Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of Arabidopsis roots - nutritional control of plant development: molecular analysis of the NO3- response pathway

ABSTRACT: Background: The Arabidopsis ANR1 gene is a key regulator of root architecture (Zhang and Forde, 1998): when ANR1 is down-regulated (by antisense or co-suppression) the resulting lines are no longer able to proliferate their lateral roots in response to localised supplies of NO3- (Zhang and Forde, 1998). ANR1 encodes a root-specific member of the MADS box family of transcription factors and is thought to be a component of a signalling pathway that links an external NO3- signal to increased meristematic activity in the lateral root meristem (Zhang et al., 1999).A major goal of our present BBSRC-funded project is to learn more about this NO3- response pathway by identifying the downstream targets of ANR1. To this end we have generated a set of transgenic lines in which ANR1 is under a novel post-translational control. This was achieved by tagging ANR1 with the steroid-binding domain of the rat glucocorticoid receptor (rGR) and expressing the fusion protein under the control of the CaMV 35S promoter. Initial results with these lines confirm that the architecture of the root system is altered in a way that correlates with the presence of the steroid inducer (dexamethasone, or DEX) and the expression of the ANR1::rGR transgene. We now wish to apply transcript analysis to one of these lines (ANGR4-12) to identify the genes whose expression is up- or down-regulated by ANR1. Sablowski and Meyerowitz (1998) successfully used an analogous approach to identify a gene that is an immediate target of the AP3 MADS-box transcription factor (although here, in the absence of microarray technology, differential display was used). Experimental: Plants of the two genotypes (ANGR4-12 and wild type) will be grown in sterile liquid culture for 3-4 weeks and then starved of N for 3 d (to quench the endogenous NO3- signalling pathway) before starting the DEX treatment. Roots will be harvested after 60 min from roots of DEX-treated and mock-treated RGR4-12 and control plants (four RNA samples). To minimise background noise due to environmental variables we will repeat the experiment three times and pool the RNA samples. Experiment Overall Design: Number of plants pooled:20 Experiment Overall Design: Biological replicates: 2

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: Nottingham Arabidopsis Stock Centre (NASC)

PROVIDER: E-GEOD-6155 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Our interest lies in how plants respond to bacterial pathogens. Over the past three years we have identified and documented reproducible, landmark biochemical and molecular events following the challenge of Arabidopsis with the phytopathogenic enterobacteria P. syringae. Significantly, our studies revealed 60% of cDNA-AFLP differentials not present on the 8,200 feature GeneChips and 20% absent from public EST databases (de Torres in press). We now seek to exploit this background using carefully defined time-points to analyse global changes in the Arabidopsis transcriptome using challenges selected to define gene targets implicated in (i) expression of basal immunity (ii) the establishment of successful parasitism (resistance) by a virulent pathogen (host). The results will provide a rationale for future functional assays of the identified pathways using transgenic knockouts and mutant analyses. Additionally, data will provide underpinning support for comparative proteomics of the defense response currently in progress with GARNet support using the same experimental parameters (BBSRC 32/P14635). We propose the following treatments: (i) Mock vs. DC3000hrpA @ 60 min: 60 minutes is subsequent to host immediate-early stress responses and will catalogue the innate responses induced by pathogen associated molecular patterns. The hrpA lesion will ensure no type III effectors influence transcriptional responses. Gene products induced at this time are predicted to potentiate latter host responses (2 treatments X 3 biological replicates = 6 chips). (ii) Mock vs. DC3000 vs. DC3000hrpA vs. DC3000::avrRpm1 @ 4 hours: A key time point previously defined where no macroscopic symptoms are visible but significant differences exist between compatible and incompatible interactions at the molecular and physiological levels. These treatments will serve to define the earliest genes induced by the complement of DC3000 type III effector and will specifically define genes/pathways suppressed by virulence factors in addition to those implicated in orchestration of the hypersensitive cell death. We will also include an incompatible interaction on a transgenic line expressing an RPM1 interacting protein, which fails to mount an HR but exhibits hyper-resistance. We predict this challenge will separate the resistance response (pathogen restriction) from that associated with hypersensitive cell death (5 treatments X 3 biological replicates = 15 chips). (iii) Mock vs. DC3000 vs. DC3000hrpA @ 14 hours: At 10 h before phenotypes are apparent in the DC3000 background, Type III effector delivery is well advanced and impact on host transcription maximal (3 treatments X 3 biological replicates = 9 chips). Experiment Overall Design: Number of plants pooled:4 leaves/plant; 18 plants/time point

