NASC's International Affymetrix Service

This table replaces much of the functionality of our NASCarrays database now that funding has ended.

Quick Index

FTP access to the NASCarrays archive is on the left (clickable link)
The submitted description of the experiment is in scrollable boxes on the right.

We suggest you use your browser search facility to find keywords (e.g. ctrl-F/cmd-F).

NASCARRAY1 Chalcone Synthase 2002-10-16	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 will take Sean's advice of the best isolation method to use. I have set the sample size at two slides for each sample. I am not sure if this will be enough and again I would be grateful for advice once Sean has the pilot work done. My lab and culture room refurbishment is nearly complete and I should shortly be in a position to grow the plants and make the RNA.
NASCARRAY2 Circadian Clock Experiments 2002-12-17	Plants have endogenous clocks which are entrained by the day-night cycle. For a seedling germinating under the soil temperature may be the most important entraining signal. Two groups have published data showing that the circadian clock regulates 2-6% of the genome in Arabidopsis (Harmer et al2000; Schaffer et al.2001). Many of the genes identified function in photosynthesis and associated metabolism because the RNA was extracted from light-grown tissues. Other clock-controlled genes included functions in cell expansion, photo-protection and regulatory genes. The circadian clock can therefore affect many studies of gene regulation including those that are not overtly related to timing. The clock is already functioning in dark-grown seedlings in which the highly abundant photosynthesis-related RNAs are not expressed (Zhong et al. 1998). The clock in dark-grown seedlings can be entrained by temperature; it rhythmically regulates CCR2 expression and the light response of CAB (our unpublished results). Aim This experiment aims to identify clock-regulated genes in dark-grown seedlings for direct comparison with the published results from light-grown tissue. We will use the same microarray the same ecotype and analysis criteria that match Harmer et al. The experiment will identify genes that are: 1. Specifically regulated by the clock only in dark-grown or in light-grown seedlings. 2. Genes that are clock-regulated in both conditionsor in neither. Our results will directly address several questions: * What functions might circadian rhythms serve in dark-grown seedlings as revealed by the clock-regulated physiological or biochemical pathways ? * Are candidate regulators that control rhythmic light responses in the light also rhythmic in darkness ? * Are all the genes that are current candidates for "central oscillator components" rhythmically regulated in both conditions ? Our results will permit future studies (with Nagy Schaefer): * characterising the transcriptome in clock mutants * identifying of a minimal set of target genes for functional studies of the rhythmic proteome. N.B. our protocol avoids the most abundant proteins of light-grown seedlings potentially revealing more low-abundance regulatory proteins. Experimental Plan: Col-0 Arabidopsis seed will be sown on MS plates _plus 3% sucrose and imbibed at 4 C in the dark for 48h. Germination will be synchronised with a 3h white light pulse. The seed will be entrained for 4 days in darkness with temperature cycles then transferred to constant 22 C. Tissue will be harvested after 24h 30h 36h 42h and 48h at 22 C to avoid direct responses to temperature.
NASCARRAY3 Snakeskin 2002-10-16	SKS mutant (snakeskin) vs wildtype. Understanding the factors that influence the cell wall properties of plants is of major importance for agriculture and food processing. We have identified an EN transposon-induced mutant known as snakeskin (sks) in which the epidermal cellÂ–cell linkages appear to be significantly perturbed. In snakeskin the epidermal cells of the leaves, cotyledons and root Â‘peelÂ’ apart from one another and from the underlying mesophyll cells. As a result of the breakdown of epidermal cellÂ–cell linkages in sks there are changes in epidermal cell architecture and distribution. These include alterations in pavement epidermal cell shape, stomatal distribution and trichome morphology. We will describe the characterisation of the snakeskin mutant using a wide range of techniques including reporter genes, cryo-scanning electron microscopy, FTIR, immunogold labelling and mapping of the sks mutation using SSLP markers.
NASCARRAY5 Meristem Activity- 23f8a locus involved in controlling the transition to flowering and leaf organoge 2002-10-16	My laboratory is interested in the pathways controlling meristem activity. We have identified a mutant that prolongs the transition from vegetative to reproductive growth although the onset of reproductive development is not delayed significantly. This mutant also has other phenotypes such as altered leaf morphology and altered development of cauline leaves. Growth arrests in the homozygous mutant during vegetative phase with a phenotype reminiscent of wuschel but without the repeated de-novo formation of meristems characteristic of the latter. This mutant was identified in an activation-tagged collection and the gene likely to be the cause of the phenotype is a likely transcription factor. Crosses of this line with other lines with defects in meristem control and patterning genes have just been set up. For this transcriptome experiment we would like to compare RNAs isolated from aerial tissues (entire shoots) of the wild-type background line with the same tissues from the mutant. The results from this experiment will allow us to better design experiments to pin down the function of the affected gene.
NASCARRAY6 Baseline_experiment_pollen Caution! This experiment has not been fully annotated, although all data 2002-10-16	No abstract available - please contact the experimenter.
NASCARRAY7 Baseline_experiment_ecotype_development 2002-10-16
NASCARRAY8 Identification of genes involved in the induction of storage reserve mobilisation 2003-06-08	Aim To identify genes specifically involved in the storage reserve mobilisation programme in Arabidopsis. Background During germination and early post-germinative growth in Arabidopsis seed storage reserves are broken down to provide energy and nutrients for the developing seedling. Genes encoding enzymes involved in the mobilisation of both storage lipid and protein are expressed strongly 1-2 days following imbibition and then fall to very low levels. The regulatory mechanisms controlling expression of these genes are poorly understood. Although germination and reserve mobilisation occur at the same time we have obtained evidence using Abscisic Acid (ABA) treated seeds that the events are regulated by two separate programmes. Arabidopsis seeds treated with 10mM ABA still express genes involved in the mobilisation of storage reserves and break down storage lipid even though germination is blocked. ABA treated seeds therefore provide a powerful system for the identification of genes involved specifically in the reserve mobilisation programme. Microarray analysis will allow us to gain a global undertanding of the processes involved in storage reserve mobilisation and should also result in the identification of regulatory genes involved in this process. Experimental Set-up All seeds are Col-4 imbibed on plates containing 1/2 MS media for 4 days in the dark. All plant material will be grown in controlled environment growth rooms under defined light and temperature regimes. There are two parts to the experimental design. 1. Time course to identify genes involved in both seed storage reserve and germination programmes Comparison of mRNAs from seeds immediately after imbibition (storage reserve and germination programme genes being induced) with 2 day (storage reserve genes maximally expressed) and 7 day old seedlings (storage reserve and germination programme genes off, photoautotrophic genes on) will allow the identification of genes exclusively presentor present at enhanced levels at the peak of reserve mobilisation (day 2). 2. ABA treatment experiment to identify genes involved in the storage reserve programme ABA treatment blocks germination but does not block reserve mobilisation. Microarray analysis will be performed on RNA isolated from 2 day old seed imbibed in the presence of 10mM ABA. Comparison of the results of this experiment with those of the time course experiment should allow us to distinguish between genes involved in the two major programmes that we have uncovered.
NASCARRAY10 Identification of global gene expression in cir1 and cir1:ein2 plants 2002-10-17	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.
NASCARRAY11 Characterisation of the genetic basis of acclimation of plants to light intensity 2002-11-11	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. Recently in collaboration with Julie Gray
NASCARRAY12 Changes in gene expression profiles under phosphate deficiency. 2002-08-20	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.
NASCARRAY13 Calcium/calmodulin-binding transcription factors in Arabidopsis 2004-01-05	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.
NASCARRAY14 Control of lignification 2004-03-01	Lignin is a carbon rich polymer that is embedded in the cell walls of specialised plant cells. The metabolic cost of making lignin is very high. Therefore lignin biosynthesis must be carefully regulated so as to balance the demands of growth and development against the availability of resources to make this compound. Little is known about the signalling mechanisms that underpin this regulation. We have devised experiments which dissect the regulation of lignification. The "base-line" for our analyses are wild-type plants that have been grown in liquid medium in the absence of sugars in the dark. These plants develop a vascular system but do not lignify. We compare plants grown in this way against mutants and transgenic plants that are able to lignify even under these conditions. By comparing these mutants and transgenic plants against the wild type we are able to uncover the regulatory mechanisms that control when and where lignins are made. Our analyses would include: 1) wild-type dark grown no sugar 7 days post germination - no lignification 2) wild-type dark grown_plus sucrose 7 days post germination - lignification 3) wild-type light grown no sugar 7 days post germination - lignification 4) wild-type 6 days dark 1 day light grown no sugar- lignification 5) de-etiolated 3 mutant dark grown no sugar 7 days post germination - some lignification 6) de-etiolated 3 mutant dark grown_plus sucrose 7 days post germination - extensive ectopic lignification 7) zebra 1 mutant dark grown no sugar 7 days post germination - strong ectopic lignification 8) zebra 2 mutant dark grown no sugar 7 days post germination - strong ectopic lignification 9) de-etiolated 3 mutant _plus co-suppressed MYB61 dark grown_plus sugar 7 days post germination - low lignification - two lines (duplicated) 10) wild-type _plus overexpressed MYB61dark grown no sugar 7 days post germination - ectopic lignification - 3 lines (duplicated) 11) de-etiolated 3 mutant _plus kanamycin dark grown_plus sugar 7 days post germination - wild-type phenotype restored (duplicated) We have already completed this analysis once using Affymetrix GeneRNAs. The results of this analysis have been spectacular and using cluster analysis we have already identified several putative "regulons". In order to validate these data we ask permission to complete a biological replicate of this experiment and to extend our analysis of the transgenic plants to include duplicates of the following: 1) 3 independent lines of MYB61 overexpressing plants 2) 2 independent lines of de-etiolated 3 mutants with MYB61 knocked-out 3) de-etiolated 3 mutant treated with spermidine - restores wild type phenotype This experiment will require 20 RNAs. All plants are in a Col-O background
NASCARRAY15 Knocking out MEK1 and MEK3 through RNAi; their function studied by transcript profiling 2003-10-22	The mitogen-activated protein kinase (MAPK) signal transduction pathway composed of three protein kinases in a cascade (MAPK - MEK - MEKK) is central to relay extracellular stimulus in eukaryotes. We decided to delineate MAPK pathways and learn about their function by altering specific MEK levels. We designed double stranded RNA-interference constructs using 3` UTRs of specific MEKs and placed it under a glucocorticoid-inducible expression system based on lac-repressor (from Ian Moore)and we have established Arabidopsis cell suspensions carrying these constructs. Here we suggest the transcriptomics analysis of two of these MEK RNAi lines: MEK1-RNAi and MEK3-RNAi. MEK1 was shown to interact with and activate the AtMPK4 MAPK. AtMPK4 insertional knockout was found to have a pleiotropic phenotype and microarray analysis suggested its involvement as a negative regulator of stress-responses. It will be interesting to compare this altered gene expression with our induced cell line where the putative upstream MEK1 is not present. We do not have information on the role of MEK3 so studying the mRNA level in MEK3 downregulated cells might provide the first clues to its function. Thus we plan to use microarray analysis with four samples MEK1-RNAi and MEK3-RNAi when their expression is induced or un-induced with glucocorticoid.
