Project description:We were interested in investigating the transcriptome responses to exogenous applications of brassinosteroid hormone when Arabidopsis seedlings are pre-stressed with a reactive oxygen species, hydrogen peroxide. We were interested in seeing which subsets of BR-responsive gene transcripts were most affected and how BR-responsive gene transcripts responded to increasing concentrations of hydrogen peroxide both as a whole and individually. Liquid culture Arabidopsis seedlings are grown under standard conditions. Hydrogen peroxide is added at various concentrations to pre-stress the seedlings. Following this pretreatment, the seedlings are then treated with brassinosteroid (BR) hormone (epi-brassinolide, BL). Following this treatment, seedlings are harvested and total RNA is extracted for genome-wide transcriptome analysis.
Project description:We were interested in investigating the transcriptome responses to exogenous applications of brassinosteroid hormone when Arabidopsis seedlings are pre-stressed with a reactive oxygen species, hydrogen peroxide. We were interested in seeing which subsets of BR-responsive gene transcripts were most affected and how BR-responsive gene transcripts responded to increasing concentrations of hydrogen peroxide both as a whole and individually.
Project description:Excessive levels of reactive oxygen species (ROS) cause cellular stress through damage to all classes of macromolecules and result in cell death. However, ROS can also act as signaling molecules in various biological processes. In plants, ROS signaling has been documented in environmental stress perception, plant development and cell death amongst others. Knowledge on the regulatory events governing ROS signal transduction is however still scratching the surface. To further elucidate the transcriptional response and regulation upon ROS accumulation we supplemented Arabidopsis seedlings with a 10mM hydrogen peroxide (H2O2) solution to trigger oxidative stress.
Project description:The growth and shape of plants is mainly defined by the properties of their cell walls, which dynamically adapt to internal and external cues. Therefore, the cell wall is under constant surveillance to relay feedback information to the cell interior. However, very little is known about how cell wall signaling is integrated with intracellular growth regulation. Here, we have identified a receptor-like protein, RLP44, which conveys information from the cell wall to the brassinosteroid hormone signaling pathway by interacting with the regulatory receptor-like kinase BAK1. This conditional RLP44-mediated signaling activation is required for normal development and stress responses, requires functional brassinosteroid receptor, but is partially independent of the hormone ligand. We sought to identify transcriptional changes in the PMEIox transformant in which an altered pectin modification leads to an induction of the brassinosteroid signalling pathway. In addition we determined which fraction of these expression changes was reverted in the cnu1 and cnu2 suppressor mutants of PMEIox, respectively. Complete transcriptome analysis was performed on Col-0 wild-type as well as the PMEIox transformant and the cnu1 and cnu2 suppressor mutant seedlings grown in the dark for four days. 3 Biological replicates of etiolated seedlings grown for 4 days in the dark were harvested on successive weeks.
Project description:Excessive levels of reactive oxygen species (ROS) cause cellular stress through damage to all classes of macromolecules and result in cell death. However, ROS can also act as signaling molecules in various biological processes. In plants, ROS signaling has been documented in environmental stress perception, plant development and cell death amongst others. Knowledge on the regulatory events governing ROS signal transduction is however still scratching the surface. To further elucidate the transcriptional response and regulation upon ROS accumulation we supplemented Arabidopsis seedlings with a 10mM hydrogen peroxide (H2O2) solution to trigger oxidative stress. After growth of 7 days, hydrogen peroxide (H2O2) was added to a final concentration of 10mM. Control plants were treated with the same volume of H2O. Seedlings were grown for 24h under the same controlled conditions. Design: 3 replicates x 2 conditions (7+1 day H2O or 7+1 day H2O2)
Project description:We used the flu mutant of Arabidopsis and a transgenic line that overexpresses the thylakoid-bound ascorbate peroxidase (tAPX) to address the interactions between different reactive oxygen species (ROS) signaling pathways. The conditional flu mutant of Arabidopsis accumulates excess protochlorophyllide in the dark within chloroplast membranes that upon illumination acts as a photosensitizer and generates singlet oxygen (1O2). Immediately after the release of singlet oxygen rapid changes in nuclear gene expression occur. Distinct sets of genes were activated that were different from those induced by other reactive oxygen species, superoxide or hydrogen peroxide (H2O2), suggesting that different types of active oxygen species activate distinct signaling pathways. It was not known whether the pathways operate separately or interact with each other. We have addressed this problem by modulating noninvasively the level of H2O2 in plastids by means of a transgenic line that overexpresses the thylakoid-bound ascorbate peroxidase (tAPX, line 14/2 PMID: 15165186). In the flu mutant overexpressing tAPX, the expression of most of the nuclear genes that were rapidly activated after the release of 1O2 was significantly higher in flu plants overexpressing tAPX, whereas in wild-type plants, overexpression of tAPX had only a very minor impact on nuclear gene expression. The results suggest that H2O2 antagonizes the 1O2-mediated signaling of stress responses as seen in the flu mutant. This cross-talk between H2O2- and 1O2-dependent signaling pathways might contribute to the overall stability and robustness of wild-type plants exposed to adverse environmental stress conditions. Keywords: Single time point comparison
Project description:The growth and shape of plants is mainly defined by the properties of their cell walls, which dynamically adapt to internal and external cues. Therefore, the cell wall is under constant surveillance to relay feedback information to the cell interior. However, very little is known about how cell wall signaling is integrated with intracellular growth regulation. Here, we have identified a receptor-like protein, RLP44, which conveys information from the cell wall to the brassinosteroid hormone signaling pathway by interacting with the regulatory receptor-like kinase BAK1. This conditional RLP44-mediated signaling activation is required for normal development and stress responses, requires functional brassinosteroid receptor, but is partially independent of the hormone ligand. We sought to identify transcriptional changes in the PMEIox transformant in which an altered pectin modification leads to an induction of the brassinosteroid signalling pathway. In addition we determined which fraction of these expression changes was reverted in the cnu1 and cnu2 suppressor mutants of PMEIox, respectively. Complete transcriptome analysis was performed on Col-0 wild-type as well as the PMEIox transformant and the cnu1 and cnu2 suppressor mutant seedlings grown in the dark for four days.
