title: Seedling transcriptome affected by a far-red light preconditioning treatment to block chloroplast development.

summary:
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)
Keywords: strain_or_line_design

overall_design: Number of plants pooled:300 seedlings

first: Alex

last: McCormac


title: Genes affected by hog1 alleviation of CHS silencing

status: Public on Jan 22 2007

city: Loughborough

last: Nottingham Arabidopsis Stock Centre (NASC)

address: Sutton Bonington Campus

department: School of Biosciences, University of Nottingham

country: United Kingdom

summary:
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.
Keywords: strain_or_line_design

overall_design:
Number of plants pooled:10
Biological replicates: 4

first: Nick

last: Jordan


title: Differential gene expression patterns in Arabidopsis mutants lacking the K+ channels, akt1, cngc1 and cngc4.

institute: Nottingham Arabidopsis Stock Centre (NASC)

summary:
Background: Release of the caesium radioisotope 137Cs during weapons testing and industrial activity has contaminated thousands of hectares of agricultural land. Ingesting 137Cs has damaging and, sometimes, fatal effects.  Most Cs enters the food chain through plants. The generation of _safe_ crops that exclude Cs and can be cultivated on contaminated land requires knowledge about the mechanisms for Cs uptake. Caesium is chemically similar to potassium (K) and might enter plants through K+ transporters in the plasma membrane of root cells.  To determine which transporters mediate Cs entry to plants, we have compared the accumulation of Cs and K by wildtype Arabidopsis with mutants lacking specific K+ transporters. Preliminary results showed that Cs concentration in the shoots of akt1-1, cngc1 and cngc4 (obtained from the Wisconsin T-DNA knockout facility) differed significantly from the Wassilewskija wildtype (Ws-2). A cursory investigation of their transcriptome, using the Affymetrix Arabidopsis 8K GeneChip, showed that the expression of several genes encoding K+ transporters differed between mutants and wildtype plants.  The aim of this GarNet project is to confirm the previous observations and to identify further genes that are differentially expressed in mutant and wildtype plants and which might impact on Cs accumulation.  Methods: Arabidopsis mutants akt1-1 (N3762), cngc1 and cngc4 and their parental ecotype Wassilewskija -2 (N1601) will be sown on MS agar and transferred to hydroponics 21 days after germination. Seedlings will be grown for a further 7 days on full nutrient solution under continuous light in a Saxcil growth cabinet. RNA will be extracted from roots of mutant and parent (control) plants at the same growth stage and twelve complete-genome Affymetrix GeneChips (3 biological replicates of material from wildtype and 3 mutants) are requested to determine the differences in their transcriptome under comparable environmental conditions.
Keywords: strain_or_line_design

overall_design: Number of plants pooled:20-40

first: Corrina

last: Hampton


title: Investigating the links between organic acid and carbohydrate regulation of gene expression.

summary:
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.
Keywords: gene_knock_out

overall_design: Number of plants pooled:3 x 1000 seedlings

first: Karen

last: Greville


title: The mechanisms involved in the interplay between dormancy and secondary growth in Arabidopsis

summary:
Plants that exhibit secondary growth, such as trees, are a prominent feature of terrestrial ecosystems.  Furthermore, secondary growth itself, particularly wood, has huge economic value. Despite the importance of secondary growth from both basic and applied science perspectives, little is known about the molecular mechanisms that underpin this facet of plant development.  The proposed microarray experiments are designed to expand our knowledge of the regulation of secondary growth by combining the power of Arabidopsis genetics with complete transcriptome analysis.  It is now well established that Arabidopsis can be grown under conditions that induce secondary growth in the hypocotyl, albeit small, wood.   We have grown 8500 Arabidopsis plants of different genotypes under these conditions and will extract RNA from the developing vascular cambia of these plants to subject them to complete transcriptome analysis.The mutants that we have chosen for these analyses are all related to each other on the basis of the fact that they impact dormancy in either seeds or shoots (abi1, aba1, max4, axr1, AtMYB61 knockout, AtMYB50 knockout).  The mutants themselves are the core group of mutants that are the focus of a three laboratory consortium, funded under the BBSRC Exploiting Genomics Initiative, to investigate the molecular basis of meristem dormancy in Arabidopsis.  The other partners in the consortium are Dr. Ottoline Leyser (York) and Dr. Michael Holdsworth (IACR).  While the Leyser and Holdsworth groups have investigated the impact of these mutations on transcriptome activity in shoot meristems and seeds respectively, our work focuses on the vascular cambium.  Thus, this work will not only provide insights into the regulation of cambial function, but, when compared with the existing datasets from the Leyser and Holdworth labs, the work should also provide insights into the relationship between dormancy-impacted  phenomena in different meristematic regions. Beyond this, the specific work on the cambium-specific regulation of genes in the MYB61KO will provide even greater insights into the functioning of this important resource allocation regulator, as it will build on a significant complete transcriptome dataset that is already available through GARNet.  In total, the proposed analyses will generate important new data that builds on existing datasets, to provide an even more comprehensive understanding of gene function, and genetic networks, in an important biological and applied context. Please note that the sample numbers that we have provided below are meant to be the 'base' number for each biological condition (mutant, etc.), and that there will be independent TRIPLICATE biological replicates produced for each condition. (ie. A-1 to A-12).
Keywords: strain_or_line_design; ecotype_analysis;  mutant;  insertion_mutant;  gene_knock_out;  tissue_organ_specificity;

overall_design: Number of plants pooled:200-400 per RNA sample

first: Christian

last: Dubos


title: The trans-differentiation of cultured Arabidopsis cells

summary:
The formation of vascular tissue occurs when cellulose, hemicellulose, lignin and other wall components are deposited within the primary cell wall.  These secondary thickened cells then undergo programmed cell death producing a network of empty cells with which water and ions can be transported throughout the plant. The hormones auxin and cytokinin are the principle signals for vascular tissue initiation. As a consequence cells cultured in-vitro can be converted into vascular tissue with the addition of exogenous auxin and cytokinin. We have created an in-vitro cell system, using callus produced from leaves that can be induced to form vascular tissue. Leaves are callused on induction media for two weeks. The callus is then transferred to liquid media and incubated under optimum conditions resulting in an increase in vascular tissue formation. Approximately 20% of cells will differentiate during the incubation period. The alteration of cytokinin concentration affects the ability of the cultured cells to undergo differentiation. Consequently callus incubated in liquid media, containing lower cytokinin concentrations, will undertake relatively little differentiation. Samples have been isolated from cell cultures at different time points and different hormone concentrations during incubation. Quantitative PCR using the marker AtCesA7, which encodes a cellulose synthase subunit specific to secondary wall deposition, was used as a guide to determine periods of high and low vascular differentiation. This system provides an opportunity to compare gene expression between differentiating and non differentiating cells and allow the identification of genes up regulated during vascular tissue formation.
Keywords: time_series_design;   compound_treatment_design

overall_design: 6 samples

first: David

last: Brown


title: Transcriptome analysis of Arabidopsis microgametogenesis

summary:
Aims We aim to use transcriptome analysis to establish on a genome-wide scale the identity and regulatory clusters of genes that specify microgametogenesis from the haploid microspore to mature functional pollen in Arabidopsis. Background Pollen as the haploid male gametophyte plays a vital role in plant fertility and crop production through the ability to deliver the male gametes in fertilisation. Despite the obvious importance for plant fertility and crop production we have a very limited understanding of the regulatory mechanisms that have evolved to specify male gametophyte development and functions and less than 150 genes have been identified that are gametophytically expressed in the anther.The availability of functional genomic resources now provides the opportunity to undertake a comprehensive approach to describing cellular development in terms of the transcriptome. This approach is particularly powerful where the complete transcriptome of a single developing cell can be analysed. The male gametophyte is a uniquely accessible cell type for such studies, enabling RNA analysis from distinct purified cell populations during development.The proposed experiments are designed to support a current application (P19208, Twell) to investigate the gametophytic transcriptome and transcription factor networks. The results obtained will extend our knowledge of the contribution of haploid gene expression to anther development and will be used directly to extend BBSRC funded work (P15086, Wilson) to investigate the role and targets the MALE STERILE 1 gene (MS1). In particular the data will be used in collaboration to extract haploid gene expression from datasets of transcriptome analysis of staged flower buds of wild type (Ler) and ms1.  This work will also complement BBSRC funded work on sporogenesis (G13338, Dickinson and Scott) and meiosis (G15941, Franklin and Jones) that are focussed on earlier steps in anther development. Biological material and methods.  Isolated microspores and pollen at 4 different developmental stages will be analysed. We will isolate spores from developmentally staged buds of Ler grown under defined growth conditions. Buds from several batches of 100 plants will be rapidly sorted into 4 groups according to developmental age, uninucleate microspores (UM), bicellular pollen (BP) tricellular pollen (TP) and mature pollen. Spores will be released by gentle mechanical tissue disruption and purified by filtration and purification of spores. We are confident that our spore isolation procedures are rigorous since we could not detect even trace expression of highly abundant sporophytic transcripts such RbcS and Cab transcripts in microarray data from pollen RNA.
Keywords: development_or_differentiation_design