NASCARRAY16 Establishing the action(s) of nitric oxide in plants. 2003-10-22	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.
NASCARRAY17 Identification of nuclear genes regulated by protoporphyrin IX-mediated retrograde signalling 2003-06-08	Background 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 flumioxazinwild-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. Aim
NASCARRAY18 Transcriptomic analysis of the plastid protein import 1 (ppi1) and ppi3 mutants 2003-06-08	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.
NASCARRAY19 Profile expression of genes involved in plant reaction to lead: uptake tolerance and defense. 2003-06-08	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 of promissing applicable aspects will also lead to better understanding plant acclimation and adaptation to antropogenic stresses. We expect to obtain mutant(s) tolerant to Pb and if sothey will be provided to NASC.
NASCARRAY20 The transcriptome of A.thaliana during UV-induced Programmed Cell Death 2004-06-23	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.
NASCARRAY21 Modulation of leaf transcriptome by phytochromes and cryptochromes 2002-11-11	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.
NASCARRAY22 Changes in gene expression specific to the depletion of major nutrients 2002-11-11	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.
NASCARRAY23 Analysis of anther development by identifying downstream genes controlled by MS1 2004-07-26	We have cloned the Arabidopsis MALE STERILITY1 (MS1) gene and shown it to have homology to the PHD-finger class of transcriptional regulators (Wilson et al 2001 Plant J. 28: 27-39). The overall importance of MS1 in pollen development has been clearly demonstrated since when mutated pollen degenerates after microspore release leaving a male sterile but otherwise phenotypically normal plant. We have demonstrated that MS1 is expressed in the tapetum around the stage of microspore release prior to tapetal degeneration. Comparisons of gene expression will be made between buds from the Ler-0 (wt) and ms1-ttg (mutant) to identifying changes in expression patterns of genes of critical importance. The use of the ttg marker will enable a prompt identification of the ms1 sterile plants amongst the segregating population. A further set of hybridisations will also be carried out using Ler-ttg (wt) to make sure that the expression changes observed in the first hybridisation experiment are not related to the ttg mutation. Staging of buds will be defined by bud size and two treatments, young and old unopened buds, will be used. Young buds will correspond from the start of pollen development until after MS1 expression and microspore mitosis I and old, from mitosis II until immediately prior to bud opening.(is this correct?). The identified genes will then be analysed for their expression patterns by RT-PCR and sequence/database homology to identify possible functions. Reverse genetic screens will be used to obtain Knockout mutants corresponding to the genes showing altered expression in the ms1 mutant.
NASCARRAY24 Cold induced changes in gene expression: differences between the Arabidopsis thaliana wild type and 2003-01-27	Arabidopsis sfr mutants are deficient in cold acclimation during exposure to cool-non-freezing temperatures. Although not visibly affected by the cold they have lost the ability to survive subsequent freezing. We plan to investigate how the sfr2 and sfr6 mutants respond to low temperature on the gene expression level. Wild type plants that have undergone identical treatments in parallel are necessary controls. The cold treatment of plants in the rosette stage (soil grown in a 8/16 hours day/night cycle) will be carried out in a cooled growth chamber at 4 degrees for 24 hours (same light regime treatment starting/ending at the 4th hour of light). The aerial parts of the treated and untreated plants will be collected and frozen immediately in liquid nitrogen for RNA extraction. Comparison of the cold response of thousands of Arabidopsis genes in the wild type to the situation in our freezing sensitive mutants will enhance our understanding of the cold response itself and illuminate the effect of the mutations on the cold acclimation process.
NASCARRAY25 Transcriptome analysis of ARRESTED DEVELOPMENT 3 mutant. 2004-10-26	Aims: Comparison of transcriptome between mutant and wild-type plant. Based on the temperature sensitive period of the mutant the gene likely acts during the earliest stages of the specification of the leaf primordium. Background: The ARRESTED DEVELOPMENT 3 mutation causes a temperature dependent loss of all spongy mesophyll and most palisade parenchyma in developing leaves. Although these leaves lack most internal tissues excepting vasculature they continue to expand away from the main axis of plant growth. Mature leaves have a small midrib and marginal regions that are large balloons of epidermis covering airspace. add3 is a temperature sensitive mutation whose most severe phenotypic response occurs at a restrictive temperature of 29 degrees C. Using BAC and TAC filters a recombinant population we have generated and available and newly developed molecular markers a high resolution (1000 chromosomes scored) physical genetic map has been completed to define the physical extent of the locus. Transformation rescue and RNAi experiments are currently underway to determine if a candidate gene identified using the approach outlined above is ADD3. Experimentalia: Wild-type and add3 tissue will be grown at 29 degrees C until the first two true leaves have emerged. Two days after emergence the aerial portions of the plants will be collected and RNA will be isolated. This developmental stage will provide plants with primordia in various stages of development. The wild-type tissue will serve as a control for exposure of developing primordia to high temperature the add3 plants will provide the experimental transcriptome. We intend to use RNA isolation protocols provided by the former AFGC facility for isolation of RNA. Conclusion:ADD3 is the first mutant from our collection of temperature sensitive mutations affecting shoot, floral and root organogenesis to be studied at the transcriptome level. Our work modeling the evolution of gene duplicates and genetic redundancy suggests that temperature conditional redundancy may be common in plants (Pickett and Meeks-Wagner Plant Cell 19957 1347-1356)(Force,Lynch,Pickett et al. Genetics 1999151 1531-1545). A highly similar gene to the ADD3 candidate gene has been identified. An exploration of the utility of transcriptomics to describe phenotypic impacts of potential conditionally redundant mutants is particularly timely. Many reverse genetics experiments in Arabidopsis result in "mutants" displaying small or no impacts on phenotype. The completion of the Arabidopsis genome sequence suggests that this experience may become common.
NASCARRAY26 Functional Genomics of Ozone Stress in Arabidopsis. 2005-01-26	Ozone is known to induce gene expression in plants. The roles of the induced genes and the molecular basis for the induction however largely remain to be elucidated. We will expose 2 week-old Arabidopsis seedlings to 200 ppb ozone for 1 h. RNA will be extracted from control and ozone-fumigated seedlings 3 h following the end of the fumigation period. The induction of the known anti-oxidant enzyme GST will be confirmed prior to submission of the RNA samples to GARNet. Changes in gene expression will be assessed by hybridising microarrays with fluorescently labelled cDNA prepared from control and ozone-fumigated seedlings. The role of selected genes will be inferred from their sequence and further established by over expression under the control of the 35S or cell-specific promoters and by searching for mutants among single transposon or T-DNA insertion collections. Plants will be analysed on the basis of traits that are known to be affected by ozone including growth, flowering and stomatal responses. In addition, the ozone calcium signature we have reported previously (Clayton et al.1999) will be studied by crossing transgenic or mutant plants with apoaequorin or yellow cameleon 2.1-transformed plants.
NASCARRAY27 Assembly of the cell wall pectic matrix 2003-01-28	The primary cell walls of land plants are composed principally of a load bearing cellulose microfibril-hemicellulose network embedded in a matrix of pectic polysaccharides. The pectic matrix is multifunctional and in additional to a directly structural role it is central to many fundamental plant processes including cell expansion, defence and cell signalling. The sequencing of the Arabidopsis genome has revealed the massive investment made by plants in modulating the pectic matrix in response to local functional requirements but despite concerted biochemical-based efforts over many years none of the genes involved in pectin biosynthesis/pectic matrix assembly have so far been identified. The pectin matrix contains some of the most complex polysaccharides found in nature and based on linkage analysis it is known that at least 53 glycosyltransferases must be involved in its construction. Our proposal to identify genes involved in pectin biosynthesis and matrix assembly exploits the well characterised phenomenon that many plants and cultured plant cells that are exposed to treatments that disrupt the synthesis of one cell wall component are capable of a compensatory increases in other components - including pectin. Specifically suspension cultured cells that are incrementally exposed to increasing concentrations of the herbicide 26-dichlorobenzonitrile (DCB) which specifically inhibits cellulose synthesis compensate for the resulting almost complete loss of cellulose from their cell walls by constructing walls made predominantly of pectin. We believe that the significant up-regulation of pectin biosynthesis in this system offers an opportunity to identify genes that function in the assembly of the pectic matrix by microarray comparison of transcripts of DCB-treated Arabidopsis cells with untreated cells. The use of Arabidopsis suspension-cultured cells rather than plants or seedlings offers the significant advantage that extracted RNA would be derived from only one cell type. It is anticipated therefore that the output from a transcriptome analysis of this system will indicate a number of genes of unknown function and lead to the identification of genes involved in pectin biosynthesis and the assembly of the pectin matrix.
NASCARRAY28 The role of the OX1 protein kinase in H2O2 signal transduction 2002-10-16	Expression of the OX1 gene coding for a putative protein kinase was shown to be induced in 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.
NASCARRAY29 Identification of genes controlling stomatal number in response to rising atmospheric carbon dioxide 2002-11-08	As part of a GUS promoter trap screen a gene exhibiting a guard cell-specific expression pattern was identified. When these plants are grown at elevated (1000ppm) CO2 they display a massive increase in stomatal density compared with the parent ecotype (C24) a response markedly different to the majority of plant species which show a decrease in stomatal density in response to elevated CO2. The disrupted gene has been identified and named HIC (for High CO2; Gray et al.2000 Nature 408: 713-716). Using the GARNet transcriptome analysis facility we shall be able to rapidly identify genes whose expression is altered between guard cells of hic and C24 (parental ecotype) plants in response to elevated CO2 levels. Control plants will be grown under identical light (240 mM m-2 s-1; 18hr light/6hr dark) and temperature (22oC light; 18oC dark) regimes but at ambient (360ppm) CO2 and guard cell enriched extracts produced from developing leaves. We will then be able to compare the expression profiles of C24 and hic guard cells grown under ambient and elevated atmospheric CO2 to identify additional components of this environmental signalling pathway. This approach should identify both genes involved in responses to rising atmospheric CO2 levels and genes involved in the control of stomatal development. This is a BBSRC collaborative project with Prof. A. Hetherington's laboratory, Institute of Environmental and Natural Sciences, University of Lancaster and Prof. F. I. Woodward, APS, University of Sheffield. Plants will be grown at Lancaster in controlled environment growth chambers (Percival Scientific, IA, USA) providing 240 mM m-2 s-1 18hr light/6hr dark regime at 22oC (light) 18oC (dark) with RH at 60 % +. Seeds will be surface sterilised using 10 % domestic bleach (<0.5 % sodium hyochlorite solution), washed with sterile RO water and sowed onto half-strength (2.2 g/l) Murashigee and Skoog basal media (Sigma chemical M5519), 10 g/l (1 %) sucrose, solidified with 6 g/l (0.6 %) plant cell tissue grade agar (Sigma chemical A1296). Media will be contained in 10 cm 3-vent petri dishes that are sealed with microporous surgical tape after sowing. All sown plates will be given a 48-hour stratification treatment at 0-4oC before going into growth chambers.After 10 days, the seedlings will be transferred to a peat based compost mixture (4 parts SHL multipurpose peat based compost (William Sinclair Horticulture, Lincoln, UK): 1 pat washed horticultural silver sand (GEM Horticulture, Accrington, Lancs., UK). After a further seven days plants are repotted (4:1 SHL: GEM silver sand) into 5 cm Arabaskets, of the Arasystem, Arabidopsis growing kit (Beta Tech, Gent, Belgium). At 5 weeks old, before inflorescence bolting had occurred, all leaf material from 10 plants (whole leaf) and 20 plants (guard cell enriched) will be harvested and cryogenically stored. RNA will be extracted in Sheffield using TRIzol reagent (GIBCo BRL) and cleaned using Qiagen minicolumns.
