Project description:Growth daylength, ambient CO2 level, and intracellular hydrogen peroxide (H2O2) availability all impact plant function by modulating signalling pathways, but interactions between them remain unclear. Using a whole-genome transcriptomics approach, we exploited the conditional photorespiratory nature of the catalase-deficient cat2 mutant to identify gene expression patterns responding to these three factors. Arabidopsis Col-0 and cat2 grown for 5 weeks in high CO2 in short days (SD) were transferred to air in SD or long days (LD), and microarray analysis was performed. Of more than 500 genes differentially expressed in Col-0 between high CO2 and transfer to air in SD, the response of about one-third was attenuated by transfer to air in LD. H2O2-responsive genes in cat2 were highly dependent on daylength. The majority of H2O2-induced genes were more strongly up-regulated after transfer to air in SD than to LD, while a smaller number showed an opposing pattern. Responses of other H2O2-dependent genes indicate redox-modulation of the daylength control of fundamental cell processes. The overall analysis provides evidence that (1) CO2 level modulates stress-associated gene expression; (2) both CO2 and H2O2 interact with daylength and photoreceptor signalling pathways; and (3) cellular signalling pathways may be primed to respond to increased H2O2 in a daylength-determined manner.
Project description:Growth daylength, ambient CO2 level, and intracellular hydrogen peroxide (H2O2) availability all impact plant function by modulating signalling pathways, but interactions between them remain unclear. Using a whole-genome transcriptomics approach, we exploited the conditional photorespiratory nature of the catalase-deficient cat2 mutant to identify gene expression patterns responding to these three factors. Arabidopsis Col-0 and cat2 grown for 5 weeks in high CO2 in short days (SD) were transferred to air in SD or long days (LD), and microarray analysis was performed. Of more than 500 genes differentially expressed in Col-0 between high CO2 and transfer to air in SD, the response of about one-third was attenuated by transfer to air in LD. H2O2-responsive genes in cat2 were highly dependent on daylength. The majority of H2O2-induced genes were more strongly up-regulated after transfer to air in SD than to LD, while a smaller number showed an opposing pattern. Responses of other H2O2-dependent genes indicate redox-modulation of the daylength control of fundamental cell processes. The overall analysis provides evidence that (1) CO2 level modulates stress-associated gene expression; (2) both CO2 and H2O2 interact with daylength and photoreceptor signalling pathways; and (3) cellular signalling pathways may be primed to respond to increased H2O2 in a daylength-determined manner. Two genotypes x five conditions experiment, including Arabidopsis Col0WT and cat2-1 plants grown in soil for 5 weeks in a 8h light/16h dark (short day) regime at high CO2 concentration (3000 ppm CO2) and subsequently transferred to air (400 ppm CO2) in a short day or long day (16h light/8h dark) regime for 2 and 4 days. Three biological replicates were used that consist each of a pool of two leaves from different plants. Each sample was hybridized to one Genechip® Arabidopsis ATH1 Genome Array (Affymetrix).
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:Purpose: The goals of this study are to compare the transcriptome profiling and alternative splicing (AS) profiling between Col-0 wild type and SFPS knockout mutant (sfps-2) through RNA-seq to determine the molecular mechanisms of how splicing factor SFPS regulates photomorphogenesis in Arabidopsis. Results: Using an optimized data analysis workflow, we mapped about 100 million sequence reads per sample to the Arabidopsis genome (TAIR10) and identified 1495 differentially expressed genes between Col-0 and mutant dark samples; 1361 differentially expressed genes between Col-0 and mutant red light treated samples; 4291 differentially expressed genes between Col-0 dark and red light treated samples; and 4479 differentially expressed genes between mutant dark and red light treated samples. Except for gene expression, we also discovered 788 differentially spliced bins between Col-0 and mutant dark samples; 827 differentially spliced bins between Col-0 and mutant red light treated samples; 610 differentially spliced bins between Col-0 dark and red light treated samples; and 405 differentially spliced bins between mutant dark and red light treated samples. Altered splicing of 9 genes was confirmed with qRT-PCR, demonstrating the high degree of sensitivity of the RNA-seq method. Conclusions: Our study represents the first detailed analysis of SFPS mutant transcriptomes, with biologic replicates, generated by RNA-seq technology. Our results show that SFPS regulates photomorphogenesis in Arabidopisis through regulating the splicing activity of light signaling genes, which helps us.