overall_design: Number of plants pooled:over 100

last: Honys


title: Gene expression and carbohydrate metabolism through the diurnal cycle

summary:
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.
Keywords: time_series_design;  growth_condition_design

overall_design:
Number of plants pooled:20
Biological replicates: 2

first: Steve

last: Smith


title: Expression data from 23 breast cancer cell lines

status: Public on Mar 05 2007

city: Princeton

first: Fei

last: Huang

state: NJ

country: USA

institute: Bristol-Myers Squibb Co

summary:
Transcriptional profiling was conducted on RNA from 23 breast cancer cell lines to identify genes whose expression level correlates with sensitivity of particular drug
Keywords: comparison of sensitive group versus resistant group of cell lines to particular drug

overall_design: Baseline gene expression profiling was performed using 23 breast cancer cell lines to identify genomic signatures highly correlated with in vitro sensitivity to a particular drug

first: F

first: K

last: Reeves

first: X

last: Han


title: Mechanisms of DNA double strand break repair in Arabidopsis non-homologous end joining mutants

summary:
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.
Keywords: strain_or_line_design; compound_treatment_design

overall_design: Number of plants pooled:20

first: Chris

last: West


title: Comparison of gene expression data from human and mouse breast cancers: Identification of conserved breast tumor genes

status: Public on Jan 12 2007

city: Berlin

first: Andreas

last: Klein

address: Charitéplatz 1

department: Institute of Biochemistry

country: Germany

institute: Charité (CCO)

summary:
The aim of our work was the comparison of human and mouse gene expression data and to identify a conserved breast tumor gene set. The results encourage the usefulness of transgenic mice as a model for human breast cancer formation and therapy.
Keywords: Comparison of gene expression data

overall_design: The aim of our work was to establish a database for breast cancer gene expression data in order to compare human and mouse breast cancer. We identified human and mouse homologues genes and compared the expression profile of 24 human breast tumors with six WAP-SVT/t breast tumors (WAP-SVT/t animals, line 8). Our studies confirmed the heterogeneity in gene expression of human as well as mouse breast cancer cells. However, 63 genes were found to be differentially expressed (upregulated: 40; downregulated: 23 genes) in at least 75% of the breast tumors of both species.

first: Susanne

last: Seitz

first: Rita

last: Schmutzler

first: Dieter

last: Niederacher

first: Norbert

last: Arnold

first: Alfons

last: Meindl

first: Siegfried

last: Scherneck

first: Adolf

last: Graessmann


title: The effects of the sfr2, sfr3 and sfr6 mutations on lyotropic stress responses

last: Nottingham Arabidopsis Stock Centre (NASC)

summary:
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.
Keywords: stimulus_or_stress_design; strain_or_line_design; time_series_design

overall_design: Number of plants pooled:18

first: Gareth

last: Warren


title: Transcriptome analysis of ARRESTED DEVELOPMENT 3 mutant.

summary:
Aims: Comparison of transcriptome between mutant and wild-type plant.
Based on the temperature sensitive period of the mutant the gene likely acts during the earliest stages of the specification of the leaf primordium.
Background: The ARRESTED DEVELOPMENT 3 mutation causes a temperature dependent loss of all spongy mesophyll and most palisade parenchyma in developing leaves. Although these leaves lack most internal tissues excepting vasculature they continue to expand away from the main axis of plant growth. Mature leaves have a small midrib and marginal regions that are large balloons of epidermis covering airspace. add3 is a temperature sensitive mutation whose most severe phenotypic response occurs at a restrictive temperature of 29 degrees C.
Using BAC and TAC filters a recombinant population we have generated and available and newly developed molecular markers a high resolution (1000 chromosomes scored) physical genetic map has been completed to define the physical extent of the locus.
Transformation rescue and RNAi experiments are currently underway to determine if a candidate gene identified using the approach outlined above is ADD3.
Experimentalia: Wild-type and add3 tissue will be grown at 29 degrees C until the first two true leaves have emerged. Two days after emergence the aerial portions of the plants will be collected and RNA will be isolated. This developmental stage will provide plants with primordia in various stages of development.
The wild-type tissue will serve as a control for exposure of developing primordia to high temperature the add3 plants will provide the experimental transcriptome.
We intend to use RNA isolation protocols provided by the former AFGC facility for isolation of RNA.
Conclusion:ADD3 is the first mutant from our collection of temperature sensitive mutations affecting shoot, floral and root organogenesis to be studied at the transcriptome level.
Our work modeling the evolution of gene duplicates and genetic redundancy suggests that temperature conditional redundancy may be common in plants (Pickett and Meeks-Wagner Plant Cell 19957 1347-1356)(Force,Lynch,Pickett et al. Genetics 1999151 1531-1545).
A highly similar gene to the ADD3 candidate gene has been identified.
An exploration of the utility of transcriptomics to describe phenotypic impacts of potential conditionally redundant mutants is particularly timely. Many reverse genetics experiments in Arabidopsis result in "mutants" displaying small or no impacts on phenotype.
The completion of the Arabidopsis genome sequence suggests that this experience may become common.

Experimenter name = F. Bryan Pickett
Experimenter institute = Loyola University of Chicago
Experimenter address = Dept. of Biology
Experimenter address = Loyola University of Chicago
Experimenter address = 6525 N. Sheridan Rd.
Experimenter address = Chicago, IL
Experimenter zip/postal_code = 60626
Experimenter country = USA
Keywords: genetic_modification_design

overall_design: 4 samples were used in this experiment

middle: B

last: Pickett

first: Henrik

last: Townsend

first: Zoe

last: Emmerson

first: Beatrice

last: Schildknecht


title: Comparison of the transcript profile of the auxin resistant axr4 mutant and wild-type Col0

status: Public on Jan 08 2007

summary:
The plant hormone auxin represents an important regulator of growth and development. Significant insight into the mechanisms of auxin action have been obtained from studies of auxin resistant mutants such as aux1 and axr3. The Arabidopsis axr4 mutant was identified in a screen for auxin resistant root growth. In addition to the root growth of axr4 being resistant to exogenous auxin, there is also a 50% reduction in the number of lateral roots that form. The double axr4/aux1 mutant shows an additive effect in reducing lateral root numbers to 10% of wild-type. Gaining further information about the potential interaction between AUX1 and AXR4 may provide important insight into auxin regulated plant growth. Mapping experiments have placed the AXR4 gene on the lower arm of chromosome 1 between the ch1 and le markers (Hobbie and Estelle 1995). However, the AXR4 gene remains to be cloned. Identifying the AXR4 gene will help in elucidating the function of the protein. A transcript analysis of axr4 mutant seedlings will be used in 2 ways. Firstly, the transcription level of genes in the locality of the axr4 map position will be examined to identify those which are absent or significantly reduced in axr4 compared to the Col0 control. If the lesion causing the axr4 mutation results in a highly unstable mRNA or abolishes transcription then the signal will be dramatically reduced. Potential candidate genes identified in this way will be further analysed using a combination of RT-PCR and sequencing to identify the AXR4 gene.  Secondly, the transcriptomics data obtained from axr4 and Col0 will be compared to identify genes which show significant transcript level differences and therefore represent targets for either direct or indirect regulation by AXR4.  Hobbie, L. and Estelle, M. (1995) The axr4 auxin-resistant mutants of Arabidopsis thaliana define a gene important for root gravitropism and lateral root initiation. Plant J. 7 211-220
Keywords: strain_or_line_design

overall_design: 2 samples

first: Alan

last: Marchant


title: Assembly of the cell wall pectic matrix.