Project description:Our analysis of the sfr6 freezing-sensitive mutant (Knight, H., Veale, E., Warren, G. J. and Knight, M. R. (1999). Plant Cell 11, 875-886.) and cls8 (unpublished) chilling-sensitive mutant of Arabidopsis, has revealed that the expression of certain cold-regulated genes is aberrant in both these mutants. In order to understand the molecular basis of chilling and freezing stress in Arabidopsis and also to determine commonalities and differences between these 2 different physiological stress-tolerance processes, we request transcriptome analysis for both of these mutants compared to wild type in one experiment, upon cold treatment and at ambient conditions. The sfr6 mutant shows the most severe phenotype with respect to cold gene expression, but is tolerant to chilling (Knight, H., Veale, E., Warren, G. J. and Knight, M. R. (1999). Plant Cell 11, 875-886.). However, it is unable to cold acclimate and hence is sensitive to freezing. The cls8 mutant, on the other hand, has a relatively mild phenotype relative to the cold-regulated genes we have examined, but is very sensitive to chilling temperatures (15 to 10 degree centigrade). It is thus likely that in cls8 we have not yet identified the genes which are most affected, and which account for the physiological phenotype. Both sfr6 and cls8 have been fine-mapped and are close to being cloned. The cls8 mutant has an altered calcium signature in response to cold which means it is likely to be affected in early signalling, e.g. cold perception itself.We will compare the expression profiles of genes in sfr6, cls8 and Columbia (parental line for both mutants), both at ambient, and after treatment with cold (5 degrees) for 3 hours. This timepoint is designed to ÂcaptureÂ both rapidly responding genes e.g. CBF/DREB1 transcription factors, and also more slow genes e.g. COR genes (KIN1/2 and LTI78). Pilot northerns confirm that this time point is suitable.This analysis will provide new insight into 2 novel genes required for tolerance to low temperature in Arabidopsis, and additionally will determine the nature of overlap between the separate processes of chilling and freezing tolerance. Experiment Overall Design: Number of plants pooled:40-60

Project description:This application is the second part of a BBSRC-funded grant to compare and contrast the plastid-signalling pathways disrupted by Norflurazon and far-red light treatment of Arabidopsis seedlings. The first application of this laboratory to GARNet's Affymetrix service (2002-08-25-17.41.49_McCormac) addressed the Norflurazon pathway; this application addresses the far-red pathway. The assembly of photosynthetic complexes in developing chloroplasts is critical to the establishment of the autotrophic plant. This requires light-mediated upregulation of both nuclear- and chloroplast-encoded genes. The expression of such photosynthetically-associated nuclear genes is also often dependant on a retrograde plastid signal which emanates from chloroplasts to modulate nuclear transcription. Extensive studies using the herbicide Norflurazon to knock-out the plastid signal (including this lab's previous Affymetrix application to GARNet) are identifying the affected gene sets. However, genetic studies have indicated the existence of more than one plastid signalling pathway. We have recently investigated a phytochrome A-mediated, far-red (FR) input pathway which blocks subsequent chloroplast development under white light (WL). This has also been found to inhibit the transcription of a small group of known nuclear-encoded plastidic proteins. Here we wish to establish how wide-reaching this FR-effect is on nuclear transcription, and will directly compare the affected gene groups with those identified from our earlier (and other's) studies with a Norflurazon treatment. In this array experiment we will compare RNA from seedlings grown in complete darkness (D) before transfer to WL, with that of seedlings preconditioned under a restricted wavelength FR source before exposure to WL. This comparison of FR- and Norflurazon-affected gene groupings will indicate whether the plastid signalling pathways are likely to be the same, overlapping or highly divergent. As well as wild-type seedlings, the gun1,gun5 mutant line is to be used as both these alleles are well established as alleviating the Norflurazon-inhibited pathway, but their affect on the FR-pathway is less clear. A single replicate of a phyA-null mutant line will also be included in order to differentiate between specific phytochromeA-mediated responses and other FR effects. It is envisaged that, in general, D- and FR-treated samples of the phyA mutant line will respond in the same way as each other and as the wild-type D-treated samples and, thus, the lack of a biological repeat in this case is not a major short-fall. The proposed experiment will consist of: wild-type: D-pretreated (x2 biological replicates) wild-type: FR-preconditioned (x2) gun1,gun5: D-pretreated (x2) gun1,gun5: FR-preconditioned (x2) phyA: D-pretreated (x1) phyA: FR-preconditioned (x1) Experiment Overall Design: Number of plants pooled:300 seedlings