NASCARRAY30 Genes Involved in Plant Programmed Cell Death (PCD) 2002-10-16	Purpose: To identify genes involved in the induction and execution of plant PCD. Rationale: PCD is a genetically-controlled form of cellular suicide that is essential in many aspects of animal and plant development. In plants the mechanisms of PCD are poorly understood and much work to date has focused on the specialised case of the hypersensitive response. To investigate the regulation of PCD in a more general manner we have established an Arabidopsis cell suspension system in which PCD may be induced by a variety of treatments. We have characterised this system extensively using molecular and biochemical approaches including the use of a custom microarray of 80-plus genes which may play a role in plant PCD (Swidzinski et al.2002 The Plant Journal; in press). We have demonstrated the feasibility of extracting sufficient quantities of RNA and performing microarray analysis during PCD - a transition during which the majority of cellular transcripts are downregulated. Using a number of different treatments to induce PCD we have been able to distinguish between genes that are apparently specific to cell death from those that are a consequence of the treatment itself. We have established that 14 day-old cell cultures undergoing PCD and 6 day-old cultures one hour post-heat treatment (55C for 10 min) provide ideal timepoints for a representative examination of PCD-related gene expression. Additionally we have characterised these systems at the level of the total cellular proteome identifying and sequencing several proteins that are commonly upregulated in both systems. Our current custom microarray data while providing valuable information on the regulation of many genes during plant PCD is limited by the small number of transcripts studied. In order to gain a fuller understanding of the molecular mechanisms of plant PCD it is necessary to examine a much larger proportion of total Arabidopsis gene sequences. Such a widely inclusive study will aid in the identification of factors which may be involved in plant PCD but would not have been identifed based on the current molecular models about this cellular event. We propose to use the large number of genes available on the Affymetrix RNAs to examine gene expression in cells induced to undergo PCD by either heat treatment or senescence. Finally by comparing this data to our existing proteome analysis it should be possible to assess whether the genes of interest are being regulated at the transcriptional or post-transcriptional level during plant PCD.
NASCARRAY32 Ethylene induced hyponastic growth in Arabidopsis thaliana 2003-03-17	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.
NASCARRAY33 Higher order transcriptional regulation conferred by the bountiful gain_of_function mutant 2002-10-16	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.
NASCARRAY35 Testing Arabidopsis for the presence of arbuscular mycorrhizal signalling pathways 2002-12-17	Around two-thirds of all plant species form arbuscular mycorrhizasa symbiosis between plant roots and glomalean fungi that leads to the formation of intraradical organs of nutrient exchange and an extraradical network of fungal hyphae effectively extending the plant root system. The mycorrhiza plays a key role in plant nutrition and in enhancing plant resistance against pathogens and improving drought resistance. At present very little is known about the molecular basis of arbuscular mycorrhiza formation. Arabidopsis thaliana (as with all Brassicaceae) does not form arbuscular mycorrhizas (AM). Arabidopsis may either have lost essential gene functions or acquired new ones that prevent a successful symbiotic interaction. However given that mycorrhizal symbiosis developed very early during the evolution of land plants and that many ectomycorrhizal plant species can be colonised by AM fungi it is likely that important components of AM signalling pathways are conserved in all plants including Arabidopsis. Possibly the lack of AM development is a multigenic trait and this would make it difficult to isolate mutants that (re-)gain the ability to interact with AM fungi. What can be done however is firstly to test which parts of one or several putative AM signalling pathways are still functional and which ones are not. Secondly we can test whether negatively acting pathways such as those involved in defence against pathogenic microorganisms are induced upon inoculation with AM fungi. Together this work is likely to give important information of why Arabidopsis (and Brassicaceae) are behaving as non-hosts for AM fungi. In other words Arabidopsis will be an ideal system to study mechanisms of non-host "resistance" to AM colonisation. Elucidating these mechanisms will obviously make a great contribution to understanding the basis of the mycorrhizal interaction. Moreover using Arabidopsis as a tool it will be possible at the end to integrate the information obtained for AM signalling with that obtained for other developmental and environmentally triggered signalling pathways such as plant hormone signalling or plant defence responses. To produce an inventory of which Arabidopsis genes respond at all to inoculation with AM fungi a genome-wide screen for AM-controlled genes is proposed. RNA will be prepared from Arabidopsis roots treated with AM fungus and mock-inoculated control plants. Arabidopsis (Col-0) will be grown in pot culture (1:1 sand/Terra-Green) at low concentrations of phosphate. Three week-old plants will be inoculated with surface-sterilised spores of Gigaspora rosea. RNA will be isolated 3 days post inoculation.
NASCARRAY38 Baseline experiment floral homeotics 2001-06-30
NASCARRAY39 Comparison of the transcript profile of the auxin resistant axr4 mutant and wild-type Col0 2004-06-16	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
NASCARRAY40 Identification of genes responsive to non-metabolised glucose analoges as an approach to hexokinase- 2003-01-27	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.
NASCARRAY41 Plant gene expression associated with susceptibility to nematodes 2002-09-01	Background Heterodera schachtii is an economically important plant parasitic nematode that forms a syncytium from a cell superficial to the formed vascular bundle by progressive recruitment of other cells into the structure. The pattern of plant gene expression changes dramatically inside the syncytium. The pathogen probably plays a major role in defining the plant response by choice of initial plant cell during precise behaviour in planta and/or by the secretions it releases. The modified plant cells enable a high feeding rate by the female nematode so enhancing its rate of development and subsequent daily egg production. Arabidopsis is widely used as a model plant to characterise molecular responses to nematodes (e.g. Sijmons et al., 1991 Plant J. 1:245-254.). A complete overview of the changes in plant gene expression when sedentary nematodes establish has not yet been gained using Arabidopsis or any other host plant. Experimental Approaches Our initial studies will focus on the H. schachtii/Arabidopsis interaction. To assure reliable microarray screening care has been taken to minimise extraneous differences between samples (see "Growth conditions" section). At 21 days (Growth stage 3.2-3.5 Boyes et al., 2001 Plant Cell 13:1499-1510) Arabidopsis plants were challenged with rigorously sterilised, infective nematodes of H. schachtii as before (Urwin et al., (1997) Plant Journal 12: 455-461.). 35 sterile J2s were pipetted onto small ~0.5mm2 squares of sterile GF/A filter paper. The GF/A paper was left in direct contact with the zone of elongation on 3 lateral roots per plant for 48 hours. Control plants were mock inoculated with sterile water. Sections of root containing syncytia have been excised from the thin and transparent roots of Arabidopsis and collected into RNAlater solution (Ambion) at 21 days post infection (Growth Stage 6.1 Boyes et al. 2001). The female nematode has been removed with watch-maker's forceps. Equivalent sections of root have been harvested from non-infected plants. Material has been collected from c. 1000 plants for each of the two samples and the uninfected material serves as an internal control. Total RNA has been prepared from the reference and test root material using an RNeasy plant RNA preparation kit (Qiagen) according to methods required by GARNET.
NASCARRAY42 The effect of mutations in AtrbohC on the pattern of gene expression in primary root tissue. 2004-03-12	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.
NASCARRAY43 Agrobacterium tumefaciens-induced tumour development of Arabidopsis thaliana 2003-01-28	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.
NASCARRAY44 Transcriptome analysis of glyoxylate cycle knock-out mutants in early stages of seedling development 2003-10-23	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.
NASCARRAY45 Identification of genes differentially expressed in dormant versus active axillary shoot apical mers 2002-12-17	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.
NASCARRAY46 Nutritional control of plant development: molecular analysis of the NO3- response pathway in Arabido 2003-10-31	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. References Sablowski, R.W.M. and Meyerowitz, E.M. (1998) Cell 92, 93-103.Zhang, H. and Forde, B.G. (1998) Science 279, 407-409.Zhang, H., Jennings, A.J., Barlow, P.W. and Forde, B.G. (1999) Proc. Natl. Acad. Sci. USA 96, 6529-6534.
NASCARRAY48 Transcriptome analysis of Arabidopsis microgametogenesis 2003-06-08	Aims We aim to use transcriptome analysis to establish on a genome-wide scale the identity and regulatory clusters of genes that specify microgametogenesis from the haploid microspore to mature functional pollen in Arabidopsis. Background Pollen as the haploid male gametophyte plays a vital role in plant fertility and crop production through the ability to deliver the male gametes in fertilisation. Despite the obvious importance for plant fertility and crop production we have a very limited understanding of the regulatory mechanisms that have evolved to specify male gametophyte development and functions and less than 150 genes have been identified that are gametophytically expressed in the anther.The availability of functional genomic resources now provides the opportunity to undertake a comprehensive approach to describing cellular development in terms of the transcriptome. This approach is particularly powerful where the complete transcriptome of a single developing cell can be analysed. The male gametophyte is a uniquely accessible cell type for such studies, enabling RNA analysis from distinct purified cell populations during development.The proposed experiments are designed to support a current application (P19208, Twell) to investigate the gametophytic transcriptome and transcription factor networks. The results obtained will extend our knowledge of the contribution of haploid gene expression to anther development and will be used directly to extend BBSRC funded work (P15086, Wilson) to investigate the role and targets the MALE STERILE 1 gene (MS1). In particular the data will be used in collaboration to extract haploid gene expression from datasets of transcriptome analysis of staged flower buds of wild type (Ler) and ms1. This work will also complement BBSRC funded work on sporogenesis (G13338, Dickinson and Scott) and meiosis (G15941, Franklin and Jones) that are focussed on earlier steps in anther development. Biological material and methods. Isolated microspores and pollen at 4 different developmental stages will be analysed. We will isolate spores from developmentally staged buds of Ler grown under defined growth conditions. Buds from several batches of 100 plants will be rapidly sorted into 4 groups according to developmental age, uninucleate microspores (UM), bicellular pollen (BP) tricellular pollen (TP) and mature pollen. Spores will be released by gentle mechanical tissue disruption and purified by filtration and purification of spores. We are confident that our spore isolation procedures are rigorous since we could not detect even trace expression of highly abundant sporophytic transcripts such RbcS and Cab transcripts in microarray data from pollen RNA.
