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	User guest, your query for Experiments   with accession  =  *NASC* produced 60 matches

1 / 60 Experiment : E-NASC-1 Submitter(s) :  Cornah Lab :  University of Edinburgh

Experiment Design Type : genetic modification (Generated description): Experiment with 12 hybridizations, using 12 samples of species [Arabidopsis thaliana], using 12 arrays of array design [Affymetrix GeneChip® Arabidopsis Genome [ATH1-121501]], producing 12 raw data files and 12 transformed and/or normalized data files. (Submitter's description 1): We aim to assess changes in gene expression in the early stages of seedling growth in glyoxylate cycle enzyme knock-outs. The analysis has two clear goals: firstly to discover those genes (and hence enzymes) whose expression changes markedly when a major switch in the pathway of seed lipid mobilisation is imposed, and secondly to use information from metabolome analysis in the same mutants, to discover the impact of well-defined changes in endogenous metabolites on the transcriptome.In wild type seedlings at approximately 2 days post-imbibiton, glyoxylate cycle activity reaches a peak associated with the mobilisation of lipid reserves for the growth of the developing seedling. We have isolated KOs in two enzymes unique to the glyoxylate cycle: malate synthase (ms) and isocitrate lyase (icl). The mutants have a visible seedling phenotype only in the absence of sugar and/or (high) light. Surprisingly, these mutants are able to mobilise their lipid reserves, apparently via a switch from glyoxylate cycle and gluconeogenesis to respiration (Eastmond et al. 2000, PNAS 97: 5669-5674). Transcriptomic analysis will allow us to establish what changes take place in expression of key genes involved in respiration and gluconeogenesis when the glyoxylate cycle is blocked in the KOs. This will in turn provide insights into the pathways of metabolism operating in these plants.In addition we are already in the process of having these mutants analysed by the GARNet metabolomics facility to quantitate changes in organic acids, amino acids and sugars. The use of the transcriptomics facility in parallel will allow us to determine how specific changes in metabolite levels affect the expression of genes involved in carbon and nitrogen metabolism, as well as in seedling development as a whole. One major strength of our approach is that both ms and icl mutants are blocked in the glyoxylate cycle and so will result in many common changes to the metabolome, but each may produce novel changes in specific metabolites which might allow correlation with specific changes in the transcriptome. The joint analysis of metabolome and transcriptome data will be carried out in collaboration with members of our School of Informatics.Whole seedlings will be grown in vitro and harvested 2 days after transfer of imbibed seed to continuous light, and used for RNA extraction. This corresponds to Principle Growth Stage 0.7 in the convention of Boyes et al. (Plant Cell 13; 1499-1510, 2001). Each mutant has a specific wild type control (Columbia in one case and Ws in the other) giving four RNA samples in total.

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2 / 60 Experiment : E-NASC-2 Submitter(s) :  Millenaar Lab :  University of Utrecht

Experiment Design Type : growth condition (Generated description): Experiment with 9 hybridizations, using 9 samples of species [Arabidopsis thaliana], using 9 arrays of array design [Affymetrix GeneChip Arabidopsis Genome [ATH1-121501]], producing 9 raw data files and 9 transformed and/or normalized data files.. (Submitter's description 1): Ethylene induced hyponastic growth in Arabidopsis thaliana F.F. Millenaar L.A.C.J. Voesenek and A.J.M. Peeters Our aim is to identify genes involved in the ethylene induced hyponastic growth. Upon submergence some plant species like Rumex palustris changes its leaf angle (hyponastic growth) and shows enhanced petiole elongation to reach the water surface. In Rumex palustris the hyponastic growth is initiated by an increased concentration of ethylene due to physical entrapment and ongoing ethylene biosynthesis. A proteomics, genomics and genetical approach to improve our understanding of above described flooding-induced responses are not feasible in Rumex palustris since genomic information about this species is limited. However it is possible to use the model plant Arabidopsis thaliana as a tool in flooding research. Natural accessions (Be0 Col Cvi Kas Ler Nd Rld Shah and Ws) show considerable genetic variation in hyponastic growth upon exposure to ethylene Col exhibiting the largest effect (maximum rate after 3 hours) and Ler no effect whatsoever. Using a computer controlled digital camera the hyponastic growth is measured in great detail. Next to ethylene addition also a transfer to low light causes hyponastic growth. This seems to be an ethylene independent pathway because etr1 and ctr1 showed hyponastic growth after transfer to low light. Ethylene and low light showed additive effects in Col. It is likely that ethylene induces more changes in gene expression than only the ones involved in hyponastic growth. By subtracting changes in the Ler expression profile from changes in the Col expression profile we expect to find why Col and Ler respond differently on ethylene by finding specific ethylene induced genes that are involved in hyponastic growth. The expression profile of Col following transfer to low light will be substracted from Col following ethylene addition to distinguish between genes that are involved in hyponastic growth but are not specific for ethylene induced hyponastic growth. There are strong indications in Rumex palustris that other hormones i.e. auxin ABAand GA are involved in the ethylene induced hyponastic growth. Currently mutants in ethylene auxin and ABA biosynthesis and/or signal transduction are screened for hyponastic growth. Preliminary results showed that also in Arabidopsis these other hormones are involved in ethylene induced hyponastic growth. Beside the mutant approach we also started a proteomics and a PCR based differential screen approach. Together with the proposed transcriptome analysis we hope to find new genes involved in ethylene induced hyponastic growth.

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3 / 60 Experiment : E-NASC-3 Submitter(s) :  Urwin Lab :  University of Leeds

Experiment Design Type : (Generated description): Experiment with 2 hybridizations, using 2 samples of species [Arabidopsis thaliana], using 2 arrays of array design [Affymetrix GeneChip® Arabidopsis Genome [ATH1-121501]], producing 2 raw data files and 2 transformed and/or normalized data files. (Submitter's description 1): Background Heterodera schachtiia 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.

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4 / 60 Experiment : E-NASC-4 Submitter(s) :  Greco Lab :  Plant Research International

Experiment Design Type : strain or line (Generated description): Experiment with 2 hybridizations, using 2 samples of species [Arabidopsis thaliana], using 2 arrays of array design [Affymetrix GeneChip® Arabidopsis Genome [ATH1-121501]], producing 2 raw data files and 1 transformed and/or normalized data files. (Submitter's description 1): In recent years our group has been constructing an activation tag library based on the En-I transposon system which resulted in the identification of novel Arabidopsis mutants (Marsch Martinez et al 2002; manuscript accepted for publication in Plant Phys). Among them the bountiful (bou) mutant showed dominant alterations in leaf size and morphology, delayed flowering, vertically oriented siliques and higher yield. Sequence analysis of the genomic region flanking the transposon insertion in combination with expression analysis indicated that the mutant phenotype observed was presumably caused by over-expression of a gene encoding a yet uncharacterised DNA binding protein. In particular over-expression in the activation tagged mutant seems to cause ectopic expression of the BOU gene in all vegetative and reproductive tissues while the endogenous pattern of expression appeared to be restricted only to root tissue. Over-expression lines in which the BOU ORF was driven by the 35S promoter displayed the bountiful phenotype confirming that the activation tagged phenotype was indeed caused by activation of the BOU gene. Protein sequence analysis indicates a putative role for BOU as a chromatin remodelling factor which in association with the expression pattern suggests a possible involvement of BOU in high-hierarchy order of regulation of gene expression. Hence microarrays could be very useful for the identification of downstream interacting factors or target geneswhich will help us to gain insights towards unravelling the biological role of the BOU gene. The specific interaction to flowering time genes will be studied independently using RT-PCR to reveal the relationship to other genes in the regulatory pathway. As experimental setup we intend to compare gene expression between bountiful mutant and wild-type arabidopsis plants using rosette leaf tissue as source for RNA. Though the BOU gene is endogenously expressed in a root-specific mannerits ectopic expression in the bountiful mutant phenotipically affects the whole plant including leaves.

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5 / 60 Experiment : E-NASC-5 Submitter(s) :  Rentel Lab :  University of Oxford

Experiment Design Type : stimulus or stress (Generated description): Experiment with 4 hybridizations, using 4 samples of species [Arabidopsis thaliana], using 4 arrays of array design [Affymetrix GeneChip® Arabidopsis Genome [ATH1-121501]], producing 0 raw data files and 4 transformed and/or normalized data files. (Submitter's description 1): Expression of the OX1 gene coding for a putative protein kinase was shown to be induced in Arabidopsis thaliana ecotypes Col-0, WS and RLD seedlings by treatment with H2O2 superoxidecolddroughtsalt and cellulase (elicitor). All of these treatments will ultimately lead to production of H2O2 either as a secondary effect of the applied stress through disturbance of electron transport processes or as a direct product of the plant NADPH oxidases (oxidative burst). However the sets of protective genes switched on (end-responses) will differ depending on the original stress. The induction characteristics of OX1 point to a role of this kinase in signal transduction downstream of H2O2; however it is not clear which primary signal is relayed. OX1 may function in the perception of oxidative stress caused by all/any one of the environmental stresses and/or it may be important in signalling downstream of the oxidative burst triggered by pathogen infection and wounding. An OX1 knock-out was isolated from the Wisconsin T-DNA lines with an insertion between the first and second conserved kinase domains rendering the protein product non-functional. Aim of the microarray experiment is to compare gene induction in the homozygous knock-out line to that in the wild-type (ecotype WS) following H2O2 treatment. To this end 7 day-old seedlings will be immersed in 10 mM solution for 1 h. Analysis of differences in expression of genes belonging to a certain end-response will disclose the signalling pathway which OX1 functions in. This is necessary for further planned experiments e.g. analysis of inducible OX1 antisense and constitutive lines as well as kinase activity assays.

