal cell metabolism and as regulatory molecules in stress perception and signal transduction. Peroxisomal catalases are an important sink for photorespiratory H2O2. Using ATH1 Affymetrix microarrays, expression profiles were compared between control and catalase-deficient Arabidopsis (Arabidopsis thaliana) plants. Reduced catalase levels already provoked differences in nuclear gene expression under ambient growth conditions, and these effects were amplified by high light exposure in a sun simulator for 3 and 8 h. This genome-wide expression analysis allowed us to reveal the expression characteristics of complete pathways and functional categories during H2O2 stress. In total, 349 transcripts were significantly up-regulated by high light in catalase-deficient plants and 88 were down-regulated. From this data set, H2O2 was inferred to play a key role in the transcriptional up-regulation of small heat shock proteins during high light stress. In addition, several transcription factors and candidate regulatory genes involved in H2O2 transcriptional gene networks were identified. Comparisons with other publicly available transcriptome data sets of abiotically stressed Arabidopsis revealed an important intersection with H2O2-deregulated genes, positioning elevated H2O2 levels as an important signal within abiotic stress-induced gene expression. Finally, analysis of transcriptional changes in a combination of a genetic (catalase deficiency) and an environmental (high light) perturbation identified a transcriptional cluster that was strongly and rapidly induced by high light in control plants, but impaired in catalase-deficient plants. This cluster comprises the complete known anthocyanin regulatory and biosynthetic pathway, together with genes of hitherto unknown function.
Project description:Reactive oxygen species (ROS) are key signalling molecules that regulate growth and development and coordinate responses to biotic and abiotic stresses. ROS homeostasis is controlled through a complex network of ROS production and scavenging enzymes. Recently, the first genes involved in ROS perception and signal transduction have been identified and, currently, we are facing the challenge to uncover the other players within the ROS regulatory gene network. The specificity of ensuing cellular responses depends on the type of ROS and their subcellular production sites. Various experimental systems, including catalase-deficient plants, in combination with genome-wide expression studies demonstrated that increased hydrogen peroxide (H2O2) levels significantly affect the transcriptome of plants and are capable of launching both defence responses and cell death events. We used microarrays to assess differential gene expression provoked by H2O2 from plastid or peroxisomal origin, respectively. Columbia-0 (Col-0, wild type), catalase-deficient Salk plants (10-15% of wild-type catalase activity; cat2-2; N576998; (Queval et al., 2007)) and A. thaliana plants expressing glycolate oxidase in chloroplasts (GO5 plants; (Fahnenstich et al., 2008)) were grown in soil under a 16h light/8h dark regime at photosynthetically active photon flux densities (PPFD) of 75 µmol quanta m-2 s-1 at 22°C day/18°C night temperatures and a CO2 concentration of 3,000 ppm. After three weeks of growth, plants were transferred to ambient CO2 concentration (380 ppm) and the same PPFD. Whole rosettes were harvested at 0h and 8h after transfer. Control samples were harvested at 8 h from plants continuously maintained in high CO2.
Project description:Hydrogen peroxide (H2O2) can act as a signaling molecule that influences various aspects of plant growth and development, including stress signaling and cell death. To unravel the molecular mechanisms that regulate the response towards an impact of increased H2O2 levels in plant cells, we focused on the photorespiration-dependent peroxisomal H2O2 production in Arabidopsis thaliana mutants lacking CATALASE2 (CAT2) activity (cat2-2) and screened for second-site mutations that attenuate the Fv'/Fm' decrease and lesion formation linked to the cat2-2 phenotype. A mutation in the transcriptional regulator SHORT-ROOT (SHR) of the GRAS family rescued the cell death phenotype of cat2-2 plants under photorespiration-promoting conditions in a SCR-independent way, and provoked perturbation of photorespiratory metabolites. SHR deficiency boosted ascorbate levels and prevented the oxidation of the glutathione pool in cat2-2 background upon exposure to photorespiratory stress. These results reveal an unanticipated role for SHR as a regulator of cellular redox homeostasis. Unstressed and stressed samples of 4 genotypes (Col-0, cat2, shr and cat2 shr) in triplicate
Project description:Hydrogen peroxide (H2O2) can act as a signaling molecule that influences various aspects of plant growth and development, including stress signaling and cell death. To unravel the molecular mechanisms that regulate the response towards an impact of increased H2O2 levels in plant cells, we focused on the photorespiration-dependent peroxisomal H2O2 production in Arabidopsis thaliana mutants lacking CATALASE2 (CAT2) activity (cat2-2) and screened for second-site mutations that attenuate the Fv'/Fm' decrease and lesion formation linked to the cat2-2 phenotype. A mutation in the transcriptional regulator SHORT-ROOT (SHR) of the GRAS family rescued the cell death phenotype of cat2-2 plants under photorespiration-promoting conditions in a SCR-independent way, and provoked perturbation of photorespiratory metabolites. SHR deficiency boosted ascorbate levels and prevented the oxidation of the glutathione pool in cat2-2 background upon exposure to photorespiratory stress. These results reveal an unanticipated role for SHR as a regulator of cellular redox homeostasis.
Project description:Transcriptional profiling of Arabidopsis thaliana 12-days old seedlings comparing Col-0 wild type with transgenic plants with altered expression of dual-targetting plastid/mitochondrial organellar RNA-polymerase RPOTmp. Transgenic plants used for experiment were: overexpressor plants obtained by transformation of Col-0 WT plants with genetic constructs created in [Tarasenko et al., 2016] contained catalytic part of RPOTmp enzyme with transit peptides of RPOTm (mitochondrial) and RPOTp (plastid) by agrobacterial transformation; plants with complementation of RPOTmp functions in mitochondria or chloroplasts obtained from transformation of GABI_286E07 rpotmp knockout-mutant plants with genetic constructs created in [Tarasenko et al., 2016]. Goal was to determine the effects of RPOTmp knockout/overexpression on global Arabidopsis thaliana gene expression.
Project description:Shortly after the release of singlet oxygen (1O2), drastic changes in nuclear gene expression occur in the conditional flu mutant of Arabidopsis that reveal a rapid transfer of signals from the plastid to the nucleus. In contrast to retrograde control of nuclear gene expression by plastid signals described earlier, the primary effect of 1O2 generation in the flu mutant is not the control of chloroplast biogenesis but the activation of a broad range of signaling pathways known to be involved in biotic and abiotic stress responses. This activity of a plastid-derived signal suggests a new function of the chloroplast, namely that of a sensor of environmental changes that activates a broad range of stress responses. Inactivation of the plastid protein EXECUTER1 attenuates the extent of 1O2-induced up-regulation of nuclear gene expression, but it does not fully eliminate these changes. A second related nuclear-encoded protein, dubbed EXECUTER2, has been identified that is also implicated with the signaling of 1O2-dependent nuclear gene expression changes. Like EXECUTER1, EXECUTER2 is confined to the plastid. Inactivation of both EXECUTER proteins in the ex1/ex2/flu triple mutant is sufficient to suppress the up-regulation of almost all 1O2-responsive genes. Retrograde control of 1O2-responsive genes requires the concerted action of both EXECUTER proteins within the plastid compartment. Keywords: biotic and abiotic stress response, nuclear gene expression, plastid-derived signal, Col-0 ecotype, continuous light and then dark-incubated plants