Project description:Extracellular adenosine 5’-triphosphate (ATP) is a signaling molecule. To define the global transcriptional response to ATP, we performed DNA microarray analysis using RNA derived from wild-type (Col-0) Arabidopsis and the dorn1-1 mutant, Lectin receptor kinase I.9 (At5g60300) carrying a EMS point mutation, seedlings after 100 μM ATP.
Project description:Col-0 floral stem was grafted on the msh1 mutant (Col-0/msh1); on the dcl2,3,4,msh1 quadruple mutant (Col-0/dcl2,3,4,msh1); on Col-0 (Col-0/Col-0). Seeds were collected from the grafted Col-0 scion after grafts were established. Seed coming from the graft then were grown on the peat mix, leaf tissue was collected at the bolting and used for the total RNA sequencing.
Project description:Col-0 floral stem was grafted on the msh1 mutant (Col-0/msh1); on the dcl2,3,4,msh1 quadruple mutant (Col-0/dcl2,3,4,msh1); on Col-0 (Col-0/Col-0). Seeds were collected from the grafted Col-0 scion after grafts were established. Seed coming from the graft then were grown on the peat mix, leaf tissue was collected at the bolting and used for the bisulfite sequencing (methylome). Tissue from the msh1 mutant and dcl2,3,4,msh1 quadruple mutants used as rootstocks was similarly collected at the bolting stage and used for the bisulfite sequencing.
Project description:Atmospheric CO2 concentrations can determine the number of stomata that form on plant leaves (Woodward & Kelly 1995 New Phyt 131: 311-327). The majority of species exhibit reduced stomatal densities at elevated CO2. However, not all plant species react in the same way to elevated CO2 levels and there is a spectrum of effects: Some species increase stomatal densities, some decrease stomatal densities, and some are unaffected. In addition to which, other environmental factors influence the number of stomata that a plant form. Light intensity has also been shown to affect stomatal numbers in various Arabidopsis ecotypes (Schluter et al. 2003 J Exp Bot 54 (383): 867-874; Lake et al. 2002 J Exp Bot 53 (367): 183-193), by increasing stomatal numbers with increasing light levels. There are many changes in gene expression under elevated CO2 conditions, so pinpointing specific genes involved in the stomatal response to CO2 is difficult. In addition, if there is crosstalk between the various signalling pathways affecting ultimate stomatal numbers this complicates further the task of finding genes specifically involved the stomatal response to CO2. Therefore we propose to look at the interaction of two known influences on stomatal numbers, light and CO2, on one specific ecotype, Col-0. We aim to test the hypothesis that light signals interact the CO2 signals that affect stomatal development. Arabidopsis thaliana Columbia-0 ecotype has previously been shown to decrease stomatal numbers in response to a doubling of ambient CO2 concentrations. Col-0 has also been shown to increase stomatal numbers in response to high light intensities. Therefore we propose to grow A. thaliana Col-0 at three light intensities (50 mmol m-2 s-1, 150 mmol m-2 s-1 and 250 mmol m-2 s-1), in both ambient and elevated (double ambient) atmospheric CO2 concentrations. By looking in more detail at how gene expression differs between plants grown at ambient and elevated CO2 at the same light intensities, and also how gene expression differs between plants grown at the same CO2 concentration but different light intensities, we aim to identify those genes involved in the stomatal developmental response to CO2 and whether genes involved in the light response can also be isolated. Experimenter name = Susannah Bird Experimenter phone = (0114) 222 4649 Experimenter address = Animal and Plant Science Department Experimenter address = Alfred Denny Building Experimenter address = Western Bank Experimenter address = Sheffield Experimenter zip/postal_code = S10 2TN Experimenter country = UK Keywords: growth_condition_design
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:rs12-08_cyp715a1 - col-0 vs cyp715a1 - The microarray analysis is part of a project aimed at characterizing the function of the cytochrome P450 CYP715A1 in Arabidopsis thaliana. - Flower buds of Arabidopsis Col-0 (wild-type) and cyp715A1 mutant were harvested for a comparative analysis of their transcriptomes.