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6 / 60 Experiment : E-NASC-6 Submitter(s) :  Casal Lab :  University of Buenos Aires

Experiment Design Type : strain or line (Generated description): Experiment with 4 hybridizations, using 4 samples of species [Arabidopsis thaliana], using 4 arrays of array design [Affymetrix GeneChip® Arabidopsis Genome [AG1]], producing 0 raw data files and 4 transformed and/or normalized data files. (Submitter's description 1): Plant growth and development are strongly affected by light signals perceived by phytochromes (phy) and cryptochromes (cry). The physical interaction between photoreceptors and the cross-talk among downstream signalling steps creates a network of interactions in which the action of one photoreceptor depends on the status of the others. The processes modulated by phy and or cry include seed germination, seedling de-etiolation, plant body formation and flowering. Compared to the wild type the leaves of the phyB mutant are characterised by their extended petioles and pale colour. These features resemble the phenotype of normal plants grown under the low red to far-red ratios typical of dense plant canopies thus providing support to the idea that phyB plays a key role in the perception of high red to far-red ratios. The triple mutant lacking phyA cry1 and cry2 shows little differences in leaf size and shape with the wild typebut the quadruple phyA phyB cry1 cry2 mutant shows severely impaired leaf expansion. In addition to the specific effects on leaf sizeshape and pigmentation the phyB mutation accelerates flowering in the wild type background. This effect can be observed as a reduced number of days between sowing and visible flower buds (time scale) and as a reduced final number of leaves (developmental scale). However in the phyA cry1 cry2 triple mutant background the phyB mutation accelerates flowering on a developmental scale but severely delays flowering on a time scale. The long photoperiods inducing flowering are perceived by the leaves. Flowering signals that could include hormones and sugars migrate from the leaves to the apex in response to the light stimulus. We propose to compare the following mRNA samples of expanding leaves: 1) wild type 2) phyB mutant 3) phyA cry1 cry2 triple mutant 4) phyA phyB cry1 cry2 quadruple mutant. The comparison between the wild type and the phyB mutant will help to uncover the changes in leaf mRNA patterns underlying enhanced petiole growthreduced pigmentation and early flowering in response to the absence of phyB activity. The comparison between the phyA cry1 cry2 triple mutant and the phyA phyB cry1 cry2 quadruple mutant should reveal the mRNA differences between photosynthetic leaves with dramatically different abilities to expand. The added value of the simultaneous analysis of the two comparisons is that effect of phyB is different in both genetic backgrounds and this should provide information on the mechanisms and significance of photoreceptor interactions.

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7 / 60 Experiment : E-NASC-7 Submitter(s) :  Murray Lab :  Dr Katherine Denby Lab

Experiment Design Type : strain or line (Generated description): Experiment with 8 hybridizations, using 8 samples of species [Arabidopsis thaliana], using 8 arrays of array design [Affymetrix GeneChip® Arabidopsis Genome [ATH1-121501]], producing 2 raw data files and 8 transformed and/or normalized data files. (Submitter's description 1): The Arabidopsis mutant cir1 displayed constitutive expression of defence genes and enhanced resistance to the virulent pathogens Pseudomonas syringae pv tomato (Pst) and Peronospora parasitica Noco2. In order to dissect the downstream signalling components constitutively activated in cir1, cir1 plants were crossed to the ethylene-insensitive mutant ein2. Resistance to Pst was abolished in cir1:ein2 plantswhile resistance to P. parasitica Noco2 was maintained. Thus CIR1-mediated resistance to Pst appears to be dependent on ethylene signalling through EIN2but resistance to P. parasitica Noco2 is EIN2-independent. The aim of the proposed experiment is to identify EIN2 dependent and independent global gene expression in cir1 plants. It is likely that expression of a sub-set of EIN2-dependent genes in cir1 are required for resistance to Pst whereas EIN2-independent genes may be required for P.parasitica Noco2 resistance. Identification and analysis of genes in these sub-sets may reveal common regulatory mechanisms and may extend the understanding of resistance to Pst and P. parasitica Noco2 in Arabidopsis. This information should be of interest to the Arabidopsis disease resistance community. Total RNA will be extracted from leaf tissue harvested from five-week old soil-grown plants.

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8 / 60 Experiment : E-NASC-8 Submitter(s) :  Villadsen Lab :  University of Edinburgh

Experiment Design Type : stimulus or stress (Generated description): Experiment with 6 hybridizations, using 6 samples of species [Arabidopsis thaliana], using 6 arrays of array design [Affymetrix GeneChip® Arabidopsis Genome [ATH1-121501]], producing 4 raw data files and 6 transformed and/or normalized data files. (Submitter's description 1): It has been strongly argued that plant cells should have a means of sensing sugars at the cell surface, so that extracellular and intracellular sugars can be sensed separately and their metabolism coordinated (Lalonde et al., Plant Cell, 11, 707-26, 2000). There is good evidence for an intracellular hexokinase-dependent pathway of hexose sensing in plants, but very little evidence for a hexokinase-independent signalling pathway, such as that provided by SNF3 or RGT2 in yeast. Many papers on sugar sensing in plants cite work from two laboratories as evidence for hexokinase-independent hexose signalling in plants. The first is that in which cell-wall invertase and sucrose synthase genes were induced by treatment of a Chenopodium suspension culture with 30 mM 6-Deoxyglucose (6DOG) for 24 h (Roitsch et al., Plant Physiol 108, 285-294, 1995; Godt et al., J. Plant Physiol 146, 231-238, 1995). The second is that in which a patatin transgene in Arabidopsis was shown to be weakly induced by growth over several days on a mixture of 30 mM glucose plus 30 mM 3-O-methylglucose (3OMG), but strongly induced by growth on 30 mM Glc plus 90 mM 3OMG (Martin et al., Plant J, 11, 53-62, 1997). We are not aware of any examples of Arabidopsis genes which respond to 6DOG or 3OMG yet this is an area of wide significance. Identification of such a gene would help to establish if a hexokinase-independent signalling system operates in plants, and would provide a basis for establishment of a genetic screen for mutants, using the gene promoter linked to a reporter such as luciferase. The aim of this proposal is to discover any genes which are either activated or repressed by glucose AND by 3OMG and/or 6DOG, but not by mannitol (an osmotic control). The use of both 3OMG and 6DOG will help to identify non-specific effects of either. All substrates will first be analysed by HPLC to confirm that they are pure. Arabidopsis Col-0 seedlings will be grown in vitro for 7 days in the absence of sugars, then treated with 30 mM glucose or glucose analogue for 8 h (these conditions are based on concentrations and time courses of Roitsch et al.). RNA will then be isolated from multiple independent plates to minimise biological variation.

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9 / 60 Experiment : E-NASC-9 Submitter(s) :  Furner Lab :  Department of Genetics

Experiment Design Type : strain or line (Generated description): Experiment with 4 hybridizations, using 4 samples of species [Arabidopsis thaliana], using 4 arrays of array design [Affymetrix GeneChip® Arabidopsis Genome [AG1]], producing 0 raw data files and 3 transformed and/or normalized data files. (Submitter's description 1): The C-line contains additional copies of chalcone synthase (CHS) and these result in silencing of the endogenous copy at tt4. The clv1-2 mutation was introduced into this line as a second marker. The line was mutagenised and several revertants obtained which show reduced silencing. The best characterised of these is hog1. The hog1 mutant shows reduced DNA methylation and more intact CHS transcript in Northern blots. I have been funded by the BBSRC gene flow initiative to look for downstream targets of DNA methylation by looking for genes which are up or down regulated by hog1. Three week old plants of the genotype C/C clv1-2 with or without the hog1 mutation will be used to prepare RNA for the study. I have set the sample size at two slides for each sample.

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10 / 60 Experiment : E-NASC-10 Submitter(s) :  Ward Lab :  University of York

Experiment Design Type : strain or line (Generated description): Experiment with 2 hybridizations, using 2 samples of species [Arabidopsis thaliana], using 2 arrays of array design [Affymetrix GeneChip® Arabidopsis Genome [ATH1-121501]], producing 0 raw data files and 2 transformed and/or normalized data files. (Submitter's description 1): The aim of this study is to identify genes differentially expressed during the transition between dormancy and activity in axillary shoot apical meristems. We have chosen to study this by comparing mRNA populations from the axillary buds of the auxin over-responding, apically dominant axr3-1 mutant of Arabidopsis,with those from the axillary buds of the auxin resistant axr1-12 bushy mutant. Preliminary investigation using cDNA AFLP has been successful in identifying differentially expressed transcripts in the buds of these two genotypes, thus demonstrating the importance of this study, however this is a time consuming procedure. Axillary buds from axr3-1 are seen to arrest at an early stage when the buds are approximately 2mm long and harvested at this point. Buds of a similar size were harvested from axr1-12 plants and the RNA extracted using Qiagen columns.These two mRNA samples will represent the dormant and active buds to be comparedin this experiment. The plants from which these buds were harvested were grown in adjacent p40 trays in a plant growth room. Between two and three buds were harvested from each plant.

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11 / 60 Experiment : E-NASC-11 Submitter(s) :  Bird Lab :  Animal and Plant Science Department

Experiment Design Type : growth condition (Generated description): Experiment with 6 hybridizations, using 6 samples of species [Arabidopsis thaliana], using 6 arrays of array design [Affymetrix GeneChip Arabidopsis Genome [ATH1-121501]], producing 6 raw data files and 6 transformed and/or normalized data files.. (Submitter's description 1): 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.

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12 / 60 Experiment : E-NASC-15 Submitter(s) :  Brown Lab :  University of Manchester

Experiment Design Type : compound treatment , time series (Generated description): Experiment with 6 hybridizations, using 6 samples of species [Arabidopsis thaliana], using 6 arrays of array design [Affymetrix GeneChip Arabidopsis Genome [ATH1-121501]], producing 6 raw data files and 6 transformed and/or normalized data files.. (Submitter's description 1): 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.

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13 / 60 Experiment : E-NASC-16 Submitter(s) :  Brueggemann Lab :  Horticulture Research International

Experiment Design Type : stimulus or stress (Generated description): Experiment with 6 hybridizations, using 6 samples of species [Arabidopsis thaliana], using 6 arrays of array design [Affymetrix GeneChip® Arabidopsis Genome [ATH1-121501]], producing 6 raw data files and 6 transformed and/or normalized data files. (Submitter's description 1): 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.

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14 / 60 Experiment : E-NASC-17 Submitter(s) :  Buchanan-Wollaston Lab :  Horticulture Research International

Experiment Design Type : development or differentiation (Generated description): Experiment with 10 hybridizations, using 10 samples of species [Arabidopsis thaliana], using 10 arrays of array design [Affymetrix GeneChip® Arabidopsis Genome [ATH1-121501]], producing 10 raw data files and 10 transformed and/or normalized data files. (Submitter's description 1): Many signalling pathways are involved in controlling gene expression during plant senescence. Pathways involving SA, JA and ethylene have a role in senescence but none are essential for the senescence process to occur. The aim of this experiment is to classify senescence-enhanced genes into groups depending on the signalling pathways that regulate them. This will provide useful information on the relative importance of each signalling pathway during senescence and allow us to separate potential senescence-specific genes and pathways from the stress response pathways.Mutants in genes in the ethylene pathway (ein2) and the jasmonate pathway (coi1) and the NahG transgenic plant which is defective in the salicylic acid pathway will be grown until the mid flowering stage. Fully developed green and partially senescent leaves will be harvested from the plants at this stage. In addition, two different lines of Arabidopsis (Col-5 glabrous and Col-0) will be grown as controls. Leaves will be harvested from the two control plants before flowering (green) and at mid flowering as above. The control plants will be harvested at two stages to identify the senescence- enhanced genes. The effects of each mutation on the senescence related expression of these genes will then be studied.Mutant RNAs will be isolated in duplicate. The two control accessions will act as replicates for the wild type. Two wild type accessions will be used to reduce possible differences that could be observed the mutants due to slight differences in background.