summary:
The primary cell walls of land plants are composed principally of a load bearing cellulose microfibril-hemicellulose network embedded in a matrix of pectic polysaccharides. The pectic matrix is multifunctional and in additional to a directly structural role it is central to many fundamental plant processes including cell expansion, defence and cell signalling. The sequencing of the Arabidopsis genome has revealed the massive investment made by plants in modulating the pectic matrix in response to local functional requirements but despite concerted biochemical-based efforts over many years none of the genes involved in pectin biosynthesis/pectic matrix assembly have so far been identified. The pectin matrix contains some of the most complex polysaccharides found in nature and based on linkage analysis it is known that at least 53 glycosyltransferases must be involved in its construction. Our proposal to identify genes involved in pectin biosynthesis and matrix assembly exploits the well characterised phenomenon that many plants and cultured plant cells that are exposed to treatments that disrupt the synthesis of one cell wall component are capable of a compensatory increases in other components - including pectin. Specifically suspension cultured cells that are incrementally exposed to increasing concentrations of the herbicide 26-dichlorobenzonitrile (DCB) which specifically inhibits cellulose synthesis compensate for the resulting almost complete loss of cellulose from their cell walls by constructing walls made predominantly of pectin. We believe that the significant up-regulation of pectin biosynthesis in this system offers an opportunity to identify genes that function in the assembly of the pectic matrix by microarray comparison of transcripts of DCB-treated Arabidopsis cells with untreated cells. The use of Arabidopsis suspension-cultured cells rather than plants or seedlings offers the significant advantage that extracted RNA would be derived from only one cell type. It is anticipated therefore that the output from a transcriptome analysis of this system will indicate a number of genes of unknown function and lead to the identification of genes involved in pectin biosynthesis and the assembly of the pectin matrix.
Keywords: compound_treatment_design

first: William

last: Willats


title: AtGenExpress: Light treatments

summary:
AtGenExpress: A multinational coordinated effort to uncover the transcriptome of the multicellular model organism Arabidopsis thaliana (Hybridisations done at NASC).
The activity of genes and their encoded products can be regulated in several ways, but transcription is the primary level, since all other modes of regulation (RNA splicing, RNA and protein stability, etc.) are dependent on a gene being transcribed in the first place. The importance of transcriptional regulation has been underscored by the recent flood of global expression analyses, which have confirmed that transcriptional co-regulation of genes that act together is the norm, not the exception. Moreover, many studies suggest that evolutionary change is driven in large part by modifications of transcriptional programs. An essential first step toward deciphering the transcriptional code is to determine the expression pattern of all genes. With this goal in mind, an international effort to develop a gene expression atlas of Arabidopsis has been underway since fall 2003. This project, dubbed AtGenExpress, is funded by the DFG, and will provide the Arabidopsis community with access to a large set of Affymetrix microarray data. As part of this collaboration, we have generated expression data from 80 biologicaly different samples in triplicate.
Series 1: Growth conditions: Sterilized seeds will be stratified at 4°C for 3 days, exposed to white light for 2 h to induce germination, and grown on MS agar plates (0.9 % agar) without sugar in total darkness for 4 days at 22°C. Seedlings will then be transferred to the light conditions described for each slide below for 1 h (to identify early induced genes) and 4 hrs (maximum expression of the first initial light response of most target genes), respectively. All light treatments will be done in parallel to minimize the number of dark controls. All samples will be done in triplicate and only with shoots.
Experimenter name = Thomas Kretsch
Experimenter institute = AtGenExpress
Keywords: genetic_modification_design;  organism_part_comparison_design; development_or_differentiation_design

overall_design: 48 samples were used in this experiment

first: Thomas

last: Kretsch


title: Differential gene expression in regulatory T-cells after activation by temporary contact with dendritic cells

status: Public on Jan 09 2007

first: Reinhard

last: Wanner

address: Arnimallee 22

department: Inst. of Molecular Biology and Bioinformatics

institute: Charite-CBF

summary:
Regulatory CD4+CD25+ T-cells were isolated from human blood using MACS and were then left untreated or were cocultured overnight with immature monocyte-derived Dendritic cells. Treg's total RNAs were prepared after moDC-removal.
Keywords: agent response

overall_design: One sample per treatment-group. No replicates.

last: Becht

first: Matthias

last: Peiser


title: An investigation into transcriptional changes in developing Arabidopsis leaf caused by novel signalling protein, SPH1.

summary:
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.
Keywords: genetic_modification_design

overall_design: Number of plants pooled:40

first: Mike

last: Wheeler


title: Expression data from human skin biposies (lesional and uninvolved) from psoriatic patients

first: Rainer

last: Ternes

address: Mullerstrasse

institute: Bayer Schering Pharma

summary:
Biopsies from uninvolved and from lesional skin of 13 patients with plaque-type psoriasis. Based on paired samples, 179 genes were more than 2-fold differentially expressed in lesional skin.
Keywords: Comparison of uninvolved and lesional skin

overall_design: Comparative RNA expression profiles from uninvolved and lesional skin of 13 patients with mild to severe plaque-type psoriasis.

first: Joachim

last: Reischl

first: Susanne

last: Schwenke

first: Johanna

middle: M

last: Beekman

first: Ulrich

last: Mrowietz

first: Steffen

last: Sturzebecher

first: Jurgen

last: Heubach

first: David

last: Bauer


title: Gibberellin and ethylene cross-talk at the level of transcriptional regulation in Arabidopsis.

last: Nottingham Arabidopsis Stock Centre (NASC)

summary:
This work is part of an existing collaboration between the two laboratories, funded by the EU (EU-RTN-INTEGA). Both parties will share the cost of this microarray experiment.  Background: We have demonstrated that ethylene-insensitive mutants and wild type(col-0) Arabidopsis plants treated with an ethylene perception inhibitor have increased levels of expression of genes, such as GASA1 and g-TIP, that are thought to be regulated by GA (Vriezen et al, unpublished results). However, this observation was based on an RNA gel blot analysis and therefore limited to few genes. Aim: To investigate whether plants with decreased ethylene perception are generally hypersensitive to GA or whether this effect is restricted to specific genes. We plan to undertake a complete transcriptome analysis of GA-treated wild type andetr1-1 plants. The aim is to identify genes that are induced directly as a result of the GA treatment, and we will therefore focus on the time window 0-3h.  Tissues to be sampled: Plants will be grown in vitroon MS/2 containing 1% sucrose, pH 5.7, at 22 C,70% RH, under white light (54 PAR) and a photoperiod of 16h light/8h dark. Plants will be treated at 14 days and harvested entirely, i.e. roots and shoots are extracted together.  Experimental set-up: Col-0 and the ethylene-insensitive mutant etr1-1 will be sprayed with 50 microM GA4 in water. GA4 is the major bio-active GA in Arabidopsis. Samples will be taken after 0, 30 min, 1h, and 3h. In order to correct for touch-induced genes a control, which is sprayed with water only and harvested at 1h, will be included for both genotypes. The total number of chips to be hybridized is 10. The time course with 4 data points is preferred to a single time point with 3 repeats, because it will allow us to follow the induction kinetics and identify early response genes. For each timepoint, RNA will be extracted from at least 40 individuals.
Keywords: compound_treatment_design

overall_design: 18 samples

first: Liesbeth

last: De Grauwe


title: Identification of genes involved in secondary cell wall development in the hypocotyls of short day grown Arabidopsis

summary:
Arabidopsis, when grown under short day conditions (16 hours dark, 8 hours light, 22oC) develop extensive secondary thickened hypocotyls with both a vascular and cork cambium (Chaffey et al, 2002, Phys. Plant., 114:594-600). It has been found that once secondary xylem development is completed within the Arabidopsis hypocotyls, it closely resembles the structure of the wood of angiosperm trees (Chaffey et al, 2002, Phys. Plant., 114:594-600).  We can utilise this model Arabidopsis tree to identify genes that are important for secondary cell wall formation in xylem cells and therefore important for wood development.  Columbia plants were grown for 3 months under short day conditions and secondary thickened hypocotyls were snap-frozen in liquid nitrogen.  RNA was isolated from these hypocotyls and submitted to NASC for probing against the ATH1-121501 full GeneChip.
Keywords: growth_condition_design

first: Cathlene

last: Eland


title: Targets of the mci genes.

