Transcription profiling of Arabidopsis wild-type and gpa1 mutant in response to ABA
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ABSTRACT:
ORGANISM(S): Arabidopsis thaliana
SUBMITTER: Nottingham Arabidopsis Stock Centre (NASC)
PROVIDER: E-GEOD-6171 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Aim To identify genes specifically involved in the storage reserve mobilisation programme in Arabidopsis. Background During germination and early post-germinative growth in Arabidopsis seed storage reserves are broken down to provide energy and nutrients for the developing seedling. Genes encoding enzymes involved in the mobilisation of both storage lipid and protein are expressed strongly 1-2 days following imbibition and then fall to very low levels. The regulatory mechanisms controlling expression of these genes are poorly understood. Although germination and reserve mobilisation occur at the same time we have obtained evidence using Abscisic Acid (ABA) treated seeds that the events are regulated by two separate programmes. Arabidopsis seeds treated with 10mM ABA still express genes involved in the mobilisation of storage reserves and break down storage lipid even though germination is blocked. ABA treated seeds therefore provide a powerful system for the identification of genes involved specifically in the reserve mobilisation programme. Microarray analysis will allow us to gain a global undertanding of the processes involved in storage reserve mobilisation and should also result in the identification of regulatory genes involved in this process. Experimental Set-up All seeds are Col-4 imbibed on plates containing 1/2 MS media for 4 days in the dark. All plant material will be grown in controlled environment growth rooms under defined light and temperature regimes. There are two parts to the experimental design. 1. Time course to identify genes involved in both seed storage reserve and germination programmes Comparison of mRNAs from seeds immediately after imbibition (storage reserve and germination programme genes being induced) with 2 day (storage reserve genes maximally expressed) and 7 day old seedlings (storage reserve and germination programme genes off, photoautotrophic genes on) will allow the identification of genes exclusively presentor present at enhanced levels at the peak of reserve mobilisation (day 2). 2. ABA treatment experiment to identify genes involved in the storage reserve programme ABA treatment blocks germination but does not block reserve mobilisation. Microarray analysis will be performed on RNA isolated from 2 day old seed imbibed in the presence of 10mM ABA. Comparison of the results of this experiment with those of the time course experiment should allow us to distinguish between genes involved in the two major programmes that we have uncovered. Experiment Overall Design: 4 samples
2008-06-14 | E-GEOD-6173 | biostudies-arrayexpress
Project description:Mutations in the heterotrimeric G-protein a-subunit of Arabidopsis, GPA1, leads to deficiency in ABA-induced stomatal closure (Wang et al., 2001). To further investigate whether GPA1 is involved in the regulation of gene expression in response to ABA, we examined the induction of known ABA-inducible genes in the gpa1 mutant and compared it to wild-type. We found significant differences in levels of ABA-induced expression between wild-type and gpa1 mutant. In order to systematically investigate GPA1 involvement in ABA signalling leading to gene expression, we are requesting the transcriptome analysis of the gpa1 mutant in response to ABA.In detail, 2 week old wild-type and gpa1 plants grown in the 16/8 hrs light and dark cycle will be treated with either ABA or with a control solution for 3 hours. 10 plants will be used per sample to produce RNA, to give 4 samples, one for each chip: gpa1-1 mutant in the presence (chip1) or absence of ABA (chip2) and wild-type (WS2) in the presence (chip3) or absence of ABA (chip4).
2022-11-21 | E-NASC-45 | biostudies-arrayexpress
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
2008-06-14 | E-GEOD-6169 | biostudies-arrayexpress
Project description:Arabidopsis acetate non-utilizing mutants were isolated based on fluoroacetate resistant germination. Interestingly, a number of these mutants exhibited altered developmental characteristics in response to exogenous sucrose supply, such as bleaching of the cotyledons. A preliminary microarray experiment has already been conducted on one of the mutants, acn1-2. The gene expression analysis was done using 3 day-old seedlings of acn1-2 and the parent, Col-7, which were germinated on agar plates with and without exogenous sucrose. A cross-comparison of acn1-2 and Col-7 revealed that the expression of a number of carbohydrate responsive genes was altered in the mutant. The request for further microarray analysis is to confirm this result. Experiment Overall Design: Number of plants pooled:3 x 1000 seedlings
2008-06-14 | E-GEOD-6158 | biostudies-arrayexpress
Project description:Our goals are to discover the basis of the stress-sensitive phenotypes of the sfr2, sfr3 and sfr6 mutants, and to distinguish damage-repair from damage-prevention-related transcription in the wild type. The effects of sfr2 and sfr3 on cold-induced gene expression will be observed. Since sfr6 causes sensitivity to drought as well as freezing, the effects of sfr6 on the transcriptional response to drought is studied; an observation of cold-induced sfr6 expression is needed for direct comparison to the effect of drought. Unstressed mutants, and equivalently-stressed wild types, are necessary controls. The above experiments are conducted on tissue-culture-grown plants grown under 24 hr illumination for maximum reproducibility and comparability to other transcriptomic experiments.Freezing causes physiological changes even in a hardy, cold-acclimated wild type. During the recovery period, gene expression will reflect the induction of damage-repair processes distinct from the damage-prevention associated with cold acclimation. This will be detected by observing the wild-type transcriptome at two time points during recovery from a freezing episode. The appropriate controls is the unfrozen, cold-acclimated wild type. Plants for this experiment will be soil-grown in an 9/15 day/night cycle to the rosette stage, and this regime will be maintained during acclimation and freezing treatments.Cold-induction will be 10 days at 4°C; drought will be imposed by placing excised leaves in a desiccator for 6 h. The youngest fully-expanded rosette leaves will be harvested for RNA extraction. Experiment Overall Design: Number of plants pooled:18
2008-06-14 | E-GEOD-6177 | biostudies-arrayexpress
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
2008-06-14 | E-GEOD-6165 | biostudies-arrayexpress
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
2008-06-14 | E-GEOD-6176 | biostudies-arrayexpress
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
2008-06-14 | E-GEOD-6170 | biostudies-arrayexpress
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
2008-06-14 | E-GEOD-6174 | biostudies-arrayexpress
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 Âcapture 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
2008-06-14 | E-GEOD-6167 | biostudies-arrayexpress