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15 / 60 Experiment : E-NASC-19 Submitter(s) :  Davies Lab :  University of Leeds

Experiment Design Type : genetic modification (Generated description): Experiment with 8 hybridizations, using 8 samples of species [Arabidopsis thaliana], using 8 arrays of array design [Affymetrix GeneChip® Arabidopsis Genome [ATH1-121501]], producing 8 raw data files and 8 transformed and/or normalized data files. (Submitter's description 1): 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.

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16 / 60 Experiment : E-NASC-20 Submitter(s) :  De Grauwe Lab :  Ghent University

Experiment Design Type : compound treatment (Generated description): Experiment with 18 hybridizations, using 18 samples of species [Arabidopsis thaliana], using 18 arrays of array design [Affymetrix GeneChip® Arabidopsis Genome [ATH1-121501]], producing 18 raw data files and 18 transformed and/or normalized data files. (Submitter's description 1): 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.

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17 / 60 Experiment : E-NASC-21 Submitter(s) :  Deeken Lab :  Group Prof. Dr. Rainer Hedrich

Experiment Design Type : pathogenicity (Generated description): Experiment with 4 hybridizations, using 4 samples of species [Arabidopsis thaliana], using 4 arrays of array design [Affymetrix GeneChip® Arabidopsis Genome [ATH1-121501]], producing 4 raw data files and 4 transformed and/or normalized data files. (Submitter's description 1): Agrobacterium tumefaciens, a bacterial species found in temperate soils world wide, is the causative agent of crown gall disease on many plants. A. tumefaciens-induced tumours are feared in orchards and vineyards because of their pathological interference with nutrient and water supply which results in crop decline. Small wounds at the crown of the plant, usually induced by wind-bending, are potential entry sites for the bacterium. The tumorous growth is initiated by the integration and expression of the T-DNA of the bacterial Ti plasmid within the plant nuclear DNA. The T-DNA encodes enzymes catalysing the synthesis of increased concentrations of auxin and cytokinin, and of opines which stimulate cell division and enlargement. The fast growing tumours have been shown to be a strong nutrient sink on their host plants. As a matter of fact, sugar and K+ content were found to be up to 10- and 5-fold, respectively, higher in this tissue and transpiration was about 15 times increased compared to normal tissue. Whereas the morphological structure as well as some physiological and biochemical parameters of the tumour have been analysed in detail, little is known about the underlying gene expression pattern. Proliferation and growth of the tumour induced by Agrobacterium tumefaciens is obviously due to the extraordinary high concentration of phytohormons, minerals and metabolites. Their influence on regulation of gene transcription will provide information on the mechanisms underlying fast tumour growth. In a project funded by the DFG we recently started to investigate the role of solute transporter for tumour development on the model plant Arabidopsis thaliana. By comparing the expression pattern of RNA preparations from Arabidopsis tumour and non-tumour tissue, we will be able to identify genes which facilitate crown gall development. For the expression analysis with an Affymetrix full genome chip we will induce tumours at the base of an injured Arabidopsis inflorescence stalk (var. Wassilewskija, WS-2). RNA will be extraxted from tumour and injured non-tumor inflorescence stalk tissue using the RNeasy Plant Mini Kit (Qiagen), followed by a DNase treatment to eliminate DNA contamination.

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18 / 60 Experiment : E-NASC-22 Submitter(s) :  Diamond Lab :  Botany Department

Experiment Design Type : compound treatment (Generated description): Experiment with 8 hybridizations, using 8 samples of species [Arabidopsis thaliana], using 8 arrays of array design [Affymetrix GeneChip® Arabidopsis Genome [ATH1-121501]], producing 8 raw data files and 8 transformed and/or normalized data files. (Submitter's description 1): 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.

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19 / 60 Experiment : E-NASC-24 Submitter(s) :  Evans Lab :  University of Oxford

Experiment Design Type : strain or line (Generated description): Experiment with 6 hybridizations, using 6 samples of species [Arabidopsis thaliana], using 6 arrays of array design [Affymetrix GeneChip® Arabidopsis Genome [ATH1-121501]], producing 6 raw data files and 6 transformed and/or normalized data files. (Submitter's description 1): 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.

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20 / 60 Experiment : E-NASC-25 Submitter(s) :  Filleur Lab :  Department of Biological Science

Experiment Design Type : strain or line (Generated description): Experiment with 8 hybridizations, using 8 samples of species [Arabidopsis thaliana], using 8 arrays of array design [Affymetrix GeneChip® Arabidopsis Genome [ATH1-121501]], producing 8 raw data files and 7 transformed and/or normalized data files. (Submitter's description 1): 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.

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21 / 60 Experiment : E-NASC-26 Submitter(s) :  Fromm Lab :  Centre for Plant Sciences

Experiment Design Type : strain or line (Generated description): Experiment with 5 hybridizations, using 5 samples of species [Arabidopsis thaliana], using 5 arrays of array design [Affymetrix GeneChip® Arabidopsis Genome [AG1]], producing 5 raw data files and 5 transformed and/or normalized data files. (Submitter's description 1): Screening of cDNA expression libraries with labelled calmodulin (CaM) as a probe resulted in the isolation of cDNA encoding proteins designated CAMTA (for calmodulin-binding transcription activators). The Arabidopsis genome contains 6 members of this protein family (AtCAMTA1-6) all containing in addition to a defined CaM-binding domain a DNA-binding domain and ankyrin-repeat motifs. RT-PCR analysis revealed that all 6 genes are expressed in all organ throughout plant development. Therefore functional regulation of AtCAMTA proteins is likely mediated by second messengers (e.g. calcium/calmodulin signalling) and protein levels rather than by or in addition to gene expression levels. Based on domain organisation and sequence homologies we identified putative members of this protein family in C. elegans and in human. A yeast system was used to express chimeric fusion proteins comprised of the DNA-binding domain of the bacterial LexA protein with various segments of AtCAMTA1. This analysis revealed a distinct domain of AtCAMTA1 capable of activating transcription. Similar results were obtained with two human CAMTA homologues. To identify the gene targets of CAMTA proteins in Arabidopsiswe plan to analyse the transcriptome in loss_of_function and gain_of_function AtCAMTA mutants. For this we have already isolated a T-DNA insertion mutant of AtCAMTA1 (two alleles) and have requested two other insertion mutants of AtCAMTA2 and AtCAMTA3 identified in other labs. Screening for insertion mutants in the three remaining genes is underway. In addition we have initiated a gain_of_function approach in which DL10 proteins are expressed under the control of the dexamethasone-inducible promoter. Genes whose expression will be found modulated in the mutant plants will be considered candidate targets of AtCAMTA proteins. This analysis will complement an in vitro study of DNA-protein interactions to identify target-binding sites (part of the BBSRC funded project). As a first step in the project proposed to GARNet we suggest to compare the transcriptome in WT whole plants (2 weeks old) with that of three T-DNA insertion AtCAMTA mutants and one line expressing AtCAMTA1 under the control of an inducible promoter.

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22 / 60 Experiment : E-NASC-27 Submitter(s) :  Gallois Lab :  Gallois Lab

Experiment Design Type : growth condition (Generated description): Experiment with 6 hybridizations, using 6 samples of species [Arabidopsis thaliana], using 6 arrays of array design [Affymetrix GeneChip® Arabidopsis Genome [AG1]], producing 6 raw data files and 6 transformed and/or normalized data files. (Submitter's description 1): We have published results demonstrating that UV-C induces apoptotic-like changes in Arabidopsis (Danon and Gallois FEBS lett (1998) 437: 131-136). The Programmed Cell Death "phenotype" of nucleus shape, DNA laddering caspase-like activity is similar to what has been described in other plant PCD. This demonstrates that UV-C is an appropriate and controllable trigger to study PCD in plants. Using selected mutants and a set of chosen conditions we are aiming at identifying genes that are part of the PCD pathways in plants and filter out the general stress response. We are asking for a medium size experiment knowing that additional microarray analysis are likely to be required to refine the results obtained. We are currently awaiting the results of Affymetrix RNAs hybridisation of PCD-induced wild type that will define the appropriate single time point of the proposed analysis. The rationale is to vary treatments in order to distinguish changes in gene transcription arising from general cellular stress responses to the trigger used from those specific to PCD: 1. We will use wild-type Arabidopsis seedlings as a negative control to provide the basal gene expression pattern. 2. A dose of UV-C radiation of 1KJ/m2. We will irradiate wild type with this non PCD-inducing dose of UV-C radiation to identify sets of genes that respond to UV-induced damage but whose expression is not linked to cell death. 3. A dose of UV-C radiation of 50 KJ/m2. We will irradiate wild type with a PCD-inducing dose of UV-C radiation to identify sets of genes that respond to UV-induced damage and cell death. 4. We have shown that the cell death induced by UV is light dependant, the control, sub-inducing dose and inducing dose treatments will be repeated and the plants kept in the dark until RNA sampling. In those conditions there is no PCD.

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23 / 60 Experiment : E-NASC-28 Submitter(s) :  Gawronski Lab :  Department of Pomology and Basic Sciences in Horticulture

Experiment Design Type : organism part comparison (Generated description): Experiment with 6 hybridizations, using 6 samples of species [Arabidopsis thaliana], using 6 arrays of array design [Affymetrix GeneChip® Arabidopsis Genome [AG1]], producing 6 raw data files and 6 transformed and/or normalized data files. (Submitter's description 1): The aim of the experiment is to study the pattern of genes expression involved in defense to heavy metals and their transport and distribution within plant organs. Most of the plant species inhibits heavy metals entrance to the roots and their transport and accumulation to/in above ground organs after toxic ions penetrate roots. Some species of Brassicacea differ in the pattern of defense gene expression in the roots in a way that allow for greater transport of toxic ions to the above ground part. If so, by harvesting such plants toxic elements will be extracted from the soil. Identification of these differences in the gene expression is crucial in development of the newly emerging environmental biotechnology phytoremediation in which plants as well as being green lungs play also a role of a green liver. Results of our studies with mustard plants showed among others that exposure for 12 hr to 12.5mg of Pb/1L of hydroponics solution is efficient to increase of constitutive genes expression and in de novo induction of genes expression coding for Pb tolerance and that this response is organ specific. Sequencing of the whole genome of A. thaliana opens the opportunity to study this phenomenon at molecular level more widely including the signaling (two-component system) and plasmolemma and tonoplast transporters genes. Therefore in our further steps we are going to use Arabidopsis as a model plant and the transcriptomics aproach is the only way for such studies. At first based on results with mustard physiological study we will conduct for elucidating Arabidopsis plant responses to lead in order to choose the most appropriate dose and time of treatment. In the trascriptome experiment we would like to study the profile gene expression in above ground parts and in the roots of arabidopsis plants in response to toxic ions of lead. Results of this study, besides promising applicable aspects, will also lead to a better understanding of plant acclimation and adaptation to antropogenic stresses. We expect to obtain mutant(s) tolerant to Pb and if so, they will be provided to NASC.