summary:
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.
Keywords: genetic_modification_design

overall_design: Number of plants pooled:10

first: Brendan

last: Davies


title: A gene expression map of the Arabidopsis root

summary:
This experiment was donated by Philip N. Benfey's lab through ArexDB (http://www.arexdb.org).
A global map of gene expression within an organ can identify genes with coordinated expression in localized domains, thereby relating gene activity to cell fate and tissue specialization. Here, we present localization of expression of more than 22,000 genes in the Arabidopsis root. Gene expression was mapped to 15 different zones of the root that correspond to cell types and tissues at progressive developmental stages. Patterns of gene expression traverse traditional anatomical boundaries and show cassettes of hormonal response. Chromosomal clustering defined some coregulated genes. This expression map correlates groups of genes to specific cell fates and should serve to guide reverse genetics.
Experimenter name = Kenneth Birnbaum
Experimenter department = Benfey Lab
Experimenter institute = Duke University
Experimenter address = Department of Biology
Experimenter address = Duke University
Experimenter address = Box 91000
Experimenter address = Durham
Experimenter zip/postal_code = NC 27708
Experimenter country = America
Keywords: cell_type_comparison_design

overall_design: 27 samples were used in this experiment

first: Kenneth

last: Birnbaum

first: Philip

middle: N

last: Benfey

first: Henrik

last: Townsend

first: Zoe

last: Emmerson

first: Beatrice

last: Schildknecht


title: Molecular basis of respiratory burst-mediated thermotolerance in Arabidopsis

summary:
We have been determining signalling components essential for heat tolerance in Arabidopsis thaliana (Larkindale, J., and Knight, M.R. (2002). Protection against heat stress-induced oxidative damage in Arabidopsis involves calcium, abscisic acid, ethylene, and salicylic acid. Plant Physiol 128, 682-695).  We have most recently found that a heat-induced respiratory burst is necessary for tolerance to high temperatures in Arabidopsis (Larkindale, Torres, Jones and Knight, unpublished).  We have observed that one of the Arabidopsis respiratory burst homologues, AtrbohB, is necessary for the generation of this AOS burst in response to heat, and consequently we have also found that an AtrbohB null mutant shows reduced tolerance to heating (Larkindale, Torres, Jones and Knight, unpublished).  This mutant also shows reduced expression of genes from the HSP90 family (Evans, Larkindale and Knight, unpublished).This application is for transcriptomic analysis of the AtrbohB null mutant in response to heat, in order to understand which genes are activated as a result of heat-induced respiratory bursts in Arabidopsis and also which genes are necessary for physiological thermotolerance in Arabidopsis.  The experiment will involve 6 samples (chips), 3 from wild type Columbia and 3 from the AtrbohB null mutant.  Seedlings will be treated at 20, 30 and 40 degrees centigrade for 1 hour, RNA extracted and submitted to microarray analysis.  One hour treatment has been shown to display clear differences in HSP90 expression and physiological damage, and the temperatures chosen because 30 degrees is a temperature at which acquired thermotolerance can be initiated (thus genes involved in this process can be monitored) and 40 degrees is a temperature at which we observe physiological damage, and gives good discrimination between mutant and wild type.
Keywords: strain_or_line_design

overall_design: Number of plants pooled:90 per sample

first: Nicky

last: Evans


title: Expression Level Polymorphism Project (ELP) - Tsu-1

summary:
This experiment was donated by The ELP Project website at elp.ucdavis.edu that was supported in part by the Arabidopsis 2010 project, NSF Division of Molecular and Cellular Biosciences, award 0115109.
The study of natural genetic variation for plant disease resistance responses is a complementary approach to utilizing mutants to elucidate genetic pathways. While some key genes involved in pathways controlling disease resistance, and signaling intermediates such as salicylic acid and jasmonic acid, have been identified through mutational analyses, the use of genetic variation in natural populations permits the identification of change-of-function alleles, which likely act in a quantitative manner. Whole genome microarrays, such as Affymetrix GeneChips, allow for molecular characterization of the disease response at a genomics level and characterization of differences in gene expression due to natural variation. Differences in the level of gene expression, or expression level polymorphisms (ELPs), can be mapped in a segregating population to identify regulatory quantitative trait loci (expression QTLs) affecting host resistance responses. In order to identify an appropriate RIL population to map QTL controlling disease resistance responses, we performed a parental survey of 7 different Arabidopsis accessions.
We treated vegetatively grown plants with either salicylic acid or a control solution, and harvested the plants at 3 different time points after chemical treatment. We present Affymetrix GeneChip microarray expression data for 3 biological replications of this parental survey.
Keywords: strain_or_line; compound_treatment; time_series

overall_design: 18 samples were used in this experiment

first: Dina

last: St. Clair


title: Expression Level Polymorphism Project (ELP) - Mt-0


title: Differential gene expression patterns in potassium-starved and caesium-treated plants

summary:
At high concentrations ceasium (Cs) is toxic to plant growth. This toxic effect may occur when Cs blocks potassium (K) uptake mechanisms in plants. Consequently, plants starved of K and plants exposed to toxic concentrations of Cs should have similar gene expression patterns. To test this hypothesis, Arabidopsis will initially be grown on agar containing 1/10 MS salts before being transferred to either 1/10 MS nutrient solution (control plants), 1/10 MS nutrient solution containing 2 mM Cs, or 1/10 MS nutrient solution with no K. Roots and shoot will then be harvested seven days after transfer and used to challenge ATH1 GeneChips.
Keywords: compound_treatment_design

overall_design: Number of plants pooled:40-50

first: John

last: Hammond


title: Growth of suspension-cultured cells

summary:
The growth of Arabidopsis cell cultures following their sub-culture into fresh media follows standard growth kinetics of a period of exponential increase associated with high rate of cell division, followed by a slowing of the rate of increase as cells approach stationary phase. For the analysis described here, MM2d cells were subcultured into fresh MSS-medium and samples were taken at day 1, day3, day 5 and day7. We have carried out transcriptional profiling analysis with the aim to follow growth stage specific gene expression during unperturbed growth using the near full genome ATH1 arrays (Menges et al., 2003).
Journal Absract:
(Plant Molecular Biology: 53, 2003)
Plant cell suspension cultures are invaluable models for the study of cellular processes. Here we develop the recently described Arabidopsis suspension culture MM2d as a transcript profiling platform by means of Affymetrix ATH1 microarrays. Analysis of gene expression profiles during normal culture growth, during synchronous cell cycle re-entry and during synchronous cell cycle progression provides a unique integrated view of gene expression responses in a higher-plant system. Particularly striking is that expression of over 14 000 genes belonging to all defined categories can be reliably detected, suggesting that integrated and comparative analysis of data sets derived from transcript profiling of cultures is a powerful approach to identify candidate components involved in a wide range of biological processes. Combinatorial analysis of independent cell cycle synchrony methods allows the identification of genes that are apparently cell-cycle-regulated but are most likely responding to the induction of synchrony. We thus present an integrated genome-wide view of the transcriptional profile of a plant suspension culture and identify a refined set of 1082 cell cycle regulated genes largely independent of synchrony method.

Experimenter name = Jim Murray
Experimenter phone = 44 1223 334166
Experimenter fax = 44 1223 334162
Experimenter department = J Murray Laboratory
Experimenter address = University of Cambridge
Experimenter address = Institute of Biotechnology
Experimenter address = Tennis Court Road
Experimenter address = Cambridge
Experimenter zip/postal_code = CB2 1QT
Experimenter country = United Kingdom
Keywords: time_series_design

first: Jim

last: Murray


title: The role of Cdpk6 in development

summary:
The research programme is focussed on determining the role of CDPKs in abiotic stress response.  We chose several CDPKs for investigation, some which have been implicated in the  response to abiotic stress (Sheen 1996), others whose function is unknown.  We constructed gene fusions with YFP as a 3_in-frame fusion to investigate cellular/tissue expression; the gene-specific DNA includes 1.5kb 5'-untranslated region. Plants carrying the Cdpk6-yfp construct show a marked alteration in phenotype: the (probable)-heterozygote is small and bushy and the (probable)-homozygote shows an excess of flower primordia and insufficient leaf development.  These plants do not survive.  The expression of Cdpk6 as measured using a GUS-promoter fusion is confined to the developing flowerheads although the Cdpk6-YFP plants show more widespread expression. We have made RNA from two separate insertion lines of Cdpk6-YFP and from Col-0 plants carrying the aequorin gene all grown on selective medium.  We cannot collect homozygote seed; the seedlings germinating from seeds collected from heterzygous plants grown on selective medium will consist of both heterozygous and homozygous individuals.
Keywords: strain_or_line_design

overall_design:
Number of plants pooled:about 40
Biological replicates: 2

first: Joy

last: Boyce


title: Expression Level Polymorphism Project (ELP) - Kin-0


title: FRI FLC combos

summary:
This experiment was annotated by TAIR (http://arabidopsis.org).FRI FLC time series

Experimenter name = Markus Schmid
Experimenter phone = ++49-7071-601-1413
Experimenter fax = ++49-7071-601-1412
Experimenter department = Detlef Weigel Laboratory Germany
Experimenter institute = Max-Planck-Institute for Developmental Biology
Experimenter address = Dept. of Molecular Biology
Experimenter address = Spemannstr. 37-39
Experimenter address = Tübingen
Experimenter zip/postal_code = 72076
Experimenter country = Germnay
Keywords: time_series_design; strain_or_line_design; innate_behavior_design

overall_design: 36 samples were used in this experiment

first: Markus

last: Schmid


title: Expression Level Polymorphism Project (ELP) - Col-0

summary:
This experiment was donated by The ELP Project website at elp.ucdavis.edu that was supported in part by the Arabidopsis 2010 project, NSF Division of Molecular and Cellular Biosciences, award 0115109.
The study of natural genetic variation for plant disease resistance responses is a complementary approach to utilizing mutants to elucidate genetic pathways. While some key genes involved in pathways controlling disease resistance, and signaling intermediates such as salicylic acid and jasmonic acid, have been identified through mutational analyses, the use of genetic variation in natural populations permits the identification of change-of-function alleles, which likely act in a quantitative manner. Whole genome microarrays, such as Affymetrix GeneChips, allow for molecular characterization of the disease response at a genomics level and characterization of differences in gene expression due to natural variation. Differences in the level of gene expression, or expression level polymorphisms (ELPs), can be mapped in a segregating population to identify regulatory quantitative trait loci (expression QTLs) affecting host resistance responses. In order to identify an appropriate RIL population to map QTL controlling disease resistance responses, we performed a parental survey of 7 different Arabidopsis accessions.
We treated vegetatively grown plants with either salicylic acid or a control solution, and harvested the plants at 3 different time points after chemical treatment. We present Affymetrix GeneChip microarray expression data for 3 biological replications of this parental survey.