NASCARRAY49 Transcriptome analysis of pho3 2003-03-17	The aim of this proposal is to understand the impact of the pho3 mutation on the transcriptome.The mutant pho3 was isolated on the basis of a failure to induce acid phosphatase activity in roots in response to low Pi. The pho3 mutant had a P deficient phenotype in vitro and in the leaves of soil grown pho3 plants the total P content was again reduced. pho3 exhibited a number of characteristics normally associated with low-P stress, including severely reduced growth, increased anthocyanin content and starch accumulation. pho3 was shown to have an absolute requirement for exogenous sucrose as pho3 seedlings were arrested in early seedling development on sucrose-free medium and was unaffected by Pi-availability. pho3 was found to be insensitive to high concentrations of exogenous sucrose (10%) but was sensitive to glucose. pho3 also exhibited reduced sensitivity of seed germination to exogenous ABA (abi-phenotype), a response which has been linked to sucrose insensitivity in both sugar-sensing and ABA mutants. Two developmental stages have been selected on the basis of the physiological characterisation of pho3. Seedlings (1.02; 2 rosette leaves) grown in agar and compost-grown plants (3.90; rosette growth complete). Agar will be supplemented with 1% sucrose and 1/2 strength MS; plants will be grown under a 16 h light/8 h dark cycle. Comparisons will be made between pho3 and Wassilewskija (the genetic background of the mutant) at each stage.
NASCARRAY51 Seedling transcriptome affected by Norflurazon-induced photobleaching of chloroplasts 2004-06-09	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.
NASCARRAY52 The effects of mutants in stress response pathways on gene expression during senescence 2003-10-28	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 (Col5 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.
NASCARRAY53 Environmental Genomics of calcicole-calcifuge physiology 2003-08-13	Aim To identify genes which are differentially expressed between calcicoles and non- calcicoles. Background Grasslands on the calcareous soils of chalk and other limestones are among the most species-rich plant communities in Europe (Rodwell 1991 et seq.). They have experienced huge losses and remain vulnerable to such impacts as neglect of traditional management, agricultural improvement and global changes in climate, nitrogen depositions and ozone levels. Our understanding of the physiological characteristics of calcicoles and calcifuges remains limited. A detailed understanding of the genetic basis of the mechanisms that enable calcicoles to thrive on calcareous soils is essential to enable us to predict how these plant communities and their constituent species will be affected by environmental change and how the biodiversity of these ecosystems can be sustained.At Lancaster we have been studying calcicole-calcifuge physiology, with particular reference to Ca2+-tolerance, for over fifteen years. Recently, our research has focused on the regulation of apoplastic Ca2+ in Arabidospsis thaliana. We have compared the response of two ecotypes of A. thaliana, the non-calcicole ecotype Columbia (Col-4) and the calcicole ecotype Cal-0, which is a genetically uniform line from an original population collected by Ratcliffe from a rocky limestone slope in 1954, to rhizospheric Ca2+. Our results show that Cal-0, exhibits a markedly higher tolerance to growth on high rhizospheric Ca2+ compared to Col-4.Our hypothesis is that adaptation to a calcareous environment will be reflected in altered gene expression. To test this hypothesis we will grow Col-4 and Cal-0 at low (1 mM) and high (12.5 mM) rhizospheric Ca2+ and compare the patterns of gene expression by microarray analysis. In the first instance, to eliminate any differences in gene expression between the Cal-0 and Col-4 ecotypes, we will compare RNA that will be extracted using the Qiagen RNEasy kits, from plants grown at 16 hour day lengths and will be harvested after 30 days of growth on sand watered with 0.5 X Long Ashton solution containing 1 mM CaCl2.
NASCARRAY54 Role of COV in vascular patterning 2003-10-28	The molecular mechanisms that control the ordered patterning of vascular tissue development in plant are not well understood. Several models propose a two component for plant vascular differentiation in which requires both an inducer of vascular tissue an also an inhibitory component that prevents the formation of vascular bundles near pre-existing bundles by a process often termed lateral inhibition. We have identified two recessive allelic mutants in Arabidopsis, designated continuous vascular (cov-1, cov-2), which display a dramatic increase in vascular tissue in the stem in place of the interfascicular region that normally separates the vascular bundles. The mutant plants exhibited normal vascular patterning in leaves and cotyledons. Analysis of the interaction of cov with known auxin signalling mutant and direct analysis of auxin concentrations suggests that cov affects vascular pattering by some mechanism that is independent of auxin. COV is expressed in all plant tissue but more highly in developing flowers and stems. The COV protein is predicted to be an integral membrane protein of unknown function, conserved in both plants and bacteria that is involved in negatively regulating the differentiation of the vascular tissue in the stem of Arabidopsis.The phenotype of COV exhibits some similarities to acl5 which is known to be a defect in the gene for spermidine synthase. Consequently, we are interested to test the idea that COV may be involved in the transport or perception of polyamines and that polyamines may be essential signals in normal vascular tissue development. Clearly, it this proves to be the case we would expect to see some differences in the transcript levels of genes associated with these pathways.
NASCARRAY55 Transcriptional profiling of laser-capture micro-dissected embryonic tissues 2005-07-21	The aim of this BBSRC-funded project is to develop laser-capture microdissection (LCMD) to isolate small cell clusters in different regions of arabidopsis embryos at different stages of development; to develop RNA amplification procedures on dissected tissue sampes; and to use DNA microarray techniques to investigate global transcriptional differences between samples. Cryosectioned embryos of ecotype Col-O of globular, heart and torpedo stage were used to isolate cell clusters from the apical and basal regions, for RNA isolation and amplification. Samples will be provided as T7-primed cDNA, with three biological replicates for each tissue to be analysed. Each replicate comprises cDNA from pooled tissue samples from ca. 15 embryos. The experimental details have been discussed with Sean May et al. at NASC.
NASCARRAY56 UV-B Responses in Light Grown Plants: Similarities to Biotic Stress 2003-07-11	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.
NASCARRAY57 Comparative transcriptome analysis between wild-type and gpa1 mutant in response to ABA 2002-11-14	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).
NASCARRAY59 Impact of Type III effectors on plant defense responses 2003-10-27	Our interest lies in how plants respond to bacterial pathogens. Over the past three years we have identified and documented reproducible, landmark biochemical and molecular events following the challenge of Arabidopsis with the phytopathogenic enterobacteria P. syringae. Significantly, our studies revealed 60% of cDNA-AFLP differentials not present on the 8,200 feature GeneChips and 20% absent from public EST databases (de Torres in press). We now seek to exploit this background using carefully defined time-points to analyse global changes in the Arabidopsis transcriptome using challenges selected to define gene targets implicated in (i) expression of basal immunity (ii) the establishment of successful parasitism (resistance) by a virulent pathogen (host). The results will provide a rationale for future functional assays of the identified pathways using transgenic knockouts and mutant analyses. Additionally, data will provide underpinning support for comparative proteomics of the defense response currently in progress with GARNet support using the same experimental parameters (BBSRC 32/P14635). We propose the following treatments: (i) Mock vs. DC3000hrpA @ 60 min: 60 minutes is subsequent to host immediate-early stress responses and will catalogue the innate responses induced by pathogen associated molecular patterns. The hrpA lesion will ensure no type III effectors influence transcriptional responses. Gene products induced at this time are predicted to potentiate latter host responses (2 treatments X 3 biological replicates = 6 chips). (ii) Mock vs. DC3000 vs. DC3000hrpA vs. DC3000::avrRpm1 @ 4 hours: A key time point previously defined where no macroscopic symptoms are visible but significant differences exist between compatible and incompatible interactions at the molecular and physiological levels. These treatments will serve to define the earliest genes induced by the complement of DC3000 type III effector and will specifically define genes/pathways suppressed by virulence factors in addition to those implicated in orchestration of the hypersensitive cell death. We will also include an incompatible interaction on a transgenic line expressing an RPM1 interacting protein, which fails to mount an HR but exhibits hyper-resistance. We predict this challenge will separate the resistance response (pathogen restriction) from that associated with hypersensitive cell death (5 treatments X 3 biological replicates = 15 chips). (iii) Mock vs. DC3000 vs. DC3000hrpA @ 14 hours: At 10 h before phenotypes are apparent in the DC3000 background, Type III effector delivery is well advanced and impact on host transcription maximal (3 treatments X 3 biological replicates = 9 chips).
NASCARRAY60 Gene expression and carbohydrate metabolism through the diurnal cycle 2003-10-24	This proposal is aimed at providing transcriptome data to underpin a long-term joint research programme of Steve Smith and Alison Smith. We are jointly studying starch synthesis and breakdown in Arabidopsis leaves, and individually studying other enzymes of carbohydrate metabolism (eg. sucrose synthases, invertases, sugar transporters). Collectively these enzymes are encoded by up to 100 known genes, but there are many others of relevance to our studies. For several years we have employed a defined set of growth conditions for this work (resulting in numerous publications). We have extensive data for changes in the amounts of starch, malto-oligosaccharides and sugars throughout the diurnal cycle in these plants, and we intend to quantitate numerous key enzymes. We now wish to profile changes in transcripts in these plants, so that this information can be correlated with changes in the amounts of key enzymes and metabolites. Analysis of the data sets will help us to relate the synthesis of certain enzymes to particular metabolic functions, and also to help identify genes encoding novel proteins involved in carbohydrate metabolism. Current research is aimed at discovering such novel proteins using proteomics approaches (BBSRC Grant: _Discovery and Functional Analysis of the Starch Proteome_). While the growth conditions (12h photoperiod, 150 umol/m2/s, 20C) and ecotype (Col-0) are specifically tailored to our experiments, they will be of value to the wider community, and could form the basis for collaborative studies with other groups. The experiment has involved sampling leaves at eleven different time points as follows: 0, 1, 2, 4, 8, 12, 13, 14, 16, 20, and 24 h (where time 0 is the onset of dark and 12 h is the onset of light). The 24 h time point is a repeat of 0 h. This sequence provides relatively more samples immediately after the light-dark transitions, when changes in metabolism are most pronounced. Plants were grown in a controlled environment chamber under defined conditions, and selected for harvest according to a random number generator. Three fully expanded leaves were harvested from each of 20 plants for each sample. Leaves were frozen at -80 C and a portion saved for metabolite and enzyme assays while RNA was isolated from the remainder. The RNA has been purified and is ready to send to Nottingham.
