Transcription profiling of Arabidopsis Cdpk6-yfp vs control to investigate the role of Cdpk6 in development
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ABSTRACT:
ORGANISM(S): Arabidopsis thaliana
SUBMITTER: Nottingham Arabidopsis Stock Centre (NASC)
PROVIDER: E-GEOD-6147 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:In Antirrhinum, the equivalent mutant to the Arabidopsis cuc1 cuc2 double is called cup. We have cloned CUP and shown that it encodes a NAC-domain transcription factor homologous to CUC1 and CUC2. Yeast two-hybrid analysis shows that CUP interacts with TIC, an Antirrhinum TCP transcription factor. Moving back to Arabidopsis, the closest homologues to TIC encode TCP factors TCP13 and TCP14 which, we have now shown, also interact in two-hybrid experiments with CUC1 and CUC2. We have identified insertions in both TCP13 and TCP14. CUP, CUC1 and CUC2 play a role in the establishment of boundaries between lateral organs. As evolutionarily conserved interactors, we expect TCP13 and TCP14 to act in the same process. Homozygous tcp13 mutant flowers show mixed cell identity (mci) with the boundaries of organ identity out of register with those of physical organ development. tcp 14 mutants show a very weak phenotype, but the double heterozygote is identical to the tcp13 homozygote. The double homozygote is underway at the moment and this application takes into account the time required to generate these plants.The aim of this microarray experiment is to identify targets of TCP13 and TCP14. We propose to compare expression levels in WT, tcp13, tcp14, tcp13/+ tcp14/+, and tcp13 tcp14 plants. Although we initially propose only duplicate experiments for each mutant, requiring a total of 10 chips, the experimental material will serve as internal replicates, since we expect to see different degrees of inactivation/activation of at least a core of conserved genes. The fact that all of the mutant combinations observed so far produce flowers with the appropriate cell types present, but in a different position, suggests that, unlike in homeotic mutants, very similar sets of floral genes will be present in each sample. This gives us hope that we might find a smaller number of genes with altered expression. Our tissue samples will be wild type and mutant inflorescences and, as such, will contain a variety of flowers at different stages of development. By taking a large number of flowers and buds we hope to overcome any sampling errors. Although we have requested 10 chips - for duplicate analyses of WT, both single mutants, the heterozygote and the double mutant - it remains a formal possibility that the double homozygote is inviable, or produces no inflorescence. In this unlikely eventuality we would only submit 8 of the 10 samples. Experiment Overall Design: Number of plants pooled:10
2008-06-14 | E-GEOD-6149 | biostudies-arrayexpress
Project description:As part of an investigation into mechanisms of HDG silencing in Arabidopsis, we have produced transgenic plants containing extra copies of the chalcone synthase (CHS) gene. The CHS gene mediates an early step in the biosynthesis of the purple pigment anthocyanin. The insertion of extra copies of CHS in Arabidopsis caused the gene to be silenced in some plants. Seeds harvested from these CHS-silenced plants were mutated by treatment with ems. The progeny of these seeds were screened for "revertants" in which the effects of CHS silencing was alleviated and plants were able to produce anthocyanin. These revertants were found to contain a single recessive mutation; the trait has been termed hog1 (for homology dependant gene silencing 1). Our previous experiment used the Affymetrix 8200 chip to make comparisons between gene expression in the two genetic variants: the CHS-silenced type (ECG) and the anthocyanin-producing revertants (15B). Results showed that there were over 100 genes with at least a 10-fold increase or decrease in expression. However, the variation between 2 reps of each genetic variant was high. We would now like to carry out a modified version of this experiment using the 25K Affymetrix chip. It is intended to increase the number of reps to 4, and take samples at an earlier stage than previously in order to decrease the effect of developmental spread. To reduce the effects of interplant variability, samples will be generated from total plant tissue from a pool of 10 plants at GS 1.04. RNA will be purified using RNeasy kits from Qiagen. Experiment Overall Design: Number of plants pooled:10 Experiment Overall Design: Biological replicates: 4
2008-06-14 | E-GEOD-6166 | 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:The proposal aims to characterise the pathways of DSB repair and recombination in Arabidopsis with the main focus of this research being the NHEJ pathway of illegitimate recombination. We will build on our expertise and resources in the field of DSB repair in plants, using the Arabidopsis NHEJ mutant atku80 which is an excellent model system for the study of DSB repair in higher eukaryotes (West et al., 2002 Plant J. 31, 517-28). Comparison of the transcriptome in NHEJ mutant and wild type plants under different experimental conditions will identify novel candidate genes involved in DNA DSB repair or damage signalling pathways.We will conduct 4 separate experiments on Arabidopsis seedlings grown on 0.5 MS media: the first will be a control consisting of Wassilewskija (WS-2) plants grown under standard conditions. In the second experiment WS plants will be exposed to the chemotherapeutic agent bleomycin (1 microgram per ml). This agent has been shown to cause both single and double strand breaks in the genome of Arabidopsis seedlings (Menke et al., 2001 Mut. Res. 493, 87-93). This agent is used in preference to gamma irradiation, which causes high levels of oxidative damage to cellular