Experimenter name = Dina St. Clair
Experimenter phone = (530) 752-1740
Experimenter department = Department of Plant Sciences
Experimenter institute = UC Davis
Experimenter address = UC Davis Campus
Experimenter address = 114 Amundson Hall
Experimenter address = Davis, California
Experimenter zip/postal_code = CA
Experimenter country = USA
Keywords: strain_or_line; compound_treatment; time_series


title: In vitro tracheary element transdifferentiation of Col-0 suspension cells.

summary:
This experiment was annotated by TAIR (http://arabidopsis.org).
Col-0 suspension cells provided by Dr. C. Koncz and Dr. M. Umeda. 15 ml of Arabidopsis Col-0 suspension cells (Mathur et al. 1998) was transferred to 35 ml of a fresh modified Murashige and Skoog (MS) medium supplemented with 1 ug /ml 2,4-dichlorphenoxyacetic acid and 3% sucrose every 7 day, and cultured on a rotary shaker at 120 rpm in the dark at 22 C for subculture. For xylem vessel element induction, a 7.5 ml aliquot of 7-day-old subcultured cells was transferred into 42.5 ml of fresh medium that included 1 uM brassinolide and 10 mM H3BO3, and cultured as described above. The frequency of xylem vessel element formation was calculated as the proportion of xylem vessel elements to the number of living cells and the vessel elements.

Experimenter name = Hiroo Fukuda
Experimenter phone = +81-3-5841-4463
Experimenter fax = +81-3-5841-4462
Experimenter department = H Fukuda Laboratory
Experimenter institute = University of Tokyo
Experimenter address = Department of Biological Science
Experimenter address = 2nd Buil.
Experimenter address = Hongo 7-3-1, Bunkyo-ku
Experimenter zip/postal_code = Tokyo 113-0033
Experimenter country = Tokyo
Keywords: compound_treatment_design; time_series_design;

overall_design: 12 samples were used in this experiment

first: Hiroo

last: Fukuda


title: Expression Level Polymorphism Project (ELP) - Van-0


title: The early post-germinative embryo and endosperm transcriptomes in Arabidopsis

summary:
Arabidopsis seed germination is coordinated with the strong induction of metabolic pathways required for the mobilisation and utilization of seed storage reserves. These are essential to support the seedling before the establishment of photoauxotrophic growth. The activity of genes encoding enzymes required for lipid mobilisation is regulated largely at the level of transcription, but our knowledge of how this regulation occurs is extremely limited. After germination the rate of lipid reserve mobilisation is determined by the carbohydrate status of the seedling and by the osmotic potential of the growth substrate. The plant response to both of these requires the action of the hormone abscisic acid (ABA).
We have shown that this regulation is tissue specific (Penfield et al., 2004 Plant Cell 16, 2705-2718), and that although lipid breakdown in the embryo is inhibited by ABA, lipid breakdown in the endosperm tissues is not. Furthermore, in many species the action of the endosperm is central to the processes controlling seed germination, yet very little is known about gene expression in this tissue, or of the function of the endosperm in mature Arabidopsis seeds. Mature Arabidopsis seeds can be dissected into embryo and endosperm/seed coat fractions, with the latter fraction containing RNA only from the endosperm as the seed coat cells undergo programmed cell death during the latter stages of seed development. In this experiment we divide seeds into embryo and endosperm tissues and transcript profile both shortly after germination, or when germination and lipid reserve mobilisation are inhibited by ABA or by the gibberellin biosynthesis inhibitor paclobutrazol. In this way we can discover the endosperm transcriptome and uncover candidate regulators of lipid mobilisation by searching for genes showing differential expression between embryo and endosperm before and after ABA treatment.

Experimenter name = Steven Penfield
Experimenter phone = 01904 328759
Experimenter fax = 01904 328762
Experimenter department = Department of Biology
Experimenter institute = Centre for Novel Agricultural Products
Experimenter address = University of York
Experimenter address = PO BOX 373
Experimenter address = York
Experimenter zip/postal_code = YO10 5YW
Experimenter country = UK
Keywords: organism_part_comparison_design

first: Steven

last: Penfield

first: Ian

last: Graham


title: Nutritional control of plant development: molecular analysis of the NO3- response pathway in Arabidopsis roots.

last: Nottingham Arabidopsis Stock Centre (NASC)

summary:
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.
Keywords: strain_or_line_design

overall_design:
Number of plants pooled:20
Biological replicates: 2

first: Sophie

last: Filleur


title: Response to potassium starvation in roots

summary:
This experiment was annotated by TAIR (http://arabidopsis.org).
This experiment studies the response of gene expression in roots of 25-35 day old plants grown on hydroponics after 6, 48 and 96 hours of potassium starvation. RNA from roots was extracted after transfer to control (control) or potassium free nutrient solution respectively (starvation).

Experimenter name = Julian Schroeder
Experimenter phone = 619-534-7759
Experimenter fax = 619-534-7108
Experimenter department = J Schroeder Laboratory
Experimenter institute = University of California-San Diego
Experimenter address = Biology Department
Experimenter address = University of California-San Diego
Experimenter address = La Jolla
Experimenter zip/postal_code = CA 92093-0116
Experimenter country = USA
Keywords: time_series_design; growth_condition_design

first: Julian

last: Schroeder


title: Expression Level Polymorphism Project (ELP) - Cvi-1


title: Expression Level Polymorphism Project (ELP) - Est


title: Mutant array

summary:
This data was annotated by TAIR (http://arabidopsis.org).
Two week-old Arabidopsis aerial tissues from Columbia-0 and cpr5, cpr5npr1, cpr5scv1, cpr5npr1svi1, and npr1 lines were collected for analysis.

Experimenter name = Jinyoung Yang
Experimenter phone = 919-613-8176, 919-61
Experimenter fax = 919-613-8177
Experimenter department = DCMB GROUP
Experimenter institute = Duke University
Experimenter address = B361 LSRC Bldg.
Experimenter address = Research Dr.
Experimenter address = Durham
Experimenter zip/postal_code = NC 27708
Experimenter country = USA
Keywords: genetic_modification_design

first: Jinyoung

last: Yang

first: Xinnian

last: Dong


title: Identification of Core Genes Regulating Plant Programmed Cell Death (PCD)

summary:
PCD is a highly organised process that is involved in development and in an organisms response to biotic stresses (toxins and avirulent pathogens) and abiotic stresses (such as temperature, water availability, etc.).  It is a genetically regulated form of cellular suicide, however in plants the underlying process is poorly understood.  Although PCD may occur in response to different stimuli; we believe once it is triggered, one core mechanism is responsible for the cellular demise.  It is our aim to identify the elements of this mechanism.  We will do this by expanding on the work of a previous user of GARNet's GeneChip microarray facility, Dr. Jodi Swidzinski.  She utilised an Arabidopsis cell suspension system; performing microarray analysis on both senescing, and heat shock induced PCD samples.  This resulted in data showing that a large number of genes were upregulated (or downregulated) in response to both treatments.  We are working under the premise that some of the genes that were similarly regulated under both treatments must be core PCD genes.  However because of the large amount of data generated it is difficult to choose appropriate candidate genes (with any degree of confidence that they are core genes) for further study.
We propose to use a third PCD-inducing treatment, involving a mycotoxin, to generate another population of microarray results.  The mycotoxin we intend to use is Fumonisin B1 (FB1).  This is an extremely potent compound that induces PCD by disrupting ceramide synthesis.  We have found Arabidopsis protoplasts to be much more sensitive than cells to the toxin at low concentrations.  Protoplasts are treated with 20mM FB1 and RNA is extracted at time points when 0%, 20% and 40% of protoplasts have died.  This RNA is then pooled.  RNA from methanol treated protoplasts is used as a control.  We intend for these RNA samples to be subjected to GeneChip microarray analysis.  This would identify genes differentially regulated due to FB1 treatment, which would be interesting in itself.  However, by combining this data with that from previous work by Swidzinski (2002) we will be able to decrease the pool of possible core genes further, and increase the chances of selecting an appropriate candidate gene.  This will both improve upon existing work and add value to an existing GARNet data set.
Experimenter name = Mark Diamond
Experimenter phone = 017162251
Experimenter fax = 017161153
Experimenter address = Botany Department
Experimenter address = University College Dublin
Experimenter address = Belfield
Experimenter zip/postal_code = Dublin 4
Experimenter country = Ireland
Keywords: compound_treatment_design

overall_design: 8 samples were used in this experiment

first: Mark

last: Diamond

first: Paul

last: McCabe

last: Townsend


title: Whole genome expression in response to herbicidal levels of 2,4-D application

summary:
This experiment was annotated by TAIR (http://www.arabidopsis.org).
In this experiment we examined whole genome response to herbicidal levels of 2,4-D application. Arabidopsis plants (14 days) grown in vitro on MS medium in petri dishes were flooded with 1 mL stock of 2,4-D to enable root uptake. Plants were treated with 1.0 mM 2,4-D for a period of 1 hour immediately after which RNA was extracted and used for the microarray based experiments. the goal of this study was to explore the herbicidal mode of action of auxinic herbicide 2,4-D.