NASCARRAY61 Carrera Bergua: Functional genomics of shoot meristem dormancy. 2002-09-14	This experiment is an integral part of BBSRC Exploiting Genomics grant: 233/EGA16203. Functional genomics shoot meristem dormancy. Dormancy is an active state in which cell division is repressed. We are interested in the regulation of the transitions of dormancy and activity in shoot meristems in seeds. Abscisic acid (ABA) is involved in the dormancy process. Different general ABA biosynthesis and sensitivity mutants have been characterised in Arabidopsis showing reduced dormancy levels (aba1-1, abi1-1 and abi3-4) (Leon-kloosterziel et al 1996, Holdsworth et al 1999), viviparous fus3-8 mutant seeds (Baumlein et al 1994), and also seed-specific no dormant mutant seed rdo2 (koornneef et al 1996). We will compare the transcriptome of wildtype dormant seed with those of freshly harvested seed of the non-dormant mutants abi1-1, aba1-1, rdo2 and viviparous mutant seeds fus3-8 and abi3-4. This will allow us to distinguish germination-related genes from genes specifically expressed during seed dormancy.
NASCARRAY62 The role of Cdpk6 in develpoment 2003-10-23	The research programme is focussed on determining the role of CDPKs in abiotic stress response. We chose several CDPKs for investigation, some which have been implicated in the response to abiotic stress (Sheen 1996), others whose function is unknown. We constructed gene fusions with YFP as a 3_in-frame fusion to investigate cellular/tissue expression; the gene-specific DNA includes 1.5kb 5'-untranslated region. Plants carrying the Cdpk6-yfp construct show a marked alteration in phenotype: the (probable)-heterozygote is small and bushy and the (probable)-homozygote shows an excess of flower primordia and insufficient leaf development. These plants do not survive. The expression of Cdpk6 as measured using a GUS-promoter fusion is confined to the developing flowerheads although the Cdpk6-YFP plants show more widespread expression. We have made RNA from two separate insertion lines of Cdpk6-YFP and from Col-0 plants carrying the aequorin gene all grown on selective medium. We cannot collect homozygote seed; the seedlings germinating from seeds collected from heterzygous plants grown on selective medium will consist of both heterozygous and homozygous individuals.
NASCARRAY63 Characterisation of the genetic basis of changes in stomatal numbers at elevated atmospheric carbon 2004-06-09	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.
NASCARRAY65 An investigation into transcriptional changes in the developing Arabidopsis leaf caused by a novel s 2003-01-13	Arabidopsis genome sequencing has revealed the presence of at least three extensive gene families that may encode protein ligands. One of these, the SPH (S-protein homologue) family, was identified as a direct result of our studies on self-incompatibility in Papaver. The Arabidopsis SPH gene family consists of 81 members. We have initiated experimental work on a subset of these. RT-PCR studies indicate that many, if not all, SPH genes are expressed. Each SPH gene encodes an N-terminal signal peptide sequence and thus SPH proteins are likely to be secreted. Until recently none of the genes in this family had known function. However we have evidence that one member of the family, SPH1 is involved in leaf vascular development. In order to determine the function of SPH1, Arabidopsis plants were transformed with an SPH1 antisense construct. Analysis of the mutant phenotype shows that whilst plants appear as wt until principal growth stage 1.04, they subsequently show severe morphological defects. Plants are severely dwarfed with twisted rosette leaves at ~ 30% wt length and width as well as shortened inflorescence stem. Closer examination revealed aberrant leaf vasculature and severe reduction in expansion of parenchyma cells surrounding the primary leaf vein. We have conducted preliminary immunolocalisation studies with antibody raised to SPH1. These suggest that SPH1 protein is secreted by cells within the developing vasculature of the immature leaf (leaves<6mm in length). The data that we have so far obtained leads us to believe that SPH1 protein acts as a signalling molecule during early leaf development. As SPH1 is likely to be a signalling protein it is assumed that its interaction with a cognate receptor results in initiation of developmental processes within leaf tissue. The purpose of the experiment is to determine the network of genes within the normally developing rosette leaf whose expression is altered by SPH1. This will be accomplished by comparing transcriptional levels in rosette leaves of antisense-SPH1 plants with wt plants. We propose to make target RNA from rosette leaves taken from plants at principal stage 1.05 (immediately after initial appearance of developmental abnormality) and from plants at principal stage 1.14 (where gross changes are apparent). We propose to use replicate slides for each hybridisation, i.e. 2 hybridised to RNA from wt leaves at 1.05, 2 antisense at 1.05, 2 wt at 1.14 and 2 antisense at 1.14.
NASCARRAY66 Investigating the links between organic acid and carbohydrate regulation of gene expression. 2003-10-29	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.
NASCARRAY67 Mechanisms of DNA double strand break repair in Arabidopsis non-homologous end joining mutants 2003-10-27	The proposal aims to characterise the pathways of DSB repair and recombination in Arabidopsis with the main focus of this research being the NHEJ pathway of illegitimate recombination. We will build on our expertise and resources in the field of DSB repair in plants, using the Arabidopsis NHEJ mutant atku80 which is an excellent model system for the study of DSB repair in higher eukaryotes (West et al., 2002 Plant J. 31, 517-28). Comparison of the transcriptome in NHEJ mutant and wild type plants under different experimental conditions will identify novel candidate genes involved in DNA DSB repair or damage signalling pathways.We will conduct 4 separate experiments on Arabidopsis seedlings grown on 0.5 MS media: the first will be a control consisting of Wassilewskija (WS-2) plants grown under standard conditions. In the second experiment WS plants will be exposed to the chemotherapeutic agent bleomycin (1 microgram per ml). This agent has been shown to cause both single and double strand breaks in the genome of Arabidopsis seedlings (Menke et al., 2001 Mut. Res. 493, 87-93). This agent is used in preference to gamma irradiation, which causes high levels of oxidative damage to cellular components. These experiments will characterise for the first time the transcriptional responses of Arabidopsis cells to the specific induction of DNA strand breaks. The transcript profile will also be determined in untreated atku80 mutants. This experiment will identify the components of novel and previously characterised DNA repair pathways up regulated in the mutant background. Finally, transcript analysis of bleomycin treated atku80 mutants and comparison with untreated mutant plants and both untreated and treated WS plants will identify novel putative DSB repair genes up regulated in response to genotoxic stress in the absence of a Ku80 mediated DSB repair pathway.The results of these studies will provide new and important information which will be instrumental in identifying novel genes and pathways involved in DNA repair and genome stability in plants and help elucidate the fundamental differences that exist between animal and plant DSB repair processes.
NASCARRAY69 A genomic approach to understanding seed dormancy 2007-04-01	Background: Physiological seed dormancy is present throughout the higher plants and has a profound impact on many aspects of crop production and the conservation of genetic resources as well as the structure and development of plant communities. Despite this, there is considerable ignorance of how dormancy is controlled at the molecular level. Greater understanding of seed dormancy therefore has considerable generic value. Aims: The long-term goal is improved understanding of seed dormancy and the identification of generic molecular markers of seed dormancy status. The specific aim of the applications was to compare gene expression patterns in Arabidopsis Cvi seeds in 13 physiological states (52 arrays) Information on selected A. thaliana ecotype: The test organism is Arabidopsis thaliana accession "Cape Verde Islands" (Cvi). Characterisation shows that mature, primary dormant seeds (at shedding) require a dry storage treatment (after-ripening) of up 6 months to induce a uniform non-dormant state in the seed population. When imbibed, germination will then ensue if the seeds are provided with appropriate conditions of light, nitrate or low temperature. Alternatively, if a non-dormant seed is imbibed in the dark they enter a state of secondary dormancy in which a moist-chilling treatment is required to re-induce a state of non-dormancy. Experimental design: A full experimental design and description of the treatments used is given in the two publications below. Table 1 in each publication summarises the 13 treatments.
NASCARRAY70 The effects of the sfr2, sfr3 and sfr6 mutations on lyotropic stress responses 2003-10-24	Our goals are to discover the basis of the stress-sensitive phenotypes of the sfr2, sfr3 and sfr6 mutants, and to distinguish damage-repair from damage-prevention-related transcription in the wild type. The effects of sfr2 and sfr3 on cold-induced gene expression will be observed. Since sfr6 causes sensitivity to drought as well as freezing, the effects of sfr6 on the transcriptional response to drought is studied; an observation of cold-induced sfr6 expression is needed for direct comparison to the effect of drought. Unstressed mutants, and equivalently-stressed wild types, are necessary controls. The above experiments are conducted on tissue-culture-grown plants grown under 24 hr illumination for maximum reproducibility and comparability to other transcriptomic experiments.Freezing causes physiological changes even in a hardy, cold-acclimated wild type. During the recovery period, gene expression will reflect the induction of damage-repair processes distinct from the damage-prevention associated with cold acclimation. This will be detected by observing the wild-type transcriptome at two time points during recovery from a freezing episode. The appropriate controls is the unfrozen, cold-acclimated wild type. Plants for this experiment will be soil-grown in an 9/15 day/night cycle to the rosette stage, and this regime will be maintained during acclimation and freezing treatments.Cold-induction will be 10 days at 4Ã‚Â°C; drought will be imposed by placing excised leaves in a desiccator for 6 h. The youngest fully-expanded rosette leaves will be harvested for RNA extraction.
NASCARRAY71 Effect of mycotoxin treatment on gene expression of wild-type and an altered sensitivity mutant 2004-06-22	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 ben, 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 tk,...), 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.;
NASCARRAY73 The molecular basis of chilling and freezing stress 2003-04-14	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 Ã‚Â“captureÃ‚Â” 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.
NASCARRAY74 Targets of the mci genes. 2004-04-21	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.
NASCARRAY76 Differential gene expression patterns in Arabidopsis mutants lacking the K+ channels, akt1, cngc1 an 2004-06-08	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.
NASCARRAY77 Identification of Core Genes Regulating Plant Programmed Cell Death (PCD) 2003-10-28	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.
NASCARRAY78 Genes affected by hog1 alleviation of CHS silencing 2003-10-27	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.
NASCARRAY79 Molecular basis of respiratory burst-mediated thermotolerance in Arabidopsis 2003-10-24	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.
NASCARRAY80 The trans-differentiation of cultured Arabidopsis cells 2003-11-10	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.