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24 / 60 Experiment : E-NASC-29 Submitter(s) :  Greville Lab :  University of Wales, Bangor

Experiment Design Type : genetic modification (Generated description): Experiment with 12 hybridizations, using 12 samples of species [Arabidopsis thaliana], using 12 arrays of array design [Affymetrix GeneChip® Arabidopsis Genome [ATH1-121501]], producing 12 raw data files and 12 transformed and/or normalized data files. (Submitter's description 1): 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.

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25 / 60 Experiment : E-NASC-31 Submitter(s) :  Hampton Lab :  Horticulture Research International

Experiment Design Type : strain or line (Generated description): Experiment with 12 hybridizations, using 12 samples of species [Arabidopsis thaliana], using 12 arrays of array design [Affymetrix GeneChip® Arabidopsis Genome [ATH1-121501]], producing 12 raw data files and 12 transformed and/or normalized data files. (Submitter's description 1): 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.

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26 / 60 Experiment : E-NASC-34 Submitter(s) :  Howard Lab :  University of Sheffield

Experiment Design Type : growth condition (Generated description): Experiment with 6 hybridizations, using 6 samples of species [Arabidopsis thaliana], using 6 arrays of array design [Affymetrix GeneChip® Arabidopsis Genome [AG1]], producing 6 raw data files and 6 transformed and/or normalized data files. (Submitter's description 1): The acclimation of plants to environmental factors (light/temperature/nutrient availability) plays a crucial role in determining their tolerance to stress their ability to compete with other plants and the efficiency with which external inputs are used for growth and productivity. Some of the clearest responses involve the major modifications in the composition of the photosynthetic apparatus in response to light intensity. Photosynthetic acclimation. The acclimation response involves changes in the abundance of a large number of proteins in different cell compartments occurring at different intensity thresholds. The signal transduction chain is complex and involves crosstalk between redox control and other pathways that control photosynthetic gene expression but is poorly understood. Over the past 7 years we have laid the foundations for a molecular genetic approach by characterising the responses of Arabidopsis thaliana to growth in and transfer between high and low light conditions(1-6). Arabidopsis exhibits all the key features of photosynthetic acclimation: changes in maximum photosynthetic rate in leaf structure and in the levels of light-harvesting complexes photosystems and enzymes of carbon metabolism. Method: Samples A-1, A-2 and A-3 were grown at a light intensity of 400 umol.m-2.s-1 until rosette growth was complete. Plants for samples A-2 and A-3 were then transferred to 100 umol.m-2.s-1. Samples A-4, A-5 and A-6 were grown at 100umol.m-2.s-1 until rosette growth was complete, when plants for samples A-5 and A-6 were transferred to 400 umol.m-2.s-1. Samples were taken 24 hours after transfer to the different light intensity and samples A-3 and A-6 were taken 72 hours after transfer.

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27 / 60 Experiment : E-NASC-35 Submitter(s) :  Jarvis Lab :  Department of Biology

Experiment Design Type : strain or line (Generated description): Experiment with 3 hybridizations, using 3 samples of species [Arabidopsis thaliana], using 3 arrays of array design [Affymetrix GeneChip® Arabidopsis Genome [AG1]], producing 3 raw data files and 3 transformed and/or normalized data files. (Submitter's description 1): Aim: To identify regulatory factors that control: (1) chloroplast protein importand (2) chloroplast-to-nucleus signalling. This project is a joint proposal from the Jarvis lab which is interested in chloroplast protein import [1] and the Moller lab which is interested in plastid-to-nucleus signalling [2]. Background: The majority of chloroplast proteins are encoded in the nucleus and imported post-translationally into chloroplasts. The abundance of chloroplast proteins may therefore be regulated at multiple levels. It is well documented that the nuclear gene expression is responsive to (largely unknown) signals from the chloroplast [23] and evidence is now emerging that protein import is also a regulated process [1]. Protein import into chloroplasts is mediated by protein complexes in the outer and inner envelope membranes called Toc and Tic respectively. Biochemical studies of pea chloroplasts identified several Toc/Tic components. These proteins are mechanistic or structural components of the import apparatus. Arabidopsis homologues of the pea Toc/Tic proteins were identified by the AGI. Pea Toc34 is represented in Arabidopsis by two genes "Toc33 and Toc34" and pea Toc75 is represented by three genes. These different Tocs have different expression patterns and are proposed to have different precursor protein recognition specificities. The factors that regulate Toc expression in concert with the needs of plastids in developmentally different cells are unknown. Proposal: Two Arabidopsis mutants will be analysed. The ppi1 mutant is null for the putative precursor protein receptor Toc33 [1]and the ppi3 mutant is null for a putative component of the protein import channel Toc75-IV (on chromosome IV). ppi1 plants are yellow-green in appearance but remarkably healthy and grow only slightly more slowly than wild type. By contrast ppi3 plants are indistinguishable from wild type by eye although analysis of the mutant's chloroplast proteome is beginning to reveal some differences (K. Lilley personal communication). Gene expression changes in ppi1 are likely to be quite extensive. Retardation of chloroplast development in ppi1 will activate retrograde signalling pathways so that many nuclear photosynthetic genes are down-regulated. Changes in the expression of photosynthetic genes and of the genes responsible for mediating these responses may therefore be observed. Any regulatory and signalling genes identified will be of interest to the Moller lab. The expression of factors that regulate Toc/Tic gene expression may also be altered in ppi1. It should be possible to distinguish these factors from those involved in the general control of chloroplast gene expression by comparing the results from the two mutants. Genes affected in both mutants are more likely to be involved in regulating chloroplast import since it is unlikely that widespread changes in gene expression will be observed in ppi3. Changes in the expression of factors that regulate import post-translationallyand of the Toc/Tic genes themselves (many are on the RNA) may also be observed. References: 1. Jarvis P. et al. (1998) Science 282: 100-103. 2. Moller S.G. et al. (2001) Genes Dev. 15:90-103. 3. Jarvis P. (2001) Curr. Biol. 11: R307-R310.

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28 / 60 Experiment : E-NASC-36 Submitter(s) :  Jordan Lab :  University of Cambridge

Experiment Design Type : strain or line (Generated description): Experiment with 8 hybridizations, using 8 samples of species [Arabidopsis thaliana], using 8 arrays of array design [Affymetrix GeneChip® Arabidopsis Genome [ATH1-121501]], producing 8 raw data files and 8 transformed and/or normalized data files. (Submitter's description 1): 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.

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29 / 60 Experiment : E-NASC-37 Submitter(s) :  Knight Lab :  University of Oxford

Experiment Design Type : strain or line (Generated description): Experiment with 6 hybridizations, using 6 samples of species [Arabidopsis thaliana], using 6 arrays of array design [Affymetrix GeneChip® Arabidopsis Genome [ATH1-121501]], producing 6 raw data files and 6 transformed and/or normalized data files. (Submitter's description 1): Our analysis of the sfr6 freezing-sensitive mutant (Knight, H., Veale, E., Warren, G. J. and Knight, M. R. (1999). Plant Cell 11, 875-886.) and cls8 (unpublished) chilling-sensitive mutant of Arabidopsis, has revealed that the expression of certain cold-regulated genes is aberrant in both these mutants. In order to understand the molecular basis of chilling and freezing stress in Arabidopsis and also to determine commonalities and differences between these 2 different physiological stress-tolerance processes, we request transcriptome analysis for both of these mutants compared to wild type in one experiment, upon cold treatment and at ambient conditions. The sfr6 mutant shows the most severe phenotype with respect to cold gene expression, but is tolerant to chilling (Knight, H., Veale, E., Warren, G. J. and Knight, M. R. (1999). Plant Cell 11, 875-886.). However, it is unable to cold acclimate and hence is sensitive to freezing. The cls8 mutant, on the other hand, has a relatively mild phenotype relative to the cold-regulated genes we have examined, but is very sensitive to chilling temperatures (15 to 10 degree centigrade). It is thus likely that in cls8 we have not yet identified the genes which are most affected, and which account for the physiological phenotype. Both sfr6 and cls8 have been fine-mapped and are close to being cloned. The cls8 mutant has an altered calcium signature in response to cold which means it is likely to be affected in early signalling, e.g. cold perception itself.We will compare the expression profiles of genes in sfr6, cls8 and Columbia (parental line for both mutants), both at ambient, and after treatment with cold (5 degrees) for 3 hours. This timepoint is designed to “capture” both rapidly responding genes e.g. CBF/DREB1 transcription factors, and also more slow genes e.g. COR genes (KIN1/2 and LTI78). Pilot northerns confirm that this time point is suitable.This analysis will provide new insight into 2 novel genes required for tolerance to low temperature in Arabidopsis, and additionally will determine the nature of overlap between the separate processes of chilling and freezing tolerance.