Experimenter name = Chitra Raghavan
Experimenter department = Trevor Stevenson Laboratory
Experimenter institute = IRRI
Experimenter address = Entomology and Plant Pathology Division IRRI
Experimenter zip/postal_code = Manila 4031
Experimenter country = Philippines
Keywords: compound_treatment_design

overall_design: 3 samples were used in this experiment

first: Chitra

last: Raghavan

first: Trevor

last: Stevenson

first: Henrik

first: Zoe

last: Emmerson

last: Schildknecht


title: UV-B Responses in Light Grown Plants: Similarities to Biotic Stress

summary:
UV-B (280-320 nm) exposure causes serious damage in plants, limiting their growth and survival, effects that are partly counteracted by repair mechanisms active in plants receiving accompanying visible radiation. Though no particular UV-B receptor has been identified to date, there is strong evidence to indicate that certain aspects of UV-B perception are receptor-mediated. Investigations of down-stream signalling events have thus far indicated broad similarities to pathogen-induced defence responses in plants. In order to identify genes in Arabidopsis that may be up- or down- regulated specifically in response to UV-B exposure and compare them to genes whose expression is altered in plants challenged by an avirulent isolate of Peronospora parasitica (downy mildew), we propose to analyse the transcriptional profiles for the following treatments:

1. UV-B Responses
"A-1" Columbia (Col-0) exposed to supplementary UV-B/UV-A* with a background of low photosynthetically active radiation (PAR of 20 micromol m-2 s-1) for 1.5 photoperiods (photoperiod = 12h). [UV-B treatment]
"A-2" Col-0 exposed to supplementary UV-A and low PAR for 1.5 photoperiods [control for UV-B treatment]
"A-3" Col-0 exposed to visible light only (low PAR) (no UV) for 1.5 photoperiods [control for UV effects in general].*  There are no pure sources of UV-B light available.

2. Pathogen Responses
"A-4" Col-0 spray-inoculated with P. parasitica isolate HIKS-1 (recognised by the R-gene RPP7). After spraying, plants were kept covered in plant propagators and transferred to an 18 degreeC growth chamber. !Samples for RNA extraction were taken 72h after inoculation.
"A-5" The viability of spores was also checked by parallel spraying of the susceptible mutant, Col-rpp7. [pathogen treatment]
"A-6" Col-0 mock treated with water, covered and transferred to an 18 degree C growth chamber, 72h prior to sampling. [control for pathogen treatment]

In all experiments, we are using RNA from leaves taken at the same time of day (6 h into the 12 h photoperiod) from 4.5-week old plants grown under 12h photoperiod. All treatments were normalised against PR-1 expression levels to ensure comparability between UV-B and pathogen treatments. Due to the difficulty in distinguishing between local and systemic induced responses in UV-B treated plants, we are using RNA from whole rosettes for both the UV-B and pathogen treatment for better comparability among treatments. The degree of similarity between these two sets of transcriptional changes will complement and help interpret our experimental data on changes in resistance to pathogens in plants pre-treated with UV-B. Moreover, the data set obtained would allow for identification of UV-B specific changes in gene expression including cis-acting UV-B-responsive promoter elements.
Experimenter name = Julia Brueggemann
Experimenter phone = 01789 470 382
Experimenter fax = 01789 470 552
Experimenter address = Horticulture Research International
Experimenter address = Wellesbourne
Experimenter address = Warwickshire
Experimenter zip/postal_code = CV35 9EF
Experimenter country = UK
Keywords: stimulus_or_stress_design

overall_design: 6 samples were used in this experiment

first: Julia

last: Brueggemann

first: Eric

last: Holub

first: Beatrice


title: Transcriptional profiling of laser-capture micro-dissected embryonic tissues

summary:
The aim of this BBSRC-funded project is to develop laser-capture microdissection (LCMD) to isolate small cell clusters in different regions of arabidopsis embryos at different stages of development; to develop RNA amplification procedures on dissected tissue sampes; and to use DNA microarray techniques to investigate global transcriptional differences between samples. Cryosectioned embryos of ecotype Col-O of globular, heart and torpedo stage were used to isolate cell clusters from the apical and basal regions, for RNA isolation and amplification. !Samples will be provided as T7-primed cDNA, with three biological replicates for each tissue to be analysed. Each replicate comprises cDNA from pooled tissue samples from ca. 15 embryos. The experimental details have been discussed with Sean May et al. at NASC.

Experimenter name = Stuart Casson and Matthew Spencer
Experimenter phone = 0191 374 7356
Experimenter fax = 0191 374 2417
Experimenter institute = Durham University
Experimenter address = Integrative Cell Biology Laboratory
Experimenter address = School of Biological Sciences
Experimenter address = Durham University
Experimenter address = South Road
Experimenter address = Durham
Experimenter zip/postal_code = DH1 3LE
Experimenter country = UK
Keywords: organism_part_comparison_design;  development_or_differentiation_design

first: Stuart

last: Casson

first: Matthew

last: Spencer

first: Keith

last: Lindsey


title: Comparison of CATMA, Affymetrix and Agilent arrays

last: Nottingham Arabidopsis Stock Centre (NASC)

summary:
Transcript profiling is crucial to study biological systems and various platforms have been implemented to survey mRNAs at the genome scale. We have assessed the characteristics of the CATMA microarray designed for Arabidopsis thaliana transcriptome analysis, and compared it with two commercial platforms from Agilent and Affymetrix. The CATMA array consists of gene-specific sequence tags of 150 to 500 base pairs, the Agilent (Arabidopsis 2) array of 60mer oligonucleotides, and the Affymetrix gene chip (ATH1) of 25mer oligonucleotide sets. We have matched each probe repertoire with the Arabidopsis genome annotation (TIGR release 5.0) and determined the correspondence between them.
Array performance was analyzed by hybridization with labeled target derived from eight RNA samples made of shoot total RNA spiked with a calibrated series of 14 control transcripts. A total of fourteen cDNA clones were thus selected and used as templates to synthesize bona fide polyadenylated spike RNAs. Each spike RNA was calibrated then mixed in equal amount with one of the other spike RNAs to obtain seven pairs at equal concentration.  These seven spike RNA pairs were then combined systematically to construct seven complex spike mixes in a design similar to an ordered Latin square, each mix containing six of the seven spike pairs in staggered concentrations covering five logs.  To prevent loss of spike RNA through adsorption to the plastic ware, the spike mixes were prepared in 0.5 µg/µl Col RNA, resulting in a range of concentration from 0.1 to 10,000 copies per cellular equivalent (cpc), assuming that the total RNA contained 1% polyadenalated mRNA and that a cell contained on average 300,000 transcripts. These seven RNA samples included equal amounts of combined spike RNA . To convert the spike hybridization signals to ratios, an eighth sample was prepared, called the reference sample, consisting of the base Col RNA completed with all spike RNAs at a concentration of 100 cpc.
The results from the eight experiments using the Affymetrix gene chips (ATH1) are available for analysis or download from this site.