Project description:Auxin is a major plant hormone for both development and environmental adaptation. Auxin responses are context dependent and highly modulated by light, temperature, the circadian clock, brassinosteroid, and gibberellin, but the underlying mechanisms remain unclear. Here, we show that auxin signaling integrates with other signals through direct interactions of AUXIN RESPONSE FACTOR6 (ARF6) with PHYTOCHROME INTERACTING FACTOR4 (PIF4), the brassinosteroid-signaling transcription factor BZR1, and the gibberellin-signaling repressor RGA. ChIP-Seq and RNA-Seq experiments show that ARF6, PIF4, and BZR1 bind to largely overlapping targets in the genome and synergistically activate gene expression. In vitro and in vivo assays show that ARF6-promoter binding is enhanced by PIF4 and BZR1 but blocked by RGA. Furthermore, a tripartite HLH/bHLH module feedback regulates PIF activity and thus modulates auxin sensitivity according to additional developmental and environmental cues. Our results demonstrate a central growth-regulation transcriptional network that coordinates hormonal, environmental, and developmental control of cell elongation and plant growth. Genome-wide identification of ARF6 DNA-binding sites in etiolated Arabidopsis seedlings.
Project description:We used the flu mutant of Arabidopsis and a transgenic line that overexpresses the thylakoid-bound ascorbate peroxidase (tAPX) to address the interactions between different reactive oxygen species (ROS) signaling pathways. The conditional flu mutant of Arabidopsis accumulates excess protochlorophyllide in the dark within chloroplast membranes that upon illumination acts as a photosensitizer and generates singlet oxygen (1O2). Immediately after the release of singlet oxygen rapid changes in nuclear gene expression occur. Distinct sets of genes were activated that were different from those induced by other reactive oxygen species, superoxide or hydrogen peroxide (H2O2), suggesting that different types of active oxygen species activate distinct signaling pathways. It was not known whether the pathways operate separately or interact with each other. We have addressed this problem by modulating noninvasively the level of H2O2 in plastids by means of a transgenic line that overexpresses the thylakoid-bound ascorbate peroxidase (tAPX, line 14/2 PMID: 15165186). In the flu mutant overexpressing tAPX, the expression of most of the nuclear genes that were rapidly activated after the release of 1O2 was significantly higher in flu plants overexpressing tAPX, whereas in wild-type plants, overexpression of tAPX had only a very minor impact on nuclear gene expression. The results suggest that H2O2 antagonizes the 1O2-mediated signaling of stress responses as seen in the flu mutant. This cross-talk between H2O2- and 1O2-dependent signaling pathways might contribute to the overall stability and robustness of wild-type plants exposed to adverse environmental stress conditions. Experiment Overall Design: Arabidopsis thaliana rosette leaves were harvested after 2 h of reillumination following a 8h dark period for RNA extraction and hybridization on Affymetrix ATH1 microarrays. The entire experiment was performed six times, providing independent biological replicates. For each of the six experiments, all four lines, wild-type, thylakoidal ascorbate peroxidase overexpressor (over tAPX, line 14/2), flu mutant and flu plants overexpressing thylakoidal ascorbate peroxidase were grown for 3 weeks under continuous light at 90 mmol. m-2 . s-1, transferred to the dark for 8 h, and reilluminated for 120 min before the rosette leaves of at least 10 plants per line were harvested. Total RNAs from three separate biological experiments were pooled (= 1 biological rep.) for the preparation of cDNA and the subsequent synthesis of biotin-labeled complementary RNA as recommended by Affymetrix.