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30 / 60 Experiment : E-NASC-39 Submitter(s) :  Marchant Lab :  Plant Science

Experiment Design Type : genetic modification (Generated description): Experiment with 2 hybridizations, using 2 samples of species [Arabidopsis thaliana], using 2 arrays of array design [Affymetrix GeneChip® Arabidopsis Genome [ATH1-121501]], producing 2 raw data files and 2 transformed and/or normalized data files. (Submitter's description 1): 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

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31 / 60 Experiment : E-NASC-40 Submitter(s) :  McCormac Lab :  University of Southampton

Experiment Design Type : compound treatment , genetic modification (Generated description): Experiment with 6 hybridizations, using 6 samples of species [Arabidopsis thaliana], using 6 arrays of array design [Affymetrix GeneChip® Arabidopsis Genome [ATH1-121501]], producing 6 raw data files and 6 transformed and/or normalized data files. (Submitter's description 1): Regulation of expression of genes encoding chloroplast components is critical to the autotrophic plant and never more so than in the cotyledons of the de-etiolating seedling. Many chloroplast proteins are nuclear-encoded and a retrograde signal from the chloroplasts (the Plastid Signal) modulates nuclear transcription. However, not all chloroplast-targeted genes are subject to this control and not all plastid-dependent nuclear genes are chloroplast-targeted. We therefore aim to provide the most comprehensive screen yet of which genes are affected by plastid-signalling. To specifically knock-out positive plastid signalling in light-grown cotyledons, the herbicide Norflurazon (NF) is supplied in the growth medium, causing a carotenoid deficiency that leaves the chloroplasts vulnerable to photobleaching. This blocks the expression of a subset of nuclear genes, such as Lhcb and HEMA1. Two pairs of RNAs will directly compare the transcription in seedlings grown under continuous white light with and without NF. A third RNA will also be compared from a mutant that shows a degree of constitutive positive plastid signalling. These two mutants act synergistically to counteract the effect of NF on nuclear transcription. The gun1,gun5 double mutant maintains a significantly higher level of Lhcb and HEMA1 expression in the presence of photobeached chloroplasts than the NF-treated wild-type. This transcriptome set will therefore complement RNA1 (wild-type+NF) and indicate which of the genes identified from the RNA1/RNA2 comparison are subject to the particular gun1/gun5 plastid signalling pathway(s). The growth of conditions of the seedlings (namely on MS medium supplemented with 1.5% sucrose, for 3 days under continuous WL following 2 days germination in darkness) has been chosen from the results of our own recent studies using Northern blotting techniques that show these conditions to maximise the respective NF and gun mutant effects on Lhcb and HEMA1 gene expression. This experiment is part one of a two-part study to compare the transcriptional output of this NF-affected pathway with that of a newly discovered FR-mediated pathway. A subsequent array experiment will assess the nuclear response as affected by this FR/ phyA-input pathway and the two sets of array data will be compared and contrasted. Note: Col-0 wild-type (NASC code N1092). gun1,gun5 double mutant(obtained from Enriquez Lopez-Juez, Royal Holloway, University of London.(Mochizuki et al. 2001 PNAS 98: 2053-2058). This line cannot be donated by us as it is the IP of Joanne Chory (SALK Institute, USA). Treatment = a herbicide (Norflurazon) application which leads to chloroplast photobleaching and hence down regulation of nuclear genes dependent on plastid signalling from intact chloroplasts.

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32 / 60 Experiment : E-NASC-41 Submitter(s) :  McCormac Lab :  University of Southampton

Experiment Design Type : genetic modification , growth condition (Generated description): Experiment with 10 hybridizations, using 10 samples of species [Arabidopsis thaliana], using 10 arrays of array design [Affymetrix GeneChip® Arabidopsis Genome [ATH1-121501]], producing 10 raw data files and 10 transformed and/or normalized data files. (Submitter's description 1): 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)

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33 / 60 Experiment : E-NASC-42 Submitter(s) :  Moller Lab :  University of Leicester

Experiment Design Type : individual genetic characteristics , strain or line , compound treatment (Generated description): Experiment with 6 hybridizations, using 6 samples of species [Arabidopsis thaliana], using 6 arrays of array design [Affymetrix GeneChip® Arabidopsis Genome [AG1]], producing 6 raw data files and 6 transformed and/or normalized data files. (Submitter's description 1): Plastids communicate with the nucleus by means of retrograde plastid signals. The far-red (FR) light insensitive Arabidopsis mutant laf6 disrupted in a plastid-localised ABC-like protein (atABC1) accumulates the plastid signal protoporphyrin IX (proto IX) and has attenuated nuclear gene expression (Moller et al.2001 Genes Dev. 15:90-103). Our data suggests that proto IX accumulation results in hypocotyl elongation in response to FR light and we have demonstrated that by inhibiting the plastid localised protoporphyrinogen IX oxidase (PPO) using flumioxazin wild-type plants phenocopy laf6 by accumulating proto IX with a concomitant loss of hypocotyl growth inhibition in a dose-dependent manner. It is at present unclear what effect increased proto IX has on nuclear gene expression and how this is integrated with photomorphogenic responses such as hypocotyl elongation.

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34 / 60 Experiment : E-NASC-43 Submitter(s) :  Beynon Lab :  Horticulture Research International

Experiment Design Type : unknown experiment design type (Generated description): Experiment with 8 hybridizations, using 8 samples of species [Arabidopsis thaliana], using 8 arrays of array design [Affymetrix GeneChip® Arabidopsis Genome [ATH1-121501]], producing 8 raw data files and 8 transformed and/or normalized data files. (Submitter's description 1): 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.

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35 / 60 Experiment : E-NASC-44 Submitter(s) :  Newbury Lab :  University of Birmingham

Experiment Design Type : stimulus or stress , species , organism part comparison , compound treatment (Generated description): Experiment with 24 hybridizations, using 24 samples of species [Arabidopsis petraea, Arabidopsis halleri], using 24 arrays of array design [Affymetrix GeneChip® Arabidopsis Genome [ATH1-121501]], producing 24 raw data files and 24 transformed and/or normalized data files. (Submitter's description 1): This application is from a NERC-funded consortium (Mark MacNair, Nick Smirnoff, Exeter) and (Brian Ford-Lloyd, John Newbury, Birmingham). Metal tolerance is one of the classic examples of micro-evolution. Despite extensive research the physiological bases of the adaptation in plants are largely unknown. Arabidopsis halleri is a zinc tolerant, zinc accumulating species whereas Arabidopsis petraea is non-accumulating and non-tolerant. The objective of our programme is to identify: a) those key genes that act to determine Zn tolerance and accumulation in Arabidopsis (and which account for the difference in performance of A. halleri and A. petraea grown in the presence of elevated Zn), and b) those _downstream_ genes that are expressed as part of the tolerance or accumulation response. Phase 1: Total of 24 chips: Material ready by May 2003. The results will: a) tell us how effectively material derived from other Arabidopsis species hybridises to the chips, and b) identify genes that are differentially expressed in the two species in the presence and absence of Zn stress (thus providing initial lists of genes that may be responsible for Zn tolerance or accumulation- (but see phase 2). A. halleri exposed to low and high Zn; root and leaf mRNAs extracted: 3 replicates of each: = 12 slides. A. petraea exposed to low and high Zn; root and leaf mRNAs extracted: 3 replicates of each: = 12 slides. Phase 2: Total of 48 chips: Material ready by September 2003. The results will tell us which genes, identified as having appropriate expression patterns, co-segregate with the Zn tolerance or accumulation phenotype and will provide firmer candidate genes for intensive study. Bulks will be produced from F3 progeny (from the halleri x petraea cross) following phentoypic analyses for Zn tolerance and accumulation. A bulk of F3 progeny all exhibiting high Zn tolerance: exposed to low and high Zn; leaf and root mRNAs: 3 replicates: = 12 slides. A bulk of F3 progeny all exhibiting low Zn tolerance: exposed to low and high Zn; leaf and root mRNAs: 3 replicates: = 12 slides. A bulk of F3 progeny all exhibiting high Zn accumulation: exposed to low and high Zn; leaf and root mRNAs: 3 replicates: = 12 slides. A bulk of F3 progeny all exhibiting low Zn accumulation: exposed to low and high Zn; leaf and root mRNAs: 3 replicates: = 12 slides.

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36 / 60 Experiment : E-NASC-45 Submitter(s) :  Okamoto Lab :  University of Oxford

Experiment Design Type : strain or line , individual genetic characteristics , compound treatment (Generated description): Experiment with 4 hybridizations, using 4 samples of species [Arabidopsis thaliana], using 4 arrays of array design [Affymetrix GeneChip® Arabidopsis Genome [ATH1-121501]], producing 4 raw data files and 4 transformed and/or normalized data files. (Submitter's description 1): Mutations in the heterotrimeric G-protein a-subunit of Arabidopsis, GPA1, leads to deficiency in ABA-induced stomatal closure (Wang et al., 2001). To further investigate whether GPA1 is involved in the regulation of gene expression in response to ABA, we examined the induction of known ABA-inducible genes in the gpa1 mutant and compared it to wild-type. We found significant differences in levels of ABA-induced expression between wild-type and gpa1 mutant. In order to systematically investigate GPA1 involvement in ABA signalling leading to gene expression, we are requesting the transcriptome analysis of the gpa1 mutant in response to ABA.In detail, 2 week old wild-type and gpa1 plants grown in the 16/8 hrs light and dark cycle will be treated with either ABA or with a control solution for 3 hours. 10 plants will be used per sample to produce RNA, to give 4 samples, one for each chip: gpa1-1 mutant in the presence (chip1) or absence of ABA (chip2) and wild-type (WS2) in the presence (chip3) or absence of ABA (chip4).

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37 / 60 Experiment : E-NASC-46 Submitter(s) :  Pritchard Lab :  University of Birmingham

Experiment Design Type : pathogenicity (Generated description): Experiment with 6 hybridizations, using 6 samples of species [Arabidopsis thaliana], using 6 arrays of array design [Affymetrix GeneChip® Arabidopsis Genome [ATH1-121501]], producing 6 raw data files and 6 transformed and/or normalized data files. (Submitter's description 1): The aim of this study is to identify Arabidopsis genes whose expression is altered by aphid feeding. An understanding of the plant aphid interaction at the level of the plant transcriptome will 1) consolidate current areas of investigation focused on the phloem composition (the aphid diet), 2) open up areas of plant aphid interactions for ourselves and other workers, 3) Contribute to understanding the use of new molecular technologies in an environmental context and 4) contribute to existing and development of novel control strategies.Our Arabidopsis/Myzus persicae system provides a valuable model for the study because of: a) the advantages of using Arabidopsis, b) The ability to use clonal insects, c) phloem feeding aphids facilitate focus on a specific cell type, d) aphid stylectomy allows collection of pure phloem sap to monitor ‘phloem phenotypeÂ’ of the plant and the insect diet, e) we have techniques to monitor the reproductive performance and feeding behaviour aphids.Our strategy has been to test the function of selected genes, particularly those regulating phloem composition (the feeding site of the aphid) based on current phloem models of phloem function. Gene choice is limited the simplicity of current models of phloem aphid interaction.We propose a simple two treatment (aphid infested vs control plants) experiment that will identify novel target genes for future analysis. Arabidopsis plants (variety Columbia) will be grown in 16/8 light/dark in temperature controlled growth rooms. At growth stage 3.90, when rosette growth is complete, 10 clonal adult Myzus persicae will be caged in clip cages on the two largest leaves on each plant. Control plants will be treated identically except that the cages will be empty. Leaves will be harvested 8 h after infestation. This time point is selected as we know that 90% of aphids are plugged into the sieve element within 2h and that a 6h lag phase has period has previously been used when examining gene expression affected by wounding. In subsequent experiments we will examine time courses of expression of relevant genes using other approaches. Pooling two leaves from each of ten plants will generate the RNA sample, ensuring that expression signals are representative of the population of plants.