Experimenter name = Jim Beynon
Experimenter phone = 01798 470382
Experimenter fax = 01789 470552
Experimenter department = Horticulture Research International
Experimenter institute = Warwick University
Experimenter address = Horticulture Research International
Experimenter address = Wellesbourne
Experimenter address = Warwick
Experimenter zip/postal_code = CV34 6QJ
Experimenter country = UK
Keywords: unknown_experiment_design_type

first: Jim

last: Beynon


title: To identify changes in gene expression during silique senescence in Arabidopsis thaliana

summary:
The aim of the experiment is to compare and contrast the profiles of gene expression during silique senescence and to identify the molecular events that regulate the timing of the process. Silique occurs over a highly predictable time frame and as a consequence it may be easier to identify the molecular events that are critical for the process to take place than in systems such as leaf senescence. The material will be collected from 20 Arabidopsis thaliana ecotype Columbia plants  (NASC order number N1093) grown under controlled conditions specified under NASC protocol: Baseline sample preparation. Two pods will be collected from each plant and pooled from 20 plants to reduce sampling variability. It is intended that 3 replicates of the experiment will be submitted.  The RNA will be extracted from mature green silique tissues of 10 days after anthesis and senescing pod tissues of 20 days after anthesis using lithium chloride extraction protocol and cleaned with Qiagen RNAeasy column.

Experimenter name = Thomas Yang
Experimenter phone = 01159516378
Experimenter institute = University of Nottingham
Experimenter address = Division of Plant Sciences
Experimenter address = University of Nottingham
Experimenter address = Sutton Bonington Campus
Experimenter address = Loughborough
Experimenter address = Leicestershire
Experimenter zip/postal_code = LE12 5RD
Experimenter country = UK
Keywords: development_or_differentiation_design

first: Thomas

first: Jerry

last: Roberts


title: Role of COV in vascular patterning

summary:
The molecular mechanisms that control the ordered patterning of vascular tissue development in plant are not well understood. Several models propose a two component for plant vascular differentiation in which requires both an inducer of vascular tissue an also an inhibitory component that prevents the formation of vascular bundles near pre-existing bundles by a process often termed lateral inhibition. We have identified two recessive allelic mutants in Arabidopsis, designated continuous vascular (cov-1, cov-2), which display a dramatic increase in vascular tissue in the stem in place of the interfascicular region that normally separates the vascular bundles.  The mutant plants exhibited normal vascular patterning in leaves and cotyledons. Analysis of the interaction of cov with known auxin signalling mutant and direct analysis of auxin concentrations suggests that cov affects vascular pattering by some mechanism that is independent of auxin. COV is expressed in all plant tissue but more highly in developing flowers and stems.  The COV protein is predicted to be an integral membrane protein of unknown function, conserved in both plants and bacteria that is involved in negatively regulating the differentiation of the vascular tissue in the stem of Arabidopsis.The phenotype of COV exhibits some similarities to acl5 which is known to be a defect in the gene for spermidine synthase. Consequently, we are interested to test the idea that COV may be involved in the transport or perception of polyamines and that polyamines may be essential signals in normal vascular tissue development. Clearly, it this proves to be the case we would expect to see some differences in the transcript levels of genes associated with these pathways.

Experimenter name = Simon Turner
Experimenter phone = 0161 275 5751
Experimenter fax = 0161 275 3938
Experimenter address = University of Manchester
Experimenter address = School of Biological Sciences
Experimenter address = 3.614 Stopford building
Experimenter address = Oxford Rd
Experimenter address = Manchester
Experimenter zip/postal_code = M13 9PT
Experimenter country = UK
Keywords: genetic_modification_design

first: Simon

last: Turner


title: Characterisation of genetic basis of changes in stomatal numbers at elevated atmospheric carbon dioxide concentrations

summary:
Atmospheric CO2 concentrations can determine the number of stomata that form on plant leaves (Woodward & Kelly 1995 New Phyt 131: 311-327). The majority of species exhibit reduced stomatal densities at elevated CO2. However, not all plant species react in the same way to elevated CO2 levels and there is a spectrum of effects:  Some species increase stomatal densities, some decrease stomatal densities, and some are unaffected.  In addition to which, other environmental factors influence the number of stomata that a plant form.  Light intensity has also been shown to affect stomatal numbers in various Arabidopsis ecotypes (Schluter et al. 2003 J Exp Bot 54 (383): 867-874; Lake et al. 2002 J Exp Bot 53 (367): 183-193), by increasing stomatal numbers with increasing light levels.
There are many changes in gene expression under elevated CO2 conditions, so pinpointing specific genes involved in the stomatal response to CO2 is difficult.  In addition, if there is crosstalk between the various signalling pathways affecting ultimate stomatal numbers this complicates further the task of finding genes specifically involved the stomatal response to CO2.  Therefore we propose to look at the interaction of two known influences on stomatal numbers, light and CO2, on one specific ecotype, Col-0.  We aim to test the hypothesis that light signals interact the CO2 signals that affect stomatal development.  Arabidopsis thaliana Columbia-0 ecotype has previously been shown to decrease stomatal numbers in response to a doubling of ambient CO2 concentrations.  Col-0 has also been shown to increase stomatal numbers in response to high light intensities.  Therefore we propose to grow A. thaliana Col-0 at three light intensities (50 mmol m-2 s-1, 150 mmol m-2 s-1 and 250 mmol m-2 s-1), in both ambient and elevated (double ambient) atmospheric CO2 concentrations.  By looking in more detail at how gene expression differs between plants grown at ambient and elevated CO2 at the same light intensities, and also how gene expression differs between plants grown at the same CO2 concentration but different light intensities, we aim to identify those genes involved in the stomatal developmental response to CO2 and whether genes involved in the light response can also be isolated.

Experimenter name = Susannah Bird
Experimenter phone = (0114) 222 4649
Experimenter address = Animal and Plant Science Department
Experimenter address = Alfred Denny Building
Experimenter address = Western Bank
Experimenter address = Sheffield
Experimenter zip/postal_code = S10 2TN
Experimenter country = UK
Keywords: growth_condition_design

first: Susannah

last: Bird

first: Ian

last: Woodward


title: Effect of mycotoxin treatment on gene expression of wild-type and an altered sensitivity mutant

summary:
Fungal secondary metabolites can not only cause toxic effects in animals and humans, but also serve as virulence factors of the producing fungi for causing plant diseases.Thus, the severity of plant diseases associated with mycotoxins depend on the sensitivity towards the toxin. In previous experiments, we have evaluated the phytotoxic effect ofa mycotoxin on root growth of Arabidopsis wild-type and mutant seedlings. Mycotoxin treatment of a new conditional root expansion mutant partially restores the expansion phenotype (JE100; Werner et al., unpublished).
AIM: This experiment aims to identify genes, in early and later phases after mycotoxin treatment in wild-type and mutant seedlings.
EXPERIMENTAL PLAN: Eight Affymetrix chips are needed for this experiment. RNA preparation will be provided from wild-type, accession Columbia, and mutant seedlings after different time points of mycotoxin treatment. As control, separate seedlings will be treated with the same concentration of solvent (DMSO). Briefly, seeds will be sterilized, stratified for 48 hours and germinated on MS agar plates containing 4.5% sucrose at 22°C and 16h/8h light/dark cycles. 10 days after germination, seedlings will be transferred to liquid MS medium and shaken for another 3 days for acclimatization. Seedlings will be harvested after 2 and 24 hours of treatment with a single concentration (50 µM) of mycotoxin. To account for experimental variations (i.e. time needed for freezing the tissues, circadian clock,...), the experiment will be repeated three times and RNA samples will be pooled.
EXPECTED RESULTS: The experiment should identify genes differentially expressed:
1) between wild-type and mutant seedlings,
2) upon mycotoxin treatment in wild-type,
3) upon mycotoxin treatment of mutant seedlings and
4) upon solvent treatment.
The results will allow us to pinpoint the mode of action of this mycotoxin. They will also allow us to better understand the function of the mutated gene which affects the sensitivity towards the mycotoxin. Furthermore, we expect to identify the signaling pathway by which the plant responses towards the mycotoxinis triggered.

Experimenter name = Ulrike Werner
Experimenter phone = +43-1-36006-6371
Experimenter fax = +43-1-36006-6392
Experimenter department = Institute of Applied Genetics and Cell Biology
Experimenter institute = BOKU
Experimenter address = Center of Applied Genetics
Experimenter address = University of Agricultural Sciences Vienna
Experimenter address = Muthgasse 18
Experimenter address = Vienna
Experimenter zip/postal_code = 1190
Experimenter country = Austria
Keywords: genetic_modification_design; compound_treatment_design;  time_series_design

first: Ulrike

last: Werner

first: Marie-Theres

last: Hauser


title: Response to ZAT12 expression

summary:
This experiment was annotated by TAIR (http://arabidopsis.org).
This experiment looks at changes in gene expression in response to constitutive expression of the transcription factor ZAT12.

