Expression data for Arabidopsis pah1 pah2 mutant plants
ABSTRACT: PHOSPHATIDIC ACID PHOSPHOHYDROLASE (PAH) catalyses the conversion of phosphatidic acid to diacylglycerol. Disruption of two gene encoding this enzyme in Arabidopsis thaliana results in increased phosphatidylcholine synthesis and proliferation of the endoplasmic reticulum (Eastmond et al.,  Plant Cell 22: 2796). We performed microarray analysis on two week old wild type Arabidopsis plants and the pah1 pah2 double mutant using the Ath1 chip to determine what effect the double mutation has on global gene expression.
Project description:Identification of diacylglycerol pyrophosphate regulated genes in ABA signaling.<br><br> The specific plant phosphorylated form of phosphatidic acid (PA), diacylglycerol pyrophosphate (DGPP) was recently shown to be a second messenger in abscisic acid (ABA) signaling. The aim of the project is to identify among the set of ABA-regulated genes the ones also regulated by DGPP and/or PA.<br> Five ml of 3 days-old suspension cells was incubated with ABA or lipids for 3 h under the conditions of culture. Lipids were emulsified by sonication for 1 mi, four times, at 4°C, in one ml of culture medium then added to 4 ml suspension cells. Cells were filtrated under vacuum, frozen in liquid nitrogen and RNA extracted. Dioleoyl PA and dioctanoyl PA are from Sigma, dioleoyl DGPP and dioctanoyl DGPP are from Avanti Polar Lipids.
Project description:Lipid remodeling is crucial for hypoxic tolerance in animals, whilst little is known about the hypoxia-induced lipid dynamics in plant cells. Here we performed a mass spectrometry-based analysis to survey the lipid profiles of Arabidopsis rosettes under various hypoxic conditions. We observed that hypoxia caused a significant increase in total amounts of phosphatidylserine, phosphatidic acid and oxylipins, but a decrease in phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Particularly, significant gains in the polyunsaturated species of PC, PE and phosphatidylinositol, and losses in their saturated and mono-unsaturated species were evident during hypoxia. Moreover, hypoxia led to a remarkable elevation of ceramides and hydroxyceramides. Depletion of ceramide synthases LOH1, LOH2, and LOH3 enhanced plant sensitivity to dark submergence (DS), but displayed more resistance to submergence under light than wild type. Consistently, levels of unsaturated ceramide species (22:1, 24:1, and 26:1) predominantly declined in the loh1, loh2, and loh3 mutants under DS. Evidence that C24:1-ceramide interacted with recombinant CTR1 protein in vitro, enhanced ER-to-nucleus translocation of EIN2-GFP and stabilization of EIN3-GFP in vivo, suggests a role of ceramides in modulating ethylene signaling. The DS-sensitive phenotypes of loh mutants were rescued by a ctr1-1 mutation. Thus, our findings demonstrate that unsaturation of very-long-chain ceramides is a protective strategy for hypoxic tolerance in Arabidopsis. Arabidopsis Affymetrix GeneChip arrays were probed with RNAs isolated from leaves of untreated plants (controls) and plants upon hypoxia under light submergence for 48 h.
Project description:Our goal is to identify Salycilic Acid responsive genes dependent on PLD activation. - The experiment is done on Arabidopsis suspension cells, ecotype Columbia : Research of phospholipase D (PLD) activity implication in the response to SA. The use of primary alcohol, like N-butanol, makes possible to derive PLD activity towards the production of phosphatidylalcohol with the detriment of phosphatidic acid, the product of the PLD. Thus, in the presence of n-butanol, the response of the genes to SA dependent on phosphatidic acid will see their response disturbed. On the contrary, in the presence of tertiary butanol, the response of the genes should not be disturbed, tertiary alcohols not acting on the PLD. Keywords: treated vs untreated comparison Overall design: 14 dye-swap - CATMA arrays Project coordinator: Eric Ruelland Physiologie Cellulaire et Moléculaire des Plantes UMR7180 CNRS/UPMC 3, rue Galilée Le Raphaël 94200 Ivry sur Seine email: firstname.lastname@example.org
Project description:Oleaginous microorganisms have considerable potential for biofuel and commodity chemical production. Under nitrogen-limitation, Rhodococcus jostii RHA1 grown on benzoate, an analog of lignin depolymerization products, accumulated triacylglycerols (TAGs) to 55% of its dry weight during transition to stationary phase, with the predominant fatty acids being C16:0 and C17:0. Transcriptomic analyses of RHA1 grown under conditions of N-limitation and N-excess revealed 1,826 dysregulated genes. Genes whose transcripts were more abundant under N-limitation included those involved in ammonium assimilation, benzoate catabolism, fatty acid biosynthesis and the methylmalonyl-CoA pathway. Of the 16 atf genes potentially encoding diacylglycerol O-acyltransferases, atf8 transcripts were the most abundant during N-limitation (~50-fold more abundant than during N-excess). Consistent with Atf8 being a physiological determinant of TAG accumulation, a Δatf8 mutant accumulated 70% less TAG than wild-type RHA1 while atf8 overexpression increased TAG accumulation 20%. Genes encoding type-2 phosphatidic acid phosphatases were not significantly expressed. By contrast, three genes potentially encoding phosphatases of the haloacid dehalogenase superfamily and that cluster with, or are fused with other Kennedy pathway genes were dysregulated. Overall, these findings advance our understanding of TAG metabolism in mycolic acid-containing bacteria and provide a framework to engineer strains for increased TAG production. Overall design: Transcriptomes of R. jostii RHA1 from nitrogen excess and nitrogen limited cultures were analysed using a Genome Analyzer IIx (Illumina®).
