Project description:This SuperSeries is composed of the following subset Series: GSE10719: Response of Arabidopsis cell culture to phytoprostane A1; GSE10732: Identification of TGA-regulated genes in response to phytoprostane A1 and OPDA Experiment Overall Design: Refer to individual Series
Project description:12-Oxo-phytodienoic acid (OPDA) and several phytoprostanes are structurally related cyclopentenone oxylipins that can be formed via the enzymatic jasmonate pathway and a non-enzymatic, free radical-catalyzed pathway, respectively. To elucidate the biological activities of phytoprostanes in comparison to OPDA as well as the metabolism we performed genome-wide expression analysis. Experiment Overall Design: In order to get a comprehensive view of the biological activity of non-enzymatically formed reactive oxylipins the regulation of gene expression in Arabidopsis thaliana was analyzed using the ATH1 chip representing 22500 genes. A mixotrophic cell culture of Arabidopsis was treated with 75 µM phytoprostane A1 or with 0.5% methanol in water (Control) and harvested after 4 h. Three independent replicates of each treatment were analyzed and the fold change of normalized signals from PPA1-treated cell culture versus control cell culture was calculated.
Project description:12-Oxo-phytodienoic acid (OPDA) and several phytoprostanes are structurally related cyclopentenone oxylipins that can be formed via the enzymatic jasmonate pathway and a non-enzymatic, free radical-catalyzed pathway, respectively. To elucidate the biological activities of phytoprostanes in comparison to OPDA as well as the metabolism we performed genome-wide expression analysis. Keywords: mixotrophic Arabidopsis cell culture, ATH1 Chip
Project description:Reproducibility issues regarding complex in vitro cell culture experiments have been mainly attributed to genetic variations within cell lines. Batch-dependent fetal calf serum variations are well known and also represent relevant influencing factors. However, the molecular constituents mediating such effects remained largely unknown. High resolution mass spectrometry is a powerful tool to identify molecular FCS constituents and investigate their effect on cultured cells. Using a differentiation and inflammatory stimulation protocol on U937 cells we observed FCS batch-dependent variations of secreted cytokines, oxylipins and other inflammatory mediators. In order to detect candidate bioactive molecules contained in FCS, the protein and oxylipin composition of FCS batches was investigated. Remarkably, the protein composition showed some, but much less batch-dependent variation when compared to the oxylipin composition. Efficient uptake of C13-labelled arachidonic acid from medium by U937 macrophages and inflammation-induced release thereof including oxylipin products was evidenced. Balancing out FCS batch-dependent nanomolar concentration differences of two selected oxylipins, 5-HETE and 15-HETE, by spiking experiments, resulted in significant proteome alterations of cultured U937 macrophages indicating PPAR activation. High-resolution microscopy demonstrated HETE-induced formation of peroxisomes, thus independently corroborating the proteome profiling results. In conclusion, the present data demonstrate a strong and previously underappreciated influence of FCS-contained oxylipins on cellular effector functions, thus representing a plausible cause for disturbing reproducibility issues.
Project description:Oxygenated unsaturated fatty acids, known as oxylipins, are signaling molecules commonly used for cell-to-cell communication in eukaryotes. However, a role for oxylipins in mediating communication in prokaryotes has not previously been described. Bacteria mainly communicate via quorum sensing (QS) , which involves the production and detection of diverse small molecules termed autoinducers. We showed that oleic acid-derived oxylipins 10-HOME and 7,10-DiHOME produced by Pseudomonas aeruginosa function as autoinducers of a novel quorum sensing system termed Oxylipin-Dependent QS Sytem (ODS). This experiment was designed to determine the genes whose expression is altered by these P. aeruginosa oxylipins. We found that the ODS system controls the cell density-dependent expression of a P. aeruginosa gene subset through the mediation of 10-HOME and 7,10-DiHOME oxylipins.
Project description:Oxylipins play a role in the response of plants to pathogens both as antimicrobial compounds and as signaling molecules. In potato, pathogen infection leads to the stimulation of the 9-lipoxygenase pathway (Göbel et al. 2001; 2002; Stumpe et al. 2001). In order to analyze whether 9-LOX-derived oxylipins such as colnele(n)ic acid (products of the 9-divinyl ether synthase reaction) act as signaling molecules for the activation of defense responses, transgenic potato plants (Solanum tuberosum cv. Désirée) were generated which express an RNAi construct directed against the pathogen-induced 9-lipoxygenase (Göbel et al. 2003) or against the pathogen-induced 9-divinyl ether synthase (Stumpe et al. 2001). Highly reduced transcript levels correlate with low levels of 9-lipoxygenase-derived oxylipins (Göbel et al. 2003). Transgenic and wild type plants were grown as sterile plants on MS medium supplemented with agar in a phytochamber with 16 h of light [200 µE] at 22 °C. After transfer to soil, plants were kept in a phytochamber with 16 h of light [200 µE], 18 °C and 60 % humidity for four weeks. Lower leaves were infiltrated with a suspension of Pseudomonas syringae pv. maculicola at a concentration of 108 cfu/ml MgCl2 or, as a control, 10 mM MgCl2-solution. RNA was isolated both from the infiltrated leaves and the upper, non-treated leaves using the trizol method. Subsequently, RNA was digested with DNase and purified via Qiagen RNeasy Kit (Qiagen, Hilden, Germany) according to the manufacturer´s instructions. Keywords: Direct comparison
Project description:Gibberella stalk rot (GSR) caused by Fusarium graminearum is one of the devastating diseases causing significant losses to maize production worldwide. Although plant oxylipins have been widely reported as potent signals to activate diverse biotic stress responses, the roles of distinct oxylipin pathway branches initiated by either 9- or 13-lipoxygenases (LOXs) in defense against GSR remain unexplored. In this study, the functional analysis showed that disruption of ZmLOX5, a maize 9-LOX gene, resulted in increased susceptibility to GSR. To identify the key genes and metabolites associated with GSR resistance, we profiled transcriptome and oxylipins in the lox5 mutant and near-isogenic wild type. The results showed that JA biosynthetic pathway genes are highly up-regulated, whereas multiple 9-LOX pathway genes down-regulated in lox5-3 mutant in response to F. graminearum infection. Furthermore, oxylipin profiling of the mutant and corresponding wild type, B73, as well as a more resistant line, W438, uncovered significantly higher contents of JA-isoleucine (JA-Ile) and other jasmonates but relatively lower levels of 9-oxylipins in lox5-3 upon infection. By contrast, resistant line W438 and B73 displayed relatively lower levels of JAs, yet considerable increase of 9-oxylipins. Taken together, these results clearly indicated that the signaling pathways of 9-oxylipins and JAs antagonize each other, and that while ZmLOX5-produced 9-oxylipins contribute to resistance, JAs are likely to function as negative regulator in maize defense against GSR.