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38 / 60 Experiment : E-NASC-47 Submitter(s) :  Pritchard Lab :  University of York

Experiment Design Type : development or differentiation , compound treatment (Generated description): Experiment with 4 hybridizations, using 4 samples of species [Arabidopsis thaliana], using 4 arrays of array design [Affymetrix GeneChip® Arabidopsis Genome [AG1]], producing 4 raw data files and 4 transformed and/or normalized data files. (Submitter's description 1): Aim To identify genes specifically involved in the storage reserve mobilisation programme in Arabidopsis. Background: During germination and early post-germinative growth in Arabidopsis seed storage reserves are broken down to provide energy and nutrients for the developing seedling. Genes encoding enzymes involved in the mobilisation of both storage lipid and protein are expressed strongly 1-2 days following imbibition and then fall to very low levels. The regulatory mechanisms controlling expression of these genes are poorly understood. Although germination and reserve mobilisation occur at the same time we have obtained evidence using Abscisic Acid (ABA) treated seeds that the events are regulated by two separate programmes. Arabidopsis seeds treated with 10mM ABA still express genes involved in the mobilisation of storage reserves and break down storage lipid even though germination is blocked. ABA treated seeds therefore provide a powerful system for the identification of genes involved specifically in the reserve mobilisation programme. Microarray analysis will allow us to gain a global undertanding of the processes involved in storage reserve mobilisation and should also result in the identification of regulatory genes involved in this process. Experimental Set-up All seeds are Col-4 imbibed on plates containing 1/2 MS media for 4 days in the dark. All plant material will be grown in controlled environment growth rooms under defined light and temperature regimes. There are two parts to the experimental design. 1. Time course to identify genes involved in both seed storage reserve and germination programmes Comparison of mRNAs from seeds immediately after imbibition (storage reserve and germination programme genes being induced) with 2 day (storage reserve genes maximally expressed) and 7 day old seedlings (storage reserve and germination programme genes off, photoautotrophic genes on) will allow the identification of genes exclusively presentor present at enhanced levels at the peak of reserve mobilisation (day 2). 2. ABA treatment experiment to identify genes involved in the storage reserve programme ABA treatment blocks germination but does not block reserve mobilisation. Microarray analysis will be performed on RNA isolated from 2 day old seed imbibed in the presence of ABA. Comparison of the results of this experiment with those of the time course experiment should allow us to distinguish between genes involved in the two major programmes that we have uncovered.

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39 / 60 Experiment : E-NASC-49 Submitter(s) :  Smith Lab :  University of Edinburgh

Experiment Design Type : time series , growth condition (Generated description): Experiment with 22 hybridizations, using 22 samples of species [Arabidopsis thaliana], using 22 arrays of array design [Affymetrix GeneChip® Arabidopsis Genome [ATH1-121501]], producing 22 raw data files and 22 transformed and/or normalized data files. (Submitter's description 1): 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.

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40 / 60 Experiment : E-NASC-52 Submitter(s) :  Werner Lab :  Institute of Applied Genetics and Cell Biology

Experiment Design Type : strain or line , dose response , genetic modification (Generated description): Experiment with 8 hybridizations, using 8 samples of species [Arabidopsis thaliana], using 8 arrays of array design [Affymetrix GeneChip® Arabidopsis Genome [ATH1-121501]], producing 8 raw data files and 8 transformed and/or normalized data files. (Submitter's description 1): 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.

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41 / 60 Experiment : E-NASC-54 Submitter(s) :  Jones Lab :  School of Biological Sciences University of Exeter

Experiment Design Type : individual genetic characteristics , strain or line (Generated description): Experiment with 6 hybridizations, using 6 samples of species [Arabidopsis thaliana], using 6 arrays of array design [Affymetrix Genechip® Arabidopsis Genome [ATH1]], producing 6 raw data files and 6 transformed and/or normalized data files. (Submitter's description 1): Aim: To determine the effect of an AtrbohC mutation on the gene expression pattern in primary root tissue, to identify candidate genes acting downstream of AtrbohC, particularly any encoding antioxidant-related proteins, signal transduction components or proteins known to be required for normal root-hair development. Background: Root-hairs are a model system for investigating plant cell polarity. The root-hair mutant rhd2 (Schiefelbein and Somerville, 1990. Plant Cell, 2:235) has short hairs that burst at their tips, (Jones and Smirnoff, unpublished). RHD2 has been cloned and is identical to AtrbohC (L. Dolan, pers. comm.), which encodes a homologue of the superoxide-generating neutrophil respiratory burst oxidase catalytic subunit gp91phox (Torres et al., 1998. Plant J., 14:365). Superoxide rapidly dismutates to hydrogen peroxide (H2O2), suggesting that the rhd2 phenotype may result from reduced H2O2 levels in root-hair cells. Low doses of exogenous antioxidants phenocopy the rhd2 root-hair phenotype in wild-type plants (Jones and Smirnoff, unpublished) further supporting a role for H2O2 in root-hair growth. Fluorescent dyes that detect H2O2 show distinct localisation patterns in growing root-hair cells, (Jones and Smirnoff, unpublished). H2O2 may be an important second messenger in plant cell signalling with proposed roles in the development of cotton fibres (Potikha et al., 1999. Plant Physiol., 119: 849) and in ABA-induced stomatal closure (Zhang et al., 2001. Plant Physiol., 126: 1438). In cultured Arabidopsis cells H2O2 induces gene expression, including that of a gp91phox homologue, (Desikan et al., 1998. J. Exp. Bot., 49: 1767; Desikan, et al., 2000. Free Rad. Biol. Med., 28: 773; Baxter-Burrell et al., 2002. Science, 296: 2026) and activates a MAP kinase cascade (Desikan et al., 1999. J. Exp Bot., 50: 1863). cDNA microarray technology has been used previously to examine the effects of H2O2 on gene expression during oxidative stress (Desikan et al., 2001. Plant Physiol., 127: 159). We wish to investigate the effects of H2O2 on gene expression during root development using the rhd2 mutant. We are currently determining the expression pattern of RHD2. By extracting RNA from the small region of the primary root (for wild-type and rhd2 plants grown in sterile conditions) where root hairs are growing we hope to enrich for root-hair RNAs. This may reveal candidate genes that could be examined more closely at the single-cell level. This approach will provide new insights into the role of H2O2 in root-hair development.

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42 / 60 Experiment : E-NASC-55 Submitter(s) :  Mur Lab :  University of Wales Aberystwyth

Experiment Design Type : compound treatment (Generated description): Experiment with 2 hybridizations, using 2 samples of species [Arabidopsis thaliana], using 2 arrays of array design [Affymetrix GeneChip® Arabidopsis Genome [AG1]], producing 2 raw data files and 2 transformed and/or normalized data files. (Submitter's description 1): Nitric oxide (NO.) is a well-established signal in mammalian cells regulating e.g. smooth muscle tone, neurotransmission and apoptosis. NO interacts with superoxide (O2-) to derive the highly reactive peroxynitrite (ONOO-) radical leading to the generation of lipid hydroxy-peroxides (ROO.) and cell death. Thus the recent reports implicating of .NO in the Hypersensitive Response (HR) a form of programmed cell death elicited by pathogens in plantsis suggestive of parallel roles in animals and plants. As part of BBSRC funded programmes which are investigating oxidative signaling and cell death in Arabidopsis we have collaborated with the Trace Gas Detection facility at the University of Nijmegen to be the first to measure NO in planta from a developing HR (Mur et al.paper in prep). The challenge remains to understand the spatio-temporal role(s) of .NO during the HR and disease development. In line with other groups e.g. Klessig et al.(2000) PNAS 978849-55 we have observed that .NO mediated event are both SA- dependent and independent. We propose a two-stage programme to investigate NO events which will lead to subsequent BBSRC grant bids. Firstly to exploit the GARNET Affymetrix transcriptomic service to identify genes which are upregulated by .NO (see programme below). These will be compared with similar data generated by other members of the consortium; Dr. Steve Neill for oxidative stress (Desikan et al. (2001). Plant Physiol 127(1): 159-72; Clarke et al.2000 Plant J.; 24:667-77); Dr. Paul Kenton who is mainly interested in signaling by the oxylipid Jasmonic acid (Kentonet al. (1999). MPMI 12(1): 74-78) as well as from other sources. Though these data in themselves will suggest modes of NO action. However as a second approach we will clone the promoter of a strongly NO-responsive gene which will be fused to positive and negative selectable markers as well as reporter genes e.g. LUC. to identify Arabidopsis EMS mutants which are perturbed in NO-mediated events. Programme. Our in planta measurement show that from a baseline production of 1nl.g fwt-1h-1 NO levels rise to 6nl. g fwt-h-1 (_plus/- 1.1 SE) prior to cell death and to 25nl g fwt-h-1 (_plus/- 4.2 SE) at cell death following which the concentration falls. At this juncture we will intend to gas Arabidopsis to ~75ppb (the head-space concentration when NO production was at 6nl. g fwt-h-1). Prior to using the DNA RNA will establish that this does not elicit cell death over the proposed treatment period and that both PR1 (SA-dependent) and PAL1 (SA-independent) genes are induced. We will compare this plant with sealed but non-treated Arabidopsis. Future targets for DNA RNAs analysis induce plant treated with both NO (Sodium Nitro Prusside) and O2- (Xanthine oxidase) generators and depending on their effectiveness (to be assessed by NO measurement) HR lesions treated with Nitric Oxide Synthase inhibitors.