NASCARRAY81 Gibberellin and ethylene cross-talk at the level of transcriptional regulation in Arabidopsis 2004-06-09	This work is part of an existing collaboration between the two laboratories, funded by the EU (EU-RTN-INTEGA). Both parties will share the cost of this microarray experiment. Background: We have demonstrated that ethylene-insensitive mutants and wild type(col-0) Arabidopsis plants treated with an ethylene perception inhibitor have increased levels of expression of genes, such as GASA1 and g-TIP, that are thought to be regulated by GA (Vriezen et al, unpublished results). However, this observation was based on an RNA gel blot analysis and therefore limited to few genes. Aim: To investigate whether plants with decreased ethylene perception are generally hypersensitive to GA or whether this effect is restricted to specific genes. We plan to undertake a complete transcriptome analysis of GA-treated wild type andetr1-1 plants. The aim is to identify genes that are induced directly as a result of the GA treatment, and we will therefore focus on the time window 0-3h. Tissues to be sampled: Plants will be grown in vitroon MS/2 containing 1% sucrose, pH 5.7, at 22 C,70% RH, under white light (54 PAR) and a photoperiod of 16h light/8h dark. Plants will be treated at 14 days and harvested entirely, i.e. roots and shoots are extracted together. Experimental set-up: Col-0 and the ethylene-insensitive mutant etr1-1 will be sprayed with 50 microM GA4 in water. GA4 is the major bio-active GA in Arabidopsis. Samples will be taken after 0, 30 min, 1h, and 3h. In order to correct for touch-induced genes a control, which is sprayed with water only and harvested at 1h, will be included for both genotypes. The total number of chips to be hybridized is 10. The time course with 4 data points is preferred to a single time point with 3 repeats, because it will allow us to follow the induction kinetics and identify early response genes. For each timepoint, RNA will be extracted from at least 40 individuals.
NASCARRAY82 To identify changes in gene expression during silique senescence in Arabidopsis thaliana 2004-08-20	The aim of the experiment is to compare and contrast the profiles of gene expression during silique senescence and to identify the molecular events that regulate the timing of the process. Silique occurs over a highly predictable time frame and as a consequence it may be easier to identify the molecular events that are critical for the process to take place than in systems such as leaf senescence. The material will be collected from 20 Arabidopsis thaliana ecotype Columbia plants (NASC order number N1093) grown under controlled conditions specified under NASC protocol: Baseline sample preparation. Two pods will be collected from each plant and pooled from 20 plants to reduce sampling variability. It is intended that 3 replicates of the experiment will be submitted. The RNA will be extracted from mature green silique tissues of 10 days after anthesis and senescing pod tissues of 20 days after anthesis using lithium chloride extraction protocol and cleaned with Qiagen RNAeasy column.
NASCARRAY84 Carbohydrate- and redox-regulation of gene expression in a TPT mutant 2003-10-27	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 Ã‚Âµmol PAR m-2 s-1. Total RNA will be extracted from pools of individual illuminated leaves from at least eight plants at growth stage 3.70. Leaves will be harvested 2 h into the photoperiod to 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.
NASCARRAY85 Molecular bases of zinc tolerance and accumulation by Arabidopsis halleri 2004-06-21	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.
NASCARRAY89 Seedling transcriptome affected by a far-red light preconditioning treatment to block chloroplast de 2004-06-09	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)
NASCARRAY92 The mechanisms involved in the interplay between dormancy and secondary growth in Arabidopsis 2003-05-15	Plants that exhibit secondary growth, such as trees, are a prominent feature of terrestrial ecosystems. Furthermore, secondary growth itself, particularly wood, has huge economic value. Despite the importance of secondary growth from both basic and applied science perspectives, little is known about the molecular mechanisms that underpin this facet of plant development. The proposed microarray experiments are designed to expand our knowledge of the regulation of secondary growth by combining the power of Arabidopsis genetics with complete transcriptome analysis. It is now well established that Arabidopsis can be grown under conditions that induce secondary growth in the hypocotyl, albeit small, wood. We have grown 8500 Arabidopsis plants of different genotypes under these conditions and will extract RNA from the developing vascular cambia of these plants to subject them to complete transcriptome analysis.The mutants that we have chosen for these analyses are all related to each other on the basis of the fact that they impact dormancy in either seeds or shoots (abi1, aba1, max4, axr1, AtMYB61 knockout, AtMYB50 knockout). The mutants themselves are the core group of mutants that are the focus of a three laboratory consortium, funded under the BBSRC Exploiting Genomics Initiative, to investigate the molecular basis of meristem dormancy in Arabidopsis. The other partners in the consortium are Dr. Ottoline Leyser (York) and Dr. Michael Holdsworth (IACR). While the Leyser and Holdsworth groups have investigated the impact of these mutations on transcriptome activity in shoot meristems and seeds respectively, our work focuses on the vascular cambium. Thus, this work will not only provide insights into the regulation of cambial function, but, when compared with the existing datasets from the Leyser and Holdworth labs, the work should also provide insights into the relationship between dormancy-impacted phenomena in different meristematic regions. Beyond this, the specific work on the cambium-specific regulation of genes in the MYB61KO will provide even greater insights into the functioning of this important resource allocation regulator, as it will build on a significant complete transcriptome dataset that is already available through GARNet. In total, the proposed analyses will generate important new data that builds on existing datasets, to provide an even more comprehensive understanding of gene function, and genetic networks, in an important biological and applied context. Please note that the sample numbers that we have provided below are meant to be the "base" number for each biological condition (mutant, etc.), and that there will be independent TRIPLICATE biological replicates produced for each condition. (ie. A-1 to A-12).
NASCARRAY94 Comparison of CATMA, Affymetrix and Agilent arrays 2004-08-11	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 Âµg/Âµl 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.
NASCARRAY95 Polycomb Binding Protein 2004-08-27	We have identified a gene encoding apolycomb-chromo domain binding protein. Complete knockout of these plants do not lead to a phenotype, but a reduction of gene expression in RNAi line produces a defined phenotype. This argues in favour of a compensation mechanism that is switched on once the concentration of the gene reaches a control level.The aim of the experiment is to search for such compensatory factors that are up-regulated in the knockout line.
NASCARRAY96 Clarification of the genetic basis of the iae1 and iae2 phenotypes 2004-06-08	The mutants iae1 and iae2 have been mapped to two distinct loci on chromosome 2. With the aid of the GetCID service, we believe iae1 has been localised to a relatively small region (~100kb); while mapping has restricted iae2 to a small interval (~50kb) with a very limited number of candidate genes. Although it has proved difficult to determine which gene is mutated in iae1, we belive that use of the transcriptomics service will help identify which genes are affected (directly and indirectly) in this background. The iae2 locus is sufficiently restricted by genetics and by molecular data that identifying a candidate (and any downstream loci affected) should be straightforward. We require the use of the full genome chips. The data from this experiment should help tie together the data we have for a paper that is currently in preparation.
NASCARRAY101 The molecular basis of plant insect interactions 2004-04-22	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 Â‘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.
NASCARRAY102 Differential gene expression patterns in the phosphate deficient mutant, pho 1 2004-06-11	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.
NASCARRAY103 Identification of genes involved in nutritional regulation of root architecture 2004-01-14	AIM: 1. To identify genes that respond to N-deficiency and N-resupply and 2. To identify the subset of these genes that are under the (direct or indirect) regulatory influence of the ANR1 MADS-box gene and which may therefore participate in the nutritional regulation of root architecture. BACKGROUND: The Arabidopsis ANR1 gene is a key regulator of root architecture (Zhang and Forde, 1998): When ANR1 expression is suppressed (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 increase meristematic activity in the lateral root meristem (Zhang et al., 1999). ANR1 expression is modulated by changes in the NO3- supply (Zhang and Forde, 1998; Y.Gan and B.G. Forde, unpublished observations). In this experiment we will study the global changes in gene expression that occur when Arabidopsis plants are exposed to three different N regimes that are known to modulate ANR1 expression. The treatments will be done on wild-type plants and on an ANR1 knockout mutant as a means of identifying the (direct and indirect) downstream targets of ANR1. This experiment is complementary to another microarray experiment (BBSRC grant no. 89/P13011) which is using a DEX-inducible ANR1 transgene to identify genes that are immediately downstream of ANR1 in the signal transduction cascade Experimental: Plants of the two genotypes will be grown in hydroponic culture in a growth cabinet for 4-5 weeks before subjecting them to the different N treatments: 1) Continuous nitrate supply; 2) N deprivation for 2.5 days; and 3) N deprivation followed by 3 h nitrate induction. All harvesting will be done simultaneously to avoid diurnal effects. Roots from 12 seedlings will be harvested and pooled with roots from 12 seedlings whose treatment has been staggered by 24 h. We propose to replicate the experiment three times. References: Zhang, H. and Forde, B.G. (1998) Science 279, 407-409. Zhang, H. et al. (1999) Proc. Natl Acad. Sci. USA 96, 6529-6534.
NASCARRAY104 Global gene expression in two species of Brassicaceae: Thlaspi caerulescens and Thlaspi arvense (cro 2004-12-01	The aim of this study is to test if Affymetrix ATH1 GeneChips can be used to study global gene expression in two species of Brassicaceae: Thlaspi caerulescens and Thlaspi arvense. These species have been chosen as they have contrasting mineral phenotypes. Plants were grown in a standard potting compost. Seeds were sown on September 16th 2003. Pots were placed in a controlled environment room set to 22Â°C and 16 h daylength. Plant shoots were harvested on December 12th. Both species had in excess of 20 leaves per plant, but had not produced flowering bolts. RNA was extracted from the entire shoots of Thlaspi caerulescens and Thlaspi arvense plants.
NASCARRAY105 Differential gene expression patterns in potassium-starved and Caesium-treated plants 2004-06-14	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.
NASCARRAY108 Circadian expression of genes: modelling the Arabidopsis circadian clock 2004-12-01	Our aim is to study the circadian expression of genes to aid in our attempt of modelling the Arabidopsis circadian clock. Circadian microarray data have previously been published for plants after white light (WL)-dark cycles, using the 8k chip (Harmer et al. 2000). We intend to repeat this experiment using the 26k chips and are coordinating with Dr. Harmer, who is pursuing complementary experiments in UC Davis. Plants will be transferred to continuous WL after entrainment to 12h:12h light dark cycles. RNAs will be harvested every 4 hours over two days, with the same accession and sampling intervals used previously by Harmer et al. The two days of sampling provide internal replication. Our experience shows that this is the most economical design: it is easier to identify rhythms over a two-day timecourse than in two replicates of a single day. Hence: 13 RNA samples on 13 chips in total. METHOD: Seed was sown on MS agar plates with 3% sucrose, imbibed at 4 C for 96 hours. Seed was then entrained for 7 days at 22C, in cycles of 12 hours white light, 12 hours darkness. After 7 days they were transferred to constant white light at 22 C: this is time 0h. Tissue harvested at the time points shown after time 0.
