Unknown,Transcriptomics,Genomics,Proteomics

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Inhibition of the mitochondrial TCA cycle by monofluoroacetate in Arabidopsis thaliana

ABSTRACT: Plant mitochondria signal to the nucleus leading to altered transcription of nuclear genes by a process called mitochondrial retrograde regulation (MRR). MRR is implicated in metabolic homeostasis and responses to stress conditions. Transcriptional consequences on nuclear gene expression of mitochondrial perturbations were examined by a microarray analyses. Expression of 606 genes was altered by monofluoroacetate (MFA) inhibition of the TCA cycle in leaves of soil grown Arabidopsis plants in the dark for 10 hours. Functional gene category (MapMan) and cluster analyses showed that genes with expression levels affected by perturbation from MFA inhibition were most similarly affected by biotic stresses such as pathogens, not oxidative stresses. Overall, the data provide further evidence for the presence of mtROS-independent MRR signaling, and support the proposed involvement of MRR and mitochondrial function in plant responses to biotic stress. Three independent (bio-replicate) experiments were done using three independent plant samples in the treatments. For each, approximately 30 plants were used for the treated sample and about 30 plants were used as the control sample. Plants were treated with 5 mM monofluoroacetate in 0.01% Tween 20 and incubated in the dark at room temperature (25C) for 6 hours. RNA was isolated from the inhibitor treated and control plants and used for microarray experiments. For each independent (bio-replicate) experiment, two microarrays were utilized using Cy3 and Cy5 dye-labeled samples and dye swapping was incorporated- 2 microarrays for each of 3 independent experiments (6 microarrays total).

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: David Rhoads

PROVIDER: E-GEOD-29268 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:We have implemented an integrated Systems Biology approach to analyze overall transcriptomic reprogramming and systems level defense responses in the model plant Arabidopsis thaliana during an insect (Brevicoryne brassicae) and a bacterial (Pseudomonas syringae pv. tomato strain DC3000) attack. The main aim of this study was to identify the attacker-specific and general defense response signatures in the model plant Arabidopsis thaliana while attacked by phloem feeding aphids or pathogenic bacteria. Defense responses and networks, unique and specific for aphid or Pseudomonas stresses were identified. Our analysis revealed a probable link between biotic stress and microRNAs in Arabidopsis and thus opened up a new direction to conduct large-scale targeted experiments to explore detailed regulatory links among them. The presented results provide a first comprehensive understanding of Arabidopsis - B. brassicae and Arabidopsis - P. syringae interactions at a systems biology level. Arabidopsis thaliana (ecotype Colombia-0) seeds were grown in 6-cm-diameter pots filled with a sterile soil mix (1.0 part soil and 0.5 part horticultural perlite), 3 plants per pot. Plants were kept in growth chambers VM-CM-6tsch VB 1514 (VM-CM-6tsch Industrietechnik GmbH, Germany) under the following conditions: a 8/16 h (light/dark) photoperiod at 22M-BM-0C/18M-BM-0C, 40%/70% relative humidity, and 70/0 M-NM-<mol m-2s-1 light intensity. After 32 days plants had 8 fully developed leaves. Each plant was infested with 32 wingless aphids [Brevicoryne Brassicae] (4 per leaf), which were transferred to leaves with a fine paintbrush. Infested plants and aphid-free controls were kept in plexiglass cylinders. Plants were harvested 72 h after infestation between the 6th and 8th hour of the light photoperiod. Four biological replicates were prepared from aphid infested and control plants, each sampled from 15 individual plants. Whole rosettes were cut at the hypocotyls and aphids were removed by washing with Milli-Q-filtered water. Differences in transcriptional responses were measured by comparing genes expression of aphid infested plants against non-infested control plants.

Project description:We have implemented an integrated Systems Biology approach to analyze overall transcriptomic reprogramming and systems level defense responses in the model plant Arabidopsis thaliana during an insect (Brevicoryne brassicae) and a bacterial (Pseudomonas syringae pv. tomato strain DC3000) attack. The main aim of this study was to identify the attacker-specific and general defense response signatures in the model plant Arabidopsis thaliana while attacked by phloem feeding aphids or pathogenic bacteria. Defense responses and networks, unique and specific for aphid or Pseudomonas stresses were identified. Our analysis revealed a probable link between biotic stress and microRNAs in Arabidopsis and thus opened up a new direction to conduct large-scale targeted experiments to explore detailed regulatory links among them. The presented results provide a first comprehensive understanding of Arabidopsis - B. brassicae and Arabidopsis - P. syringae interactions at a systems biology level. Arabidopsis thaliana (ecotype Colombia-0) seeds were sown into 6-cm-diameter pots filled with a sterile soil mix (1.0 part soil and 0.5 part horticultural perlite). Plants were kept in growth chambers VM-CM-6tsch VB 1514 (VM-CM-6tch Industrietechnik GmbH, Germany) with a 16/8 h (light/dark) photoperiod at 22/18 M-BM-0C, 40/70% relative humidity, and 70/0 mmol m-2 s-1 light intensity. The Pseudomonas syringae pv. tomato strain DC3000 culture was grown overnight in 10 ml of Kings B solution supplemented with antibiotics rifampicin (50 M-NM-<g mlM-bM-^HM-^R1) and kanamycin (25 M-NM-<g mlM-bM-^HM-^R1). Overnight culture was washed once in 10 mM MgCl2 and final cell densities were adjusted to approximately 0.20 at 600 nm (approximately 1.5 M-CM-^W 108 cfu mlM-bM-^HM-^R1) in 10 mM MgCl2. Plants were mock-challenged with 10 mM MgCl2 or inoculated with DC3000 strain, 3-4 leaves were infiltrated on the abaxial surface with a needleless 1-ml syringe.Whole rosettes were cut at the hypocotyls and harvested from Pseudomonas infested and mock-infected plants after 72 hours treatment. 4 biological replicates were prepared from each treatment, each containing rosettes from 15 individual plants. Differences in transcriptional responses were measured by comparing genes expression of Pseudomonas infected plants against mock-infected control plants.

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Inhibition of the mitochondrial TCA cycle by monofluoroacetate in Arabidopsis thaliana

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