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43 / 60 Experiment : E-NASC-56 Submitter(s) :  Broadley Lab :  University of Nottingham

Experiment Design Type : growth condition (Generated description): Experiment with 6 hybridizations, using 6 samples of species [Brassica oleracea], using 6 arrays of array design [Affymetrix GeneChip® Arabidopsis Genome [ATH1-121501]], producing 6 raw data files and 6 transformed and/or normalized data files. (Submitter's description 1): The aim of this study is to study gene expression in Brassica oleracea in shoot tissues of plants grown under contrasting P supplies (see Hammond JP et al., 2003, Plant Physiology, 132, 578-596 for background). Seeds of B. oleracea (var. alboglabra, A12dH) were first washed in 70% (v/v) ethanol/water, rinsed in distilled water and surface sterilised using 50% (v/v) domestic bleach/water. Seeds were rinsed and imbibed for 3 to 5 days in sterile distilled water at 4°C to break dormancy. Following imbibition, B. oleracea seeds were sown in un-vented, polycarbonate culture boxes (Sigma-Aldrich Company Ltd., Dorset UK). Seedlings were grown for 21 days on perforated polycarbonate discs (diameter 91 mm by 5 mm) placed on 75 ml of 0.8% (w/v) agar containing 1% (w/v) sucrose and a basal salt mix. Roots grew into the agar, but shoots remained on the opposite side of the disc. After 21 days, seedlings were transferred, still on polycarbonate discs, to a hydroponics system situated in a Saxcil growth cabinet (16 h daylength, set to 22°C and 80% humidity). Each polycarbonate disc was placed on a light-proof 500 ml beaker over 450 ml of nutrient solution. After 7 days, half the plants were transferred to nutrient solution containing no phosphate and the other half remained on full nutrient solution (control plants). Shoots were harvested 100 h after the withdrawal of P. Samples were snap frozen in liquid nitrogen. RNA was extracted using the TRIzol extraction method and cleaned through a Qiagen RNeasy column.

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44 / 60 Experiment : E-NASC-57 Submitter(s) :  Eland Lab :  Plant Sciences

Experiment Design Type : strain or line , individual genetic characteristics (Generated description): Experiment with 2 hybridizations, using 2 samples of species [Arabidopsis thaliana], using 2 arrays of array design [Affymetrix GeneChip® Arabidopsis Genome [AG1]], producing 2 raw data files and 2 transformed and/or normalized data files. (Submitter's description 1): SKS mutant (snakeskin) vs wildtype

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45 / 60 Experiment : E-NASC-58 Submitter(s) :  Eland Lab :  Plant Sciences

Experiment Design Type : growth condition (Generated description): Experiment with 2 hybridizations, using 2 samples of species [Arabidopsis thaliana], using 2 arrays of array design [Affymetrix GeneChip® Arabidopsis Genome [ATH1-121501]], producing 2 raw data files and 2 transformed and/or normalized data files. (Submitter's description 1): 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.

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46 / 60 Experiment : E-NASC-59 Submitter(s) :  Hammond Lab :  Horticulture Research International

Experiment Design Type : growth condition , stimulus or stress (Generated description): Experiment with 4 hybridizations, using 4 samples of species [Arabidopsis thaliana], using 4 arrays of array design [Affymetrix GeneChip® Arabidopsis Genome [AG1]], producing 4 raw data files and 4 transformed and/or normalized data files. (Submitter's description 1): Background: Since chemical assays of soil nutrients are unreliable the UK horticultural and agricultural industries routinely apply large amounts of inorganic fertiliser to maintain crop yields and quality. Excessive fertiliser applications are both costly and can lead to unnecessary pollution. A possible solution to this problem is to use sensor (GM or non-GM) technologies that exploit the changes in plant gene expression profiles under incipient nutrient deficiency. The aim of this project is to identify genes upregulated in the early stages of nutrient depletion. Methods: Arabidopsis ecotype Col-5 (N1644) will be grown hydroponically using established techniques. In parallel experiments NP and K will be withdrawn individually after 21 d growth. RNA will be extracted from shoots of nutrient replete (control) and nutrient depleted plants 24 h after the removal of nutrients. Shoot nutrient content will be assayed by ICP-EMS as a reference. By comparing expression profiles we will be able to differentiate between genes that are upregulated by lack of specific nutrients and those upregulated by a universal stress-response system. Promoters and transcripts of these genes will underpin the development of novel sensor technologies and knowledge of the gene expression profiles will improve our understanding of the physiology of plant mineral nutrition.

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47 / 60 Experiment : E-NASC-60 Submitter(s) :  Hammond Lab :  University of Nottingham

Experiment Design Type : individual genetic characteristics , strain or line (Generated description): Experiment with 6 hybridizations, using 6 samples of species [Arabidopsis thaliana], using 6 arrays of array design [Affymetrix GeneChip® Arabidopsis Genome [ATH1-121501]], producing 6 raw data files and 6 transformed and/or normalized data files. (Submitter's description 1): Background: The UK horticultural and agricultural industries routinely apply large amounts of inorganic fertiliser to maintain crop yield and quality, since chemical assays of soil nutrients are unreliable. Excessive fertiliser applications are costly and can lead to unnecessary pollution. A possible solution is to use sensor (GM or non-GM) technologies that exploit the changes in plant gene expression under incipient nutrient deficiency. Aim: The aim of this project is to use mutants with reduced leaf phosphate contents to identify genes upregulated in response to phosphate stress. Preliminary gene expression analysis has identified several phosphate responsive genes to be upregulated in the pho1 mutant. However, further replicates of the experiment are required to confirm these changes. Methods: Arabidopsis mutant pho1 (N8507) and its parent ecotype Columbia 2 (N907) will be grown on MS agar under identical conditions. RNA will be extracted from the rosette leaves of both parent and mutant and the same growth stage. By comparing the expression profiles, we will be able to differentiate between genes that are upregulated in leaves experiencing phosphate stress. Previously, two GeneChips have been used (mutant and parent) to provide preliminary data. A further two biological replicates are now required to confirm these results. Promoters and transcripts of these genes will underpin the development of novel sensor technologies, and knowledge of the gene expression profiles will improve our understanding of the physiology of plant mineral nutrition.

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48 / 60 Experiment : E-NASC-61 Submitter(s) :  Hammond Lab :  Warwick University

Experiment Design Type : compound treatment , organism part comparison , growth condition (Generated description): Experiment with 18 hybridizations, using 18 samples of species [Arabidopsis thaliana], using 18 arrays of array design [Affymetrix GeneChip® Arabidopsis Genome [ATH1-121501]], producing 18 raw data files and 18 transformed and/or normalized data files. (Submitter's description 1): 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.

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49 / 60 Experiment : E-NASC-65 Submitter(s) :  Capper Lab :  University of Oxford

Experiment Design Type : compound treatment , genetic modification (Generated description): Experiment with 12 hybridizations, using 12 samples of species [Arabidopsis thaliana], using 12 arrays of array design [Affymetrix GeneChip® Arabidopsis Genome [ATH1-121501]], producing 12 raw data files and 12 transformed and/or normalized data files. (Submitter's description 1): Effect of CaM overexpression on Arabidopsis transcriptome. Unlike animals, plants are immobile and cannot simply move away from unfavourable environments and thus have developed complex mechanisms to respond to and sense biotic and abiotic signals. These stimuli often lead to tightly controlled changes in cytoplasmic free calcium concentration [Ca2+]cyt termed "calcium signatures" which are thought to be, at least partly, responsible for the specificity of plant responses to the environment. However little is known about how exactly these calcium signatures are decoded into specific end-responses. Calmodulin (CaM) is the most well characterised Ca2+ binding protein and is the primary sensor of changing [Ca2+]. Upon binding Ca2+ CaM undergoes a conformational change allowing binding and activation of a wide variety of target proteins. In plants CaM exists in gene families encoding multiple isoforms. The expression of individual CaM genes can be differentially regulated and isoforms may be differentially localised. Furthermore specific isoforms can bind and activate different target proteins. These features of plant CaM allow the possibility of specificity during calcium signalling in response to specific stimuli. The effect of overexpression of four CaM protein isoforms on the Arabidopsis thaliana transcriptome will be investigated. Ten day old transgenic Arabidopsis seedlings (containing estradiol inducible CaM overexpression constructs) were induced for 9hrs in 5uM estradiol with appropriate water (0.025% DMSO) and empty vector controls.

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50 / 60 Experiment : E-NASC-66 Submitter(s) :  Evans Lab :  University of Oxford

Experiment Design Type : genetic modification , growth condition (Generated description): Experiment with 12 hybridizations, using 12 samples of species [Arabidopsis thaliana], using 12 arrays of array design [Affymetrix GeneChip® Arabidopsis Genome [ATH1-121501]], producing 12 raw data files and 12 transformed and/or normalized data files. (Submitter's description 1): The aim of this work is to investigate whether A. thaliana senses low temperature by perceiving changes in membrane fluidity. To this end, we have performed an experiment to test whether mutant or transgenic plants with altered membrane lipid composition, regulate their gene expression in the same manner as wild type plants in response to cold. Previous work has demonstrated that a change in the expression levels of a number of genes is important in acquiring tolerance to low temperatures. Chemicals which rigidify cell membranes in such a way as to mimic the effects of cold have been shown to be able to induce the expression of such genes. However, because of the non-specific nature of such chemical treatments, it has not been possible to demonstrate unequivocally that the changes in gene expression observed were the result of changes in membrane fluidity. All of the mutants used in our experiment, fab1, fad2-2 and the fad3/fad7/fad8 mutant, have increased lipid saturation levels compared to wild type plants and are thought to have reduced membrane fluidity. The fab1 mutant is also known to be sensitive to chilling. In the fab1 mutant the elongation of 16:0 fatty acids to 18:0 is reduced. The fad2-2 mutant has reduced 18:1 desaturase activity and hence reduced amounts of polyunsaturated phospholipids. The fad3/fad7/fad8 triple mutant is deficient in 18:2 desaturase activity and consequently unable to synthesise trienoic fatty acids. The transgenic line used contained a 35S::FAD3 transgene and in contrast to the mutants tested, should have increased lipid desaturation and increased membrane fluidity. A. thaliana ecotype Col-0 was used as the wild type control for the fab1 and fad2-2 mutants, in addition to the 35S::FAD3 line. The fad3/fad7/fad8 mutant had previously been transformed with the 35S::apoaequorin transgene and a Columbia line expressing apoaequorin under the control of the same promoter, was included to control for the presence of aequorin. Nine day old seedlings grown in petri-dishes on MS were transferred from their growth room (20 oC, 16 h photoperiod, 100 E m-2 s-1) to a growth cabinet (20 oC, 16 h photoperiod,160 m-2 s-1) 24 hours before the experiment began. The next day, one petri-dish of seedlings from each line of plants used was transferred to a cabinet running at 5 oC (16 h photoperiod,160 E m-2 s-1). Control plates remained at 20 oC. Seedlings were harvested after three hours and frozen in liquid nitrogen.