components. These experiments will characterise for the first time the transcriptional responses of Arabidopsis cells to the specific induction of DNA strand breaks. The transcript profile will also be determined in untreated atku80 mutants. This experiment will identify the components of novel and previously characterised DNA repair pathways up regulated in the mutant background. Finally, transcript analysis of bleomycin treated atku80 mutants and comparison with untreated mutant plants and both untreated and treated WS plants will identify novel putative DSB repair genes up regulated in response to genotoxic stress in the absence of a Ku80 mediated DSB repair pathway.The results of these studies will provide new and important information which will be instrumental in identifying novel genes and pathways involved in DNA repair and genome stability in plants and help elucidate the fundamental differences that exist between animal and plant DSB repair processes. Experiment Overall Design: Number of plants pooled:20
2008-06-14 | E-GEOD-6178 | biostudies-arrayexpress
Project description:The acclimation of plants to environmental factors (light/temperature/nutrient availability) plays a crucial role in determining their tolerance to stress their ability to compete with other plants and the efficiency with which external inputs are used for growth and productivity. Some of the clearest responses involve the major modifications in the composition of the photosynthetic apparatus in response to light intensity. Photosynthetic acclimation. The acclimation response involves changes in the abundance of a large number of proteins in different cell compartments occurring at different intensity thresholds. The signal transduction chain is complex and involves crosstalk between redox control and other pathways that control photosynthetic gene expression but is poorly understood. Over the past 7 years we have laid the foundations for a molecular genetic approach by characterising the responses of Arabidopsis thaliana to growth in and transfer between high and low light conditions(1-6). Arabidopsis exhibits all the key features of photosynthetic acclimation: changes in maximum photosynthetic rate in leaf structure and in the levels of light-harvesting complexes photosystems and enzymes of carbon metabolism. Method: Samples A-1, A-2 and A-3 were grown at a light intensity of 400 umol.m-2.s-1 until rosette growth was complete. Plants for samples A-2 and A-3 were then transferred to 100 umol.m-2.s-1. Samples A-4, A-5 and A-6 were grown at 100umol.m-2.s-1 until rosette growth was complete, when plants for samples A-5 and A-6 were transferred to 400 umol.m-2.s-1. Samples were taken 24 hours after transfer to the different light intensity and samples A-3 and A-6 were taken 72 hours after transfer. Experiment Overall Design: Number of plants pooled:
2008-06-14 | E-GEOD-6163 | 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: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: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). Experiment Overall Design: Number of plants pooled:10 plants
2008-06-14 | E-GEOD-6171 | biostudies-arrayexpress
Project description:Arabidopsis genome sequencing has revealed the presence of at least three extensive gene families that may encode protein ligands. One of these, the SPH (S-protein homologue) family, was identified as a direct result of our studies on self-incompatibility in Papaver. The Arabidopsis SPH gene family consists of 81 members. We have initiated experimental work on a subset of these. RT-PCR studies indicate that many, if not all, SPH genes are expressed. Each SPH gene encodes an N-terminal signal peptide sequence and thus SPH proteins are likely to be secreted. Until recently none of the genes in this family had known function. However we have evidence that one member of the family, SPH1 is involved in leaf vascular development. In order to determine the function of SPH1, Arabidopsis plants were transformed with an SPH1 antisense construct. Analysis of the mutant phenotype shows that whilst plants appear as wt until principal growth stage 1.04, they subsequently show severe morphological defects. Plants are severely dwarfed with twisted rosette leaves at ~ 30% wt length and width as well as shortened inflorescence stem. Closer examination revealed aberrant leaf vasculature and severe reduction in expansion of parenchyma cells surrounding the primary leaf vein. We have conducted preliminary immunolocalisation studies with antibody raised to SPH1. These suggest that SPH1 protein is secreted by cells within the developing vasculature of the immature leaf (leaves<6mm in length). The data that we have so far obtained leads us to believe that SPH1 protein acts as a signalling molecule during early leaf development. As SPH1 is likely to be a signalling protein it is assumed that its interaction with a cognate receptor results in initiation of developmental processes within leaf tissue. The purpose of the experiment is to determine the network of genes within the normally developing rosette leaf whose expression is altered by SPH1. This will be accomplished by comparing transcriptional levels in rosette leaves of antisense-SPH1 plants with wt plants. We propose to make target RNA from rosette leaves taken from plants at principal stage 1.05 (immediately after initial appearance of developmental abnormality) and from plants at principal stage 1.14 (where gross changes are apparent). We propose to use replicate slides for each hybridisation, i.e. 2 hybridised to RNA from wt leaves at 1.05, 2 antisense at 1.05, 2 wt at 1.14 and 2 antisense at 1.14. Experiment Overall Design: Number of plants pooled:40
2008-06-14 | E-GEOD-6179 | biostudies-arrayexpress
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
2008-06-14 | E-GEOD-6172 | biostudies-arrayexpress