Project description:Aim: To determine the effect of an AtrbohC mutation on the gene expression pattern in primary root tissue, to identify candidate genes acting downstream of AtrbohC, particularly any encoding antioxidant-related proteins, signal transduction components or proteins known to be required for normal root-hair development. Background: Root-hairs are a model system for investigating plant cell polarity. The root-hair mutant rhd2 (Schiefelbein and Somerville, 1990. Plant Cell, 2:235) has short hairs that burst at their tips, (Jones and Smirnoff, unpublished). RHD2 has been cloned and is identical to AtrbohC (L. Dolan, pers. comm.), which encodes a homologue of the superoxide-generating neutrophil respiratory burst oxidase catalytic subunit gp91phox (Torres et al., 1998. Plant J., 14:365). Superoxide rapidly dismutates to hydrogen peroxide (H2O2), suggesting that the rhd2 phenotype may result from reduced H2O2 levels in root-hair cells. Low doses of exogenous antioxidants phenocopy the rhd2 root-hair phenotype in wild-type plants (Jones and Smirnoff, unpublished) further supporting a role for H2O2 in root-hair growth. Fluorescent dyes that detect H2O2 show distinct localisation patterns in growing root-hair cells, (Jones and Smirnoff, unpublished). H2O2 may be an important second messenger in plant cell signalling with proposed roles in the development of cotton fibres (Potikha et al., 1999. Plant Physiol., 119: 849) and in ABA-induced stomatal closure (Zhang et al., 2001. Plant Physiol., 126: 1438). In cultured Arabidopsis cells H2O2 induces gene expression, including that of a gp91phox homologue, (Desikan et al., 1998. J. Exp. Bot., 49: 1767; Desikan, et al., 2000. Free Rad. Biol. Med., 28: 773; Baxter-Burrell et al., 2002. Science, 296: 2026) and activates a MAP kinase cascade (Desikan et al., 1999. J. Exp Bot., 50: 1863). cDNA microarray technology has been used previously to examine the effects of H2O2 on gene expression during oxidative stress (Desikan et al., 2001. Plant Physiol., 127: 159). We wish to investigate the effects of H2O2 on gene expression during root development using the rhd2 mutant. We are currently determining the expression pattern of RHD2. By extracting RNA from the small region of the primary root (for wild-type and rhd2 plants grown in sterile conditions) where root hairs are growing we hope to enrich for root-hair RNAs. This may reveal candidate genes that could be examined more closely at the single-cell level. This approach will provide new insights into the role of H2O2 in root-hair development. Experiment Overall Design: Number of plants pooled:100

Project description:The aim of this study is to identify Arabidopsis genes whose expression is altered by aphid feeding. An understanding of the plant aphid interaction at the level of the plant transcriptome will 1) consolidate current areas of investigation focused on the phloem composition (the aphid diet), 2) open up areas of plant aphid interactions for ourselves and other workers, 3) Contribute to understanding the use of new molecular technologies in an environmental context and 4) contribute to existing and development of novel control strategies.Our Arabidopsis/Myzus persicae system provides a valuable model for the study because of: a) the advantages of using Arabidopsis, b) The ability to use clonal insects, c) phloem feeding aphids facilitate focus on a specific cell type, d) aphid stylectomy allows collection of pure phloem sap to monitor ?phloem phenotype?? of the plant and the insect diet, e) we have techniques to monitor the reproductive performance and feeding behaviour aphids.Our strategy has been to test the function of selected genes, particularly those regulating phloem composition (the feeding site of the aphid) based on current phloem models of phloem function. Gene choice is limited the simplicity of current models of phloem aphid interaction.We propose a simple two treatment (aphid infested vs control plants) experiment that will identify novel target genes for future analysis. Arabidopsis plants (variety Columbia) will be grown in 16/8 light/dark in temperature controlled growth rooms. At growth stage 3.90, when rosette growth is complete, 10 clonal adult Myzus persicae will be caged in clip cages on the two largest leaves on each plant. Control plants will be treated identically except that the cages will be empty. Leaves will be harvested 8 h after infestation. This time point is selected as we know that 90% of aphids are plugged into the sieve element within 2h and that a 6h lag phase has period has previously been used when examining gene expression affected by wounding. In subsequent experiments we will examine time courses of expression of relevant genes using other approaches. Pooling two leaves from each of ten plants will generate the RNA sample, ensuring that expression signals are representative of the population of plants. Experiment Overall Design: Number of plants pooled:10