NASCARRAY114 Molecular bases of zinc tolerance and accumulation by Arabidopsis halleri 2004-04-29	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)Â· A. halleri exposed to low and high Zn; root and leaf mRNAs extracted: 3 replicates of each: = 12 slidesÂ· 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.Â· A bulk of F3 progeny all exhibiting high Zn tolerance: exposed to low and high Zn; leaf and root mRNAs: 3 replicates: = 12 slidesÂ· A bulk of F3 progeny all exhibiting low Zn tolerance: exposed to low and high Zn; leaf and root mRNAs: 3 replicates: = 12 slidesÂ· A bulk of F3 progeny all exhibiting high Zn accumulation: exposed to low and high Zn; leaf and root mRNAs: 3 replicates: = 12 slidesÂ· A bulk of F3 progeny all exhibiting low Zn accumulation: exposed to low and high Zn; leaf and root mRNAs: 3 replicates: = 12 slides
NASCARRAY117 Identification of genes involved in secondary cell wall development in the hypocotyls of short day g 2004-07-20	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.
NASCARRAY120 AtGenExpress: Response to virulent, avirulent, typeIII-secretion system deficient and nonhost bacter 2004-07-09	AtGenExpress: A multinational coordinated effort to uncover the transcriptome of the multicellular model organism Arabidopsis thaliana (Hybridisations done at NASC) The activity of genes and their encoded products can be regulated in several ways, but transcription is the primary level, since all other modes of regulation (RNA splicing, RNA and protein stability, etc.) are dependent on a gene being transcribed in the first place. The importance of transcriptional regulation has been underscored by the recent flood of global expression analyses, which have confirmed that transcriptional co-regulation of genes that act together is the norm, not the exception. Moreover, many studies suggest that evolutionary change is driven in large part by modifications of transcriptional programs. An essential first step toward deciphering the transcriptional code is to determine the expression pattern of all genes. With this goal in mind, an international effort to develop a gene expression atlas of Arabidopsis has been underway since fall 2003. This project, dubbed AtGenExpress, is funded by the DFG, and will provide the Arabidopsis community with access to a large set of Affymetrix microarray data. As part of this collaboration, we have generated expression data from 80 biologicaly different samples in triplicate. Series 2: Base line experiment for pathogen infection. Growth and infection conditions: Seeds will be stratified for 3 days at 4Â°C and sown on soil. Plants are grown at 22Â°C under a 8/16 hour light/dark regime in Percival growth chambers. All pathogen treatments will be performed on leaves of 5-weeks old plants. Bacterial infiltrations will be performed with 10-8 cfu/ml in 10 mM MgCl2. 5x105 spores of Phytophthora infestans in water will be applied to leaf surfaces. LPS (100 Âµg/ml), HrpZ (1 ÂµM), NPP1 (2 ÂµM) or flagellin (1 ÂµM flg22) will be applied in water. Experiments should be performed in triplicate at the time points indicated below.
NASCARRAY121 Changes in Gene Expression in Brassica oleracea Shoots during Phosphate Starvation (Cross-species st 2004-07-19	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 4Â°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 22Â°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.
NASCARRAY122 AtGenExpress: Response to bacterial-(LPS, HrpZ, Flg22) and oomycete-(NPP1) derived elicitors 2004-07-09	AtGenExpress: A multinational coordinated effort to uncover the transcriptome of the multicellular model organism Arabidopsis thaliana (Hybridisations done at NASC) The activity of genes and their encoded products can be regulated in several ways, but transcription is the primary level, since all other modes of regulation (RNA splicing, RNA and protein stability, etc.) are dependent on a gene being transcribed in the first place. The importance of transcriptional regulation has been underscored by the recent flood of global expression analyses, which have confirmed that transcriptional co-regulation of genes that act together is the norm, not the exception. Moreover, many studies suggest that evolutionary change is driven in large part by modifications of transcriptional programs. An essential first step toward deciphering the transcriptional code is to determine the expression pattern of all genes. With this goal in mind, an international effort to develop a gene expression atlas of Arabidopsis has been underway since fall 2003. This project, dubbed AtGenExpress, is funded by the DFG, and will provide the Arabidopsis community with access to a large set of Affymetrix microarray data. As part of this collaboration, we have generated expression data from 80 biologicaly different samples in triplicate. Series 2: Base line experiment for pathogen infection. Growth and infection conditions: Seeds will be stratified for 3 days at 4Â°C and sown on soil. Plants are grown at 22Â°C under a 8/16 hour light/dark regime in Percival growth chambers. All pathogen treatments will be performed on leaves of 5-weeks old plants. Bacterial infiltrations will be performed with 10-8 cfu/ml in 10 mM MgCl2. 5x105 spores of Phytophthora infestans in water will be applied to leaf surfaces. LPS (100 Âµg/ml), HrpZ (1 ÂµM), NPP1 (2 ÂµM) or flagellin (1 ÂµM flg22) will be applied in water. Experiments should be performed in triplicate at the time points indicated below.
NASCARRAY123 AtGenExpress: Response to Phytophthora infestans 2004-07-09	AtGenExpress: A multinational coordinated effort to uncover the transcriptome of the multicellular model organism Arabidopsis thaliana (Hybridisations done at NASC) The activity of genes and their encoded products can be regulated in several ways, but transcription is the primary level, since all other modes of regulation (RNA splicing, RNA and protein stability, etc.) are dependent on a gene being transcribed in the first place. The importance of transcriptional regulation has been underscored by the recent flood of global expression analyses, which have confirmed that transcriptional co-regulation of genes that act together is the norm, not the exception. Moreover, many studies suggest that evolutionary change is driven in large part by modifications of transcriptional programs. An essential first step toward deciphering the transcriptional code is to determine the expression pattern of all genes. With this goal in mind, an international effort to develop a gene expression atlas of Arabidopsis has been underway since fall 2003. This project, dubbed AtGenExpress, is funded by the DFG, and will provide the Arabidopsis community with access to a large set of Affymetrix microarray data. As part of this collaboration, we have generated expression data from 80 biologicaly different samples in triplicate. Series 2: Base line experiment for pathogen infection. Growth and infection conditions: Seeds will be stratified for 3 days at 4Â°C and sown on soil. Plants are grown at 22Â°C under a 8/16 hour light/dark regime in Percival growth chambers. All pathogen treatments will be performed on leaves of 5-weeks old plants. Bacterial infiltrations will be performed with 10-8 cfu/ml in 10 mM MgCl2. 5x105 spores of Phytophthora infestans in water will be applied to leaf surfaces. LPS (100 Âµg/ml), HrpZ (1 ÂµM), NPP1 (2 ÂµM) or flagellin (1 ÂµM flg22) will be applied in water. Experiments should be performed in triplicate at the time points indicated below.
NASCARRAY124 AtGenExpress: Light treatments 2004-07-09	AtGenExpress: A multinational coordinated effort to uncover the transcriptome of the multicellular model organism Arabidopsis thaliana (Hybridisations done at NASC). The activity of genes and their encoded products can be regulated in several ways, but transcription is the primary level, since all other modes of regulation (RNA splicing, RNA and protein stability, etc.) are dependent on a gene being transcribed in the first place. The importance of transcriptional regulation has been underscored by the recent flood of global expression analyses, which have confirmed that transcriptional co-regulation of genes that act together is the norm, not the exception. Moreover, many studies suggest that evolutionary change is driven in large part by modifications of transcriptional programs. An essential first step toward deciphering the transcriptional code is to determine the expression pattern of all genes. With this goal in mind, an international effort to develop a gene expression atlas of Arabidopsis has been underway since fall 2003. This project, dubbed AtGenExpress, is funded by the DFG, and will provide the Arabidopsis community with access to a large set of Affymetrix microarray data. As part of this collaboration, we have generated expression data from 80 biologicaly different samples in triplicate. Series 1: Growth conditions: Sterilized seeds will be stratified at 4Â°C for 3 days, exposed to white light for 2 h to induce germination, and grown on MS agar plates (0.9 % agar) without sugar in total darkness for 4 days at 22Â°C. Seedlings will then be transferred to the light conditions described for each slide below for 1 h (to identify early induced genes) and 4 hrs (maximum expression of the first initial light response of most target genes), respectively. All light treatments will be done in parallel to minimize the number of dark controls. All samples will be done in triplicate and only with shoots.
NASCARRAY127 Comparative transcriptome analysis of wild type and two knockout alleles of At-CAMTA 2004-11-19	Calcium has been shown to be an important signalling molecule in the transduction of abiotic stress signals in Arabidopsis thaliana. Alteration of the calcium signature through the use of inhibitors has been shown to result in changes in the expression of certain downstream abiotic stress-induced genes. However, the communication of this signal through specific calcium-sensitive intermediate molecules has remained poorly elucidated. Various candidate molecules exist and including calcinurin, calcium-dependent protein kinases and calmodulin. Recently, a screen of an Arabidopsis thaliana cDNA expression library with calmodulin resulted in the isolation of the protein designated calmodulin-binding transcriptional activator or AtCAMTA that may function as one of these calcium-sensitive intermediate molecules. The Arabidopsis genome contains six CAMTA genes (AtCAMTA1-6), all of which are expressed in varying tissues throughout development. AtCAMTA1 was shown to function as a transcriptional activator through the expression of a chimeric protein consisting of the bacteria LexA protein fused to various segments of AtCAMTA expressed in yeast. In order to identify putative target genes of AtCAMTA1 in Arabidopsis we plan to analyse the transcriptome of AtCAMTA1 loss-of-function mutants. The gene expression pattern of two previously characterised alleles of AtCAMTA1 will be compared to wild type.
NASCARRAY130 Functional studies of a new Arabidopsis SH2 domain-containing gene 2004-11-17	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.
NASCARRAY132 Effect of CaM overexpression on Arabidopsis transcriptome 2005-03-16	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.
NASCARRAY133 The role of membrane fluidity in low temperature perception 2005-03-15	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.
NASCARRAY136 Expression patterns of genes induced by sugar accumulation during early leaf senescence 2005-04-04	The aim of our experiment is to analyse the expression pattern of genes induced by sugar accumulation during early leaf senescence.We have recently shown that senescence is induced by glucose in combination with low, but not with high nitrogen supply (Wingler et al. (2004), New Phytol, in press). We would like to use Affymetrix analysis to compare senescence-specific gene expression in plants grown on (i) low nitrogen without glucose (LNM), (ii) low nitrogen with 2% glucose (LNG) and (iii) high nitrogen with 2% glucose (HNG). Comparison of (i) and (ii) will allow us to determine the effect of glucose, while comparison of (ii) and (iii) will reveal effects of nitrogen supply. For this experiment, plants will be harvested on day 30, when first signs of senescence become visible on the LNG medium. All samples will be taken around mid-day. For each sample, 12 plants from 3 different petri dishes will be pooled. We are planning to analyse 3 independent samples per treatment, i.e. 9 samples in total. RNA will be extracted from whole leaf rosettes (without roots and inflorescences) using the RNeasy Plant Kit (Qiagen). At a later stage, we are also intending to compare gene expression in wild-type plants with expression in the abi5-1 mutant (Ws-2 background), which shows a delay in sugar-induced senescence (Wingler, Astrid, MarÃ¨s, Magali & Pourtau, Nathalie (2004) Spatial patterns and metabolic regulation of photosynthetic parameters during leaf senescence. New Phytologist 161 (3), 781-789). We have therefore chosen to use Ws-2 for the experiment proposed here. The experiment with abi5-1 will be conducted once we have obtained data on treatment-specific effects in Ws-2.