Experimenter name = Jonathan Vogel
Experimenter phone = 517-355-2299
Experimenter fax = 517-353-5174
Experimenter department = MSU-DOE Plant Research Lab
Experimenter institute = Michigan State University
Experimenter address = East Lansing
Experimenter zip/postal_code = MI 48824
Experimenter country = USA
Keywords: genetic_modification_design

overall_design: 4 samples were used in this experiment

first: Jonathan

last: Vogel

first: Michael

last: Thomashow

last: Townsend


title: Carbohydrate- and redox-regulation of gene expression in a TPT mutant

summary:
The triose-phosphate/phosphate translocator (TPT) of the chloroplast inner envelope membrane mediates the counter-exchange of stromal triose phosphates derived from CO2 fixation with cytosolic phosphate, thus providing the cytosol with precursors for sucrose synthesis. We have isolated an Arabidopsis mutant (tpt-1) in which the gene encoding TPT is disrupted by a T-DNA insertion. During growth in low light tpt-1 plants are phenotypically normal, but in high light photosynthesis is inhibited and growth is retarded relative to wildtype. This mutant compensates for the absence of TPT by diverting photosynthate into starch which is hydrolysed and exported from the chloroplast as glucose that is subsequently phosphorylated by hexokinase. In low light the capacity of the pathway of starch synthesis is sufficient to accommodate the normal rate of CO2 fixation, but in high light it is unable to match the potential rate of CO2 fixation. Consequently, in high light-grown plants there are measurable effects on the redoxstate of several components. Thus, the tpt-1 mutation influences the carbohydrate status within the cell, alters the form in which carbon is received by the cytosol, and changes the redox signals that are important in photosynthetic acclimation.Plants will be grown in a 8h light:16h dark regime at both 400 and 100 Âµmol PAR m-2 s-1. Total RNA will be extracted from pools of individual illuminated leaves from at least eight plants at growth stage 3.70. Leaves will be harvested 2 h into the photoperiod to maximises differences between plant lines in the expression of genes of photosynthesis and carbohydrate metabolism. We anticipate that expression of many genes will be affected by the absence of TPT and by changes in light intensity. However, by comparing differences in transcript levels between wildtype and TPT mutant grown in high light with the differences that occur in plants grown in low light we will discriminate between genes whose expression is affected by changes in the pattern of carbohydrate metabolism and those influenced by redox poise of the thylakoid photochemical components. These comparisons will also highlight genes affected by recently identified regulatory interactions between sugar-sensing and redox-sensing. A genome-wide expression study will establish the extent to which gene expression is altered by the absence of TPT in leaves, and will provide the basis for more detailed analysis of a selected range of transcripts whose levels of expression differ between plant lines.

Experimenter name = Robin Walters
Experimenter phone = 01865 275000
Experimenter fax = 01865 275074
Experimenter address = Dept of Plant Sciences
Experimenter address = University of Oxford
Experimenter address = South Parks Road
Experimenter address = Oxford
Experimenter zip/postal_code = OX1 3RB
Experimenter country = UK
Keywords: growth_condition_design;  genetic_modification_design;

first: Robin

last: Walters

first: Nicholas

last: Kruger

first: Henrik

first: Zoe

last: Emmerson

last: Schildknecht


title: Plant gene expression associated with susceptibility to nematodes

summary:
Background
Heterodera schachtii is an economically important plant parasitic nematode that forms a syncytium from a cell superficial to the formed vascular bundle by progressive recruitment of other cells into the structure. The pattern of plant gene expression changes dramatically inside the syncytium. The pathogen probably plays a major role in defining the plant response by choice of initial plant cell during precise behaviour in planta and/or by the secretions it releases. The modified plant cells enable a high feeding rate by the female nematode so enhancing its rate of development and subsequent daily egg production. Arabidopsis is widely used as a model plant to characterise molecular responses to nematodes (e.g. Sijmons et al., 1991 Plant J. 1:245-254.). A complete overview of the changes in plant gene expression when sedentary nematodes establish has not yet been gained using Arabidopsis or any other host plant.
Experimental Approaches
Our initial studies will focus on the H. schachtii/Arabidopsis interaction. To assure reliable microarray screening care has been taken to minimise extraneous differences between samples (see "Growth conditions" section). At 21 days (Growth stage 3.2-3.5 Boyes et al., 2001 Plant Cell 13:1499-1510) Arabidopsis plants were challenged with rigorously sterilised, infective nematodes of H. schachtii as before (Urwin et al., (1997) Plant Journal 12: 455-461.). 35 sterile J2s were pipetted onto small ~0.5mm2 squares of sterile GF/A filter paper. The GF/A paper was left in direct contact with the zone of elongation on 3 lateral roots per plant for 48 hours. Control plants were mock inoculated with sterile water. Sections of root containing syncytia have been excised from the thin and transparent roots of Arabidopsis and collected into RNAlater solution (Ambion) at 21 days post infection (Growth Stage 6.1 Boyes et al. 2001). The female nematode has been removed with watch-maker's forceps. Equivalent sections of root have been harvested from non-infected plants. Material has been collected from c. 1000 plants for each of the two samples and the uninfected material serves as an internal control. Total RNA has been prepared from the reference and test root material using an RNeasy plant RNA preparation kit (Qiagen) according to methods required by GARNET.Some questions on the form are omitted as we are not using mutant or transgenic lines. This is our first application.

Experimenter name = Peter Edward Urwin
Experimenter phone = 0113 343 3035/2909
Experimenter fax = 0113 343 3144
Experimenter address = Centre for Plant Science
Experimenter address = University of Leeds
Experimenter address = Leeds
Experimenter zip/postal_code = LS2 9JT
Experimenter country = UK
Keywords: pathogenicity_design

overall_design: 2 samples were used in this experiment

first: Peter

middle: E

last: Urwin

first: Howard

middle: J

last: Atkinson

first: Beatrice


title: Polycomb Binding Protein

last: Nottingham Arabidopsis Stock Centre (NASC)

summary:
We have identified a gene encoding apolycomb-chromo domain binding protein. Complete knockout of these plants do not lead to a phenotype, but a reduction of gene expression in RNAi line produces a defined phenotype. This argues in favour of a compensation mechanism that is switched on once the concentration of the gene reaches a control level.The aim of the experiment is to search for such compensatory factors that are up-regulated in the knockout line.

Experimenter name = Katharine Cain
Experimenter phone = 01133433146
Experimenter department = University of Leeds
Experimenter address = University of Leeds
Experimenter address = Clarendon Way
Experimenter address = Leeds
Experimenter zip/postal_code = LS29JT
Experimenter country = UK
Keywords: genetic_modification_design

first: Katharine

last: Cain

last: Meyer


title: Environmental Genomics of calcicole-calcifuge physiology

summary:
Aim
To identify genes which are differentially expressed between calcicoles and non- calcicoles.
Background
Grasslands on the calcareous soils of chalk and other limestones are among the most species-rich plant communities in Europe (Rodwell 1991 et seq.). They have experienced huge losses and remain vulnerable to such impacts as neglect of traditional management, agricultural improvement and global changes in climate, nitrogen depositions and ozone levels. Our understanding of the physiological characteristics of calcicoles and calcifuges remains limited. A detailed understanding of the genetic basis of the mechanisms that enable calcicoles to thrive on calcareous soils is essential to enable us to predict how these plant communities and their constituent species will be affected by environmental change and how the biodiversity of these ecosystems can be sustained.At Lancaster we have been studying calcicole-calcifuge physiology, with particular reference to Ca2+-tolerance, for over fifteen years. Recently, our research has focused on the regulation of apoplastic Ca2+ in Arabidospsis thaliana.  We have compared the response of two ecotypes of A. thaliana, the non-calcicole ecotype Columbia (Col-4) and the calcicole ecotype Cal-0, which is a genetically uniform line from an original population collected by Ratcliffe from a rocky limestone slope in 1954, to rhizospheric Ca2+. Our results show that Cal-0, exhibits a markedly higher tolerance to growth on high rhizospheric Ca2+ compared to Col-4.Our hypothesis is that adaptation to a calcareous environment will be reflected in altered gene expression. To test this hypothesis we will grow Col-4 and Cal-0 at low (1 mM) and high (12.5 mM) rhizospheric Ca2+ and compare the patterns of gene expression by microarray analysis. In the first instance, to eliminate any differences in gene expression between the Cal-0 and Col-4 ecotypes, we will compare RNA that will be extracted using the Qiagen RNEasy kits, from plants grown at 16 hour day lengths and will be harvested after 30 days of growth on sand watered with 0.5 X Long Ashton solution containing 1 mM CaCl2.
Experimenter name = Bev Abram
Experimenter phone = 01524 65201ext93524
Experimenter fax = 01524 843854
Experimenter address = Biological Sciences
Experimenter address = Lancaster University
Experimenter address = Bailrigg
Experimenter address = Lancaster
Experimenter zip/postal_code = LA1 4YQ
Experimenter country = UK
Keywords: strain_or_line_design

first: Bev

last: Abram

first: Alan

last: Shirras