Project description:Nitric oxide (NO.) is a well-established signal in mammalian cells regulating e.g. smooth muscle tone, neurotransmission and apoptosis. NO interacts with superoxide (O2-) to derive the highly reactive peroxynitrite (ONOO-) radical leading to the generation of lipid hydroxy-peroxides (ROO.) and cell death. Thus the recent reports implicating of .NO in the Hypersensitive Response (HR) a form of programmed cell death elicited by pathogens in plantsis suggestive of parallel roles in animals and plants. As part of BBSRC funded programmes which are investigating oxidative signaling and cell death in Arabidopsis we have collaborated with the Trace Gas Detection facility at the University of Nijmegen to be the first to measure NO in planta from a developing HR (Mur et al.paper in prep). The challenge remains to understand the spatio-temporal role(s) of .NO during the HR and disease development. In line with other groups e.g. Klessig et al.(2000) PNAS 978849-55 we have observed that .NO mediated event are both SA- dependent and independent. We propose a two-stage programme to investigate NO events which will lead to subsequent BBSRC grant bids. Firstly to exploit the GARNET Affymetrix transcriptomic service to identify genes which are upregulated by .NO (see programme below). These will be compared with similar data generated by other members of the consortium; Dr. Steve Neill for oxidative stress (Desikan et al. (2001). Plant Physiol 127(1): 159-72; Clarke et al.2000 Plant J.; 24:667-77); Dr. Paul Kenton who is mainly interested in signaling by the oxylipid Jasmonic acid (Kentonet al. (1999). MPMI 12(1): 74-78) as well as from other sources. Though these data in themselves will suggest modes of NO action. However as a second approach we will clone the promoter of a strongly NO-responsive gene which will be fused to positive and negative selectable markers as well as reporter genes e.g. LUC. to identify Arabidopsis EMS mutants which are perturbed in NO-mediated events. Programme. Our in planta measurement show that from a baseline production of 1nl.g fwt-1h-1 NO levels rise to 6nl. g fwt-h-1 (_plus/- 1.1 SE) prior to cell death and to 25nl g fwt-h-1 (_plus/- 4.2 SE) at cell death following which the concentration falls. At this juncture we will intend to gas Arabidopsis to ~75ppb (the head-space concentration when NO production was at 6nl. g fwt-h-1). Prior to using the DNA RNA will establish that this does not elicit cell death over the proposed treatment period and that both PR1 (SA-dependent) and PAL1 (SA-independent) genes are induced. We will compare this plant with sealed but non-treated Arabidopsis. Future targets for DNA RNAs analysis induce plant treated with both NO (Sodium Nitro Prusside) and O2- (Xanthine oxidase) generators and depending on their effectiveness (to be assessed by NO measurement) HR lesions treated with Nitric Oxide Synthase inhibitors. Experiment Overall Design: Number of plants pooled:8

Project description:This proposal is aimed at providing transcriptome data to underpin a long-term joint research programme of Steve Smith and Alison Smith. We are jointly studying starch synthesis and breakdown in Arabidopsis leaves, and individually studying other enzymes of carbohydrate metabolism (eg. sucrose synthases, invertases, sugar transporters). Collectively these enzymes are encoded by up to 100 known genes, but there are many others of relevance to our studies. For several years we have employed a defined set of growth conditions for this work (resulting in numerous publications). We have extensive data for changes in the amounts of starch, malto-oligosaccharides and sugars throughout the diurnal cycle in these plants, and we intend to quantitate numerous key enzymes. We now wish to profile changes in transcripts in these plants, so that this information can be correlated with changes in the amounts of key enzymes and metabolites. Analysis of the data sets will help us to relate the synthesis of certain enzymes to particular metabolic functions, and also to help identify genes encoding novel proteins involved in carbohydrate metabolism. Current research is aimed at discovering such novel proteins using proteomics approaches (BBSRC Grant: _Discovery and Functional Analysis of the Starch Proteome_). While the growth conditions (12h photoperiod, 150 umol/m2/s, 20C) and ecotype (Col-0) are specifically tailored to our experiments, they will be of value to the wider community, and could form the basis for collaborative studies with other groups. The experiment has involved sampling leaves at eleven different time points as follows: 0, 1, 2, 4, 8, 12, 13, 14, 16, 20, and 24 h (where time 0 is the onset of dark and 12 h is the onset of light). The 24 h time point is a repeat of 0 h. This sequence provides relatively more samples immediately after the light-dark transitions, when changes in metabolism are most pronounced. Plants were grown in a controlled environment chamber under defined conditions, and selected for harvest according to a random number generator. Three fully expanded leaves were harvested from each of 20 plants for each sample. Leaves were frozen at -80 C and a portion saved for metabolite and enzyme assays while RNA was isolated from the remainder. The RNA has been purified and is ready to send to Nottingham. Experiment Overall Design: Number of plants pooled:20 Experiment Overall Design: Biological replicates: 2

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Transcription profiling of Arabidopsis roots - nutritional control of plant development: molecular analysis of the NO3- response pathway

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