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51 / 60 Experiment : E-NASC-67 Submitter(s) :  Knight Lab :  University of Oxford

Experiment Design Type : growth condition , genetic modification (Generated description): Experiment with 4 hybridizations, using 4 samples of species [Arabidopsis thaliana], using 4 arrays of array design [Affymetrix GeneChip® Arabidopsis Genome [ATH1-121501]], producing 4 raw data files and 4 transformed and/or normalized data files. (Submitter's description 1): The sfr6 mutant was identified on the basis of its failure to cold acclimate, and exhibits a marked deficiency in cold-and osmotic stress-inducible gene expression (Knight et al., 1999). We have demonstrated that genes of this type (so-called COR genes) are misregulated if they contain the DRE (drought-responsive element, or CRT; C-repeat) cis acting element in their promoter (Boyce et al., 2003). Micro-array analysis has allowed us to identify a number of COR genes misregulated in sfr6, all of which contain the DRE element. However, these experiments have indicated that other genes, not of the COR group, are also misregulated in the mutant and these do not contain the DRE element. We chose the three non-COR genes that were most clearly down-regulated in sfr6 on our previous micro-array, and identified each as of these as dark-inducible. We now seek to address two questions: (1) Can we discover more dark-regulated genes that are down-regulated in the mutant, and thus identify a second cis-acting element with which SFR6 may be interacting? (2) Do all of the non-COR genes that are misregulated in sfr6 fall into the category of dark-inducible, or is SFR6 likely to be interacting with three or more regulons? For this micro-array experiment, we will subject sfr6 and wild type Arabidopsis, grown in a 16/8-h light/dark cycle, to darkness for 3h or 6h at ambient growth temperature. Under these conditions we see strong up-regulation of the three genes we have identified as dark-inducible, and we see clear down-regulation of expression in sfr6. References. Knight H, Veale E, Warren GJ, Knight MR (1999) The sfr6 mutation in Arabidopsis suppresses low-temperature induction of genes dependent on the CRT/DRE sequence motif. Plant Cell 11: 875-886. Boyce JM, Knight H, Deyholos M, Openshaw MR, Galbraith DW, Warren G, Knight MR (2003). The sfr6 mutant of Arabidopsis is defective in transcriptional activation via CBF/DREB1 and DREB2 and shows sensitivity to osmotic stress. Plant Journal 34: 395-406.

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52 / 60 Experiment : E-NASC-68 Submitter(s) :  Kadalayil Lab :  University of Southampton

Experiment Design Type : genetic modification (Generated description): Experiment with 2 hybridizations, using 2 samples of species [Arabidopsis thaliana], using 2 arrays of array design [Affymetrix GeneChip® Arabidopsis Genome [ATH1-121501]], producing 2 raw data files and 2 transformed and/or normalized data files. (Submitter's description 1): The protein modules known as SH2 (Src-homology-2) domains are key players in the signal transduction of animals. Two questions arise: Do such modules exist in plants, and when did SH2 domains evolve? Here I show that the Arabidopsis genome contains three strong candidates for plant SH2 proteins (referred to as PASTA1, 2 and 3 : GI:25513455, At1g78540, At1g17040 respectively) with homology to the SH2 domains and the adjacent linker region of STAT proteins (Signal Transducer and Activator of Transcription). The three characteristics features of a STAT protein sequence1, namely, (i) the SH2 domain with a conserved arginine residue crucial for binding to a phospho-tyrosine residue (ii) a tyrosine residue outside the C-terminus of the SH2-domain for phosphorylation during signalling and (iii) a DNA-binding domain, are conserved in the PASTA3 protein. However, PASTA 1 and 2 proteins lack a tyrosine in a similar position. PASTA proteins are not homologous to STAT proteins outside the SH2 and linker regions. The three PASTA proteins are 70 to 80 % identical to one another. Gene expression studies with PASTA2 reveal that it is expressed in roots, stem, leaves, flowers and green siliques. Preliminary indications are that plants homozygous for PASTA2 do not have any obvious phenotype, most likely due to redundancies. This microarray experiment is an attempt to compare the gene expression of a mutant plant homozygous for PASTA2 with that of the wild type plant. This might give clues about the possible function of PASTA2 in Arabidopsis.

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53 / 60 Experiment : E-NASC-69 Submitter(s) :  Walters Lab :  University of Oxford

Experiment Design Type : genetic modification , growth condition (Generated description): Experiment with 12 hybridizations, using 12 samples of species [Arabidopsis thaliana], using 12 arrays of array design [Affymetrix GeneChip® Arabidopsis Genome [ATH1-121501]], producing 12 raw data files and 12 transformed and/or normalized data files. (Submitter's description 1): 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. Method: Plants will be grown in a 8h light:16h dark regime at both 400 and 100 µmol 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 maximise 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.

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54 / 60 Experiment : E-NASC-70 Submitter(s) :  Marocco Lab :  Istituto di Agronomia

Experiment Design Type : genetic modification (Generated description): Experiment with 2 hybridizations, using 2 samples of species [Arabidopsis thaliana], using 2 arrays of array design [x deprecated], producing 2 raw data files and 2 transformed and/or normalized data files. (Submitter's description 1): The samples are derived from experiments aimed to describe changes in gene expression under different temperature conditions by using wild type and virescent mutants.

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55 / 60 Experiment : E-NASC-73 Submitter(s) :  Schildknecht Lab :  Nottingham Arabidopsis Stock Centre

Experiment Design Type : strain or line (Generated description): Experiment with 11 hybridizations, using 11 samples of species [Arabidopsis thaliana], using 11 arrays of array design [Affymetrix GeneChip® Arabidopsis Genome [ATH1-121501]], producing 11 raw data files and 11 transformed and/or normalized data files. (Submitter's description 1): By reciprocally crossing 2x and 4x C24 ecotype plants, we have generated 4 types of offspring with various ploidy (2x; 3x; 4x) or parent-of-origin genome dosage (3x from 4xper2x; 3x from 2xper4x). For each offspring generated, total RNA was extracted using Trizol from 8 seedlings 9 days after germination (developmental stage1.02, 2 leaves). Sample names:

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56 / 60 Experiment : E-NASC-74 Submitter(s) :  Coates ,  Schildknecht Lab :  Nottingham Arabidopsis Stock Centre

Experiment Design Type : genetic modification (Generated description): Experiment with 12 hybridizations, using 12 samples of species [Arabidopsis thaliana], using 12 arrays of array design [Affymetrix GeneChip® Arabidopsis Genome [ATH1-121501]], producing 12 raw data files and 12 transformed and/or normalized data files. (Submitter's description 1): Arabidopsis has two genes, Arabidillo-1 and -2, related to animal Armadillo/ beta-catenin (Coates, 2003). Armadillo/beta-catenin directly activates the expression of developmental and cell proliferation genes, and also independently regulates cell-cell adhesion. Arabidillo proteins are nuclear and promote lateral root development.

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57 / 60 Experiment : E-NASC-75 Submitter(s) :  SAKAKIBARA ,  Schildknecht Lab :  Nottingham Arabidopsis Stock Centre

Experiment Design Type : compound treatment (Generated description): Experiment with 12 hybridizations, using 12 samples of species [Arabidopsis thaliana], using 12 arrays of array design [Affymetrix GeneChip® Arabidopsis Genome [ATH1-121501]], producing 12 raw data files and 12 transformed and/or normalized data files. (Submitter's description 1): In Arabidopsis thaliana, the immediate early response of plants to cytokinin is formulated as the multistep AHK-AHP-ARR phosphorelay signaling circuitry, which is initiated by the cytokinin-receptor histidine protein kinases. In the hope of finding components (or genes) that function downstream of the cytokinin-mediated His-Asp phosphorelay signaling circuitry, we carried out genome-wide microarray analyses. To this end, we focused on a pair of highly homologous ARR10 and ARR12 genes by constructing an arr10 arr12 double null mutant. The mutant alleles used in this study were arr10-5 and arr12-1. arr10-5 is the SALK_098604 T-DNA insertion line, whose mutation was determined to be located in the fifth exon of the ARR10 coding sequence. Arr12-1 is the SALK_054752 T-DNA insertion line, whose mutation was determined to be located in the third exon of the ARR12 coding sequence. The resulting mutant exhibits a characteristic phenotype with regard to the cytokinin-mediated His-Asp phosphorelay. Here we, therefore, compared response to cytokinin in wild type with that in arr10 arr12 double mutant. In this study, wild type and the arr10 arr12 double mutant grown vertically on MS agar plates for 2 weeks were treated with 20uM t-zeatin or 0.02% DMSO (solvent for t-zetion solution) for 1h. These treated plant samples were divided into three portions, from which RNA samples were prepared separately from aerial parts of seedlings with use of RNeasy Plant Mini Kit (Qiagen, Valencia, CA, U.S.A.). The Quality of RNAs prepared was analyzed by Bioanalyzer 2100 (Agilent Technologies). These RNA samples were processed as recommended by the Affymetrix instruction (Affymetrix GeneChip Expression Analysis Technical Manual, Affymetrix). These dataset will provide us with bases for understanding the early response to cytokinin on aerial parts of seedlings in Arabidopsis thaliana.

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58 / 60 Experiment : E-NASC-76 Submitter(s) :  Dewdney ,  Schildknecht Lab :  Nottingham Arabidopsis Stock Centre

Experiment Design Type : time series , compound treatment (Generated description): Experiment with 18 hybridizations, using 18 samples of species [Arabidopsis thaliana], using 18 arrays of array design [Affymetrix GeneChip® Arabidopsis Genome [ATH1-121501]], producing 18 raw data files and 18 transformed and/or normalized data files. (Submitter's description 1): To assess the contribution to defenses against necrotrophic fungal pathogens that may be mediated by recognition of oligogalacturonides (OGs), cell wall fragments released by the activity of fungal polygalacturonases, we treated seedlings with OGs and assayed transcript levels 1 and 6 hours after addition of OGs to culture medium. For each sample, approximately 30 seedlings were grown in shallow liquid MS medium for 10 days. Plants were then treated with either 200 ug/ml OGs or, for control plants, an equal volume of water. Three replicate samples were assayed for each treatment.

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59 / 60 Experiment : E-NASC-77 Submitter(s) :  Schildknecht ,  Birnbaum Lab :  Nottingham Arabidopsis Stock Centre

Experiment Design Type : cell type comparison (Generated description): Experiment with 27 hybridizations, using 27 samples of species [Arabidopsis thaliana], using 27 arrays of array design [Affymetrix GeneChip® Arabidopsis Genome [ATH1-121501]], producing 27 raw data files and 27 transformed and/or normalized data files. (Submitter's description 1): This experiment was donated by Philip N. Benfey's lab through ArexDB (http://www.arexdb.org).

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