NASCARRAY137 AtGenExpress: Stress Treatments (Control plants) 2004-10-01	AtGenExpress: A multinational coordinated effort to uncover the transcriptome of the multicellular model organism Arabidopsis thaliana The activity of genes and their encoded products can be regulated in several ways, but transcription is the primary level, since all other modes of regulation (RNA splicing, RNA and protein stability, etc.) are dependent on a gene being transcribed in the first place. The importance of transcriptional regulation has been underscored by the recent flood of global expression analyses, which have confirmed that transcriptional co-regulation of genes that act together is the norm, not the exception. Moreover, many studies suggest that evolutionary change is driven in large part by modifications of transcriptional programs. An essential first step toward deciphering the transcriptional code is to determine the expression pattern of all genes. With this goal in mind, an international effort to develop a gene expression atlas of Arabidopsis has been underway since fall 2003. This project, dubbed AtGenExpress, is funded by the DFG, and will provide the Arabidopsis community with access to a large set of Affymetrix microarray data. As part of this collaboration, we have generated expression data from 80 biologicaly different samples in triplicate. Seeds of Arabidopsis thaliana Wild Type (col-0) were sown on rafts in Magenta boxes containing MS-Agar-media. After 2 days in the cold room (4Â°C, dark) the boxes were transferred to the long day chamber. Long day conditions were 16/8 hrs light/dark, 24Â°C, 50% humidity and 150 ÂµEinstein/cm2 sec light intensity. At day 11 the rafts were transferred in Magenta boxes containing MS-liquid-media. At day 16 stress treatment started at 3 hrs of light period; samples taken at 0.5, 1, 3, 6, 12, 24 h after treatment (in selected indicated cases 0.25h and 4.0h too), control samples include 0h; roots and shoots were prepared separately; all treatments and preparations were done on the same batch of seedlings in one place (Lab J.Kudla/Ulm, Germany) by coworkers from the indicated groups. AtGenExpress: Stress treatments Seeds of Arabidopsis thaliana Wilde Type (col-0) were sown on rafts in Magenta boxes containing MS-Agar-media. After 2 days in the cold room (4Â°C, dark) the boxes were transferred to the long day chamber. Long day conditions were 16/8 hrs light/dark, 24Â°C, 50% humidity and 150 ÂµEinstein/cm2 sec light intensity. At day 11 the rafts were transferred in Magenta boxes containing MS-liquid-media. At day 16 stress treatment started at 3 hrs of light period; samples taken at 0.5, 1, 3, 6, 12, 24 h after treatment (in selected indicated cases 0.25h and 4.0h too), control samples include 0h; roots and shoots were prepared separately; all treatments and preparations were done on the same batch of seedlings in one place (Lab J.Kudla/Ulm, Germany) by coworkers from the indicated groups.
NASCARRAY138 AtGenExpress: Stress Treatments (Cold stress) 2004-10-01	AtGenExpress: A multinational coordinated effort to uncover the transcriptome of the multicellular model organism Arabidopsis thaliana The activity of genes and their encoded products can be regulated in several ways, but transcription is the primary level, since all other modes of regulation (RNA splicing, RNA and protein stability, etc.) are dependent on a gene being transcribed in the first place. The importance of transcriptional regulation has been underscored by the recent flood of global expression analyses, which have confirmed that transcriptional co-regulation of genes that act together is the norm, not the exception. Moreover, many studies suggest that evolutionary change is driven in large part by modifications of transcriptional programs. An essential first step toward deciphering the transcriptional code is to determine the expression pattern of all genes. With this goal in mind, an international effort to develop a gene expression atlas of Arabidopsis has been underway since fall 2003. This project, dubbed AtGenExpress, is funded by the DFG, and will provide the Arabidopsis community with access to a large set of Affymetrix microarray data. As part of this collaboration, we have generated expression data from 80 biologicaly different samples in triplicate. Seeds of Arabidopsis thaliana Wilde Type (col-0) were sown on rafts in Magenta boxes containing MS-Agar-media. After 2 days in the cold room (4Â°C, dark) the boxes were transferred to the long day chamber. Long day conditions were 16/8 hrs light/dark, 24Â°C, 50% humidity and 150 ÂµEinstein/cm2 sec light intensity. At day 11 the rafts were transferred in Magenta boxes containing MS-liquid-media. At day 16 stress treatment started at 3 hrs of light period; samples taken at 0.5, 1, 3, 6, 12, 24 h after treatment (in selected indicated cases 0.25h and 4.0h too), control samples include 0h; roots and shoots were prepared separately; all treatments and preparations were done on the same batch of seedlings in one place (Lab J.Kudla/Ulm, Germany) by coworkers from the indicated groups.
NASCARRAY139 AtGenExpress: Stress Treatments (Osmotic stress) 2004-10-01	AtGenExpress: A multinational coordinated effort to uncover the transcriptome of the multicellular model organism Arabidopsis thaliana. The activity of genes and their encoded products can be regulated in several ways, but transcription is the primary level, since all other modes of regulation (RNA splicing, RNA and protein stability, etc.) are dependent on a gene being transcribed in the first place. The importance of transcriptional regulation has been underscored by the recent flood of global expression analyses, which have confirmed that transcriptional co-regulation of genes that act together is the norm, not the exception. Moreover, many studies suggest that evolutionary change is driven in large part by modifications of transcriptional programs. An essential first step toward deciphering the transcriptional code is to determine the expression pattern of all genes. With this goal in mind, an international effort to develop a gene expression atlas of Arabidopsis has been underway since fall 2003. This project, dubbed AtGenExpress, is funded by the DFG, and will provide the Arabidopsis community with access to a large set of Affymetrix microarray data. As part of this collaboration, we have generated expression data from 80 biologicaly different samples in triplicate. Seeds of Arabidopsis thaliana Wild Type (col-0) were sown on rafts in Magenta boxes containing MS-Agar-media. After 2 days in the cold room (4Â°C, dark) the boxes were transferred to the long day chamber. Long day conditions were 16/8 hrs light/dark, 24Â°C, 50% humidity and 150 uEinstein/cm2 sec light intensity. At day 11 the rafts were transferred in Magenta boxes containing MS-liquid-media. At day 16 stress treatment started at 3 hrs of light period; samples taken at 0.5, 1, 3, 6, 12, 24 h after treatment (in selected indicated cases 0.25h and 4.0h too), control samples include 0h; roots and shoots were prepared separately; all treatments and preparations were done on the same batch of seedlings in one place (Lab J.Kudla/Ulm, Germany) by coworkers from the indicated groups.
NASCARRAY140 AtGenExpress: Stress Treatments (Salt stress) 2004-10-01	AtGenExpress: A multinational coordinated effort to uncover the transcriptome of the multicellular model organism Arabidopsis thaliana The activity of genes and their encoded products can be regulated in several ways, but transcription is the primary level, since all other modes of regulation (RNA splicing, RNA and protein stability, etc.) are dependent on a gene being transcribed in the first place. The importance of transcriptional regulation has been underscored by the recent flood of global expression analyses, which have confirmed that transcriptional co-regulation of genes that act together is the norm, not the exception. Moreover, many studies suggest that evolutionary change is driven in large part by modifications of transcriptional programs. An essential first step toward deciphering the transcriptional code is to determine the expression pattern of all genes. With this goal in mind, an international effort to develop a gene expression atlas of Arabidopsis has been underway since fall 2003. This project, dubbed AtGenExpress, is funded by the DFG, and will provide the Arabidopsis community with access to a large set of Affymetrix microarray data. As part of this collaboration, we have generated expression data from 80 biologicaly different samples in triplicate. Seeds of Arabidopsis thaliana Wild Type (col-0) were sown on rafts in Magenta boxes containing MS-Agar-media. After 2 days in the cold room (4Â°C, dark) the boxes were transferred to the long day chamber. Long day conditions were 16/8 hrs light/dark, 24Â°C, 50% humidity and 150 ÂµEinstein/cm2 sec light intensity. At day 11 the rafts were transferred in Magenta boxes containing MS-liquid-media. At day 16 stress treatment started at 3 hrs of light period; samples taken at 0.5, 1, 3, 6, 12, 24 h after treatment (in selected indicated cases 0.25h and 4.0h too), control samples include 0h; roots and shoots were prepared separately; all treatments and preparations were done on the same batch of seedlings in one place (Lab J.Kudla/Ulm, Germany) by coworkers from the indicated groups.
NASCARRAY141 AtGenExpress: Stress Treatments (Drought stress) 2004-10-01	AtGenExpress: A multinational coordinated effort to uncover the transcriptome of the multicellular model organism Arabidopsis thaliana. The activity of genes and their encoded products can be regulated in several ways, but transcription is the primary level, since all other modes of regulation (RNA splicing, RNA and protein stability, etc.) are dependent on a gene being transcribed in the first place. The importance of transcriptional regulation has been underscored by the recent flood of global expression analyses, which have confirmed that transcriptional co-regulation of genes that act together is the norm, not the exception. Moreover, many studies suggest that evolutionary change is driven in large part by modifications of transcriptional programs. An essential first step toward deciphering the transcriptional code is to determine the expression pattern of all genes. With this goal in mind, an international effort to develop a gene expression atlas of Arabidopsis has been underway since fall 2003. This project, dubbed AtGenExpress, is funded by the DFG, and will provide the Arabidopsis community with access to a large set of Affymetrix microarray data. As part of this collaboration, we have generated expression data from 80 biologicaly different samples in triplicate. Seeds of Arabidopsis thaliana Wilde Type (col-0) were sown on rafts in Magenta boxes containing MS-Agar-media. After 2 days in the cold room (4Â°C, dark) the boxes were transferred to the long day chamber. Long day conditions were 16/8 hrs light/dark, 24Â°C, 50% humidity and 150 ÂµEinstein/cm2 sec light intensity. At day 11 the rafts were transferred in Magenta boxes containing MS-liquid-media. At day 16 stress treatment started at 3 hrs of light period; samples taken at 0.5, 1, 3, 6, 12, 24 h after treatment (in selected indicated cases 0.25h and 4.0h too), control samples include 0h; roots and shoots were prepared separately; all treatments and preparations