Project description:It has been reported that repeated intra-tracheal instillation of S. chartarum spores induced significant pulmonary arterial remodeling in mice, which resulted in pathological changes like human pulmonary arterial hypertension (PAH) and elevation right ventricle systolic pressure. Then, we used microarrays to know the complex molecular mechanisms that underlie pathogenesis of PAH. Isolates of Stachybotrys chartarum were used. Ddy mice were anesthetized and the spore suspension was intratracheally injected 12 times, i.e. 1×104 spores into each mouse at 4-5 day intervals for 8 weeks. Mice were sacrificed one week after the final injection and then examined. Lung tissue specimens from mice model of PAH (n=3) and normal controls (n=3) were obtained. RNA targets preparation were performed according to the manufacturer's protocol using GeneChip(R) 3' IVT Express Kit (Affymetrix). One hundred nanograms of total RNA was converted into double-stranded cDNA template for transcription. In vitro transcription synthesized amplified RNA (aRNA) and incorporated a biotin-conjugated nucleotide. After purification and fragmentation of aRNA, 12.5 ug of them was hybridized to GeneChip(R) Mouse Genome 430 2.0 Array (Affymetrix).The Probe Array was scanned using a GeneChip(R) Scanner 3000 7G.
Project description:Arabidopsis seeds expressing the castor fatty acid hydroxylase accumulate hydroxylated fatty acids up to 17% of total fatty acids in seed triacylglycerols, however total seed oil is also reduced up to 50%. Investigations into the cause of the reduced oil phenotype through in vivo [14C]acteate and [3H]2O metabolic labeling of developing seeds surprisingly revealed that the rate of de novo fatty acid synthesis within the transgenic seeds was approximately half that of control seeds. Addition of castor phospholipid:diacylglycerol acyltransferase (PDAT) increased hydroxylated fatty acid content of the seed oil, increased the rate of fatty acid synthesis, and mostly restored seed oil levels. RNAseq analysis indicated no changes in expression of fatty acid synthesis genes in hydroxylase-expressing plants. Transcript profiles of Arabidopsis developing seeds of three lines, at three stages of development were generated by deep sequencing, in triplicate, using Illumina.
Project description:Although multiple gene and protein expression have been extensively profiled in human pulmonary arterial hypertension (PAH), the mechanism for the development and progression of pulmonary hypertension remains elusive. Analysis of the global metabolomic heterogeneity within the pulmonary vascular system leads to a better understanding of disease progression. Using a combination of high-throughput liquid-and-gas-chromatography-based mass spectrometry, we showed unbiased metabolomic profiles of disrupted glycolysis, increased TCA cycle, and fatty acid metabolites with altered oxidation pathways in the severe human PAH lung. The results suggest that PAH has specific metabolic pathways contributing to increased ATP synthesis for the vascular remodeling process in severe pulmonary hypertension. These identified metabolites may serve as potential biomarkers for the diagnosis of severe PAH. By profiling metabolomic alterations of the PAH lung, we reveal new pathogenic mechanisms of PAH in its later stage, which may differ from the earlier stage of PAH, opening an avenue of exploration for therapeutics that target metabolic pathway alterations in the progression of PAH. Global profiles were determined in human lung tissue and compared across 11 normal and 12 severe pulmonary arterial hypertension patients. Using a combination of microarray and high-throughput liquid-and-gas-chromatography-based mass spectrometry, we showed unbiased metabolomic profiles of disrupted glycolysis, increased TCA cycle, and fatty acid metabolites with altered oxidation pathways in the severe human PAH lung.
Project description:Background: Polycyclic aromatic hydrocarbons (PAHs) are toxic, widely-distributed, environmentally persistent, and carcinogenic byproducts of carbon-based fuel combustion. Previously, plant studies have shown that PAHs induce oxidative stress, reduce growth, and cause leaf deformation as well as tissue necrosis. To understand the transcriptional changes that occur during these processes, we performed microarray experiments on Arabidopsis thaliana L. under phenanthrene treatment, and compared the results to published Arabidopsis microarray data representing a variety of stress and hormone treatments. In addition, to probe hormonal aspects of PAH stress, we assayed transgenic ethylene-inducible reporter plants as well as ethylene pathway mutants under phenanthrene treatment. Results: Microarray results revealed numerous perturbations in signaling and metabolic pathways that regulate reactive oxygen species (ROS) and responses related to pathogen defense. A number of glutathione S-transferases that may tag xenobiotics for transport to the vacuole were upregulated. Comparative microarray analyses indicated that the phenanthrene response was closely related to other ROS conditions, including pathogen defense conditions. The ethylene-inducible transgenic reporters were activated by phenanthrene. Mutant experiments showed that PAH inhibits growth through an ethylene-independent pathway, as PAH-treated ethylene-insensitive etr1-4 mutants exhibited a greater growth reduction than WT. Further, phenanthrene-treated, constitutive ethylene signaling mutants had longer roots than the untreated control plants, indicating that the PAH inhibits parts of the ethylene signaling pathway. Conclusions: This study identified major physiological systems that participate in the PAH-induced stress response in Arabidopsis. At the transcriptional level, the results identify specific gene targets that will be valuable in finding lead compounds and engineering increased tolerance. Collectively, the results open a number of new avenues for researching and improving plant resilience and PAH phytoremediation. Overall design: Arabidopsis thaliana (ecotype Columbia) plants were long-day grown with +/- 0.25 mM phenanthrene in sterile plates at 23C for 21d before harvest. At least 20 plants were pooled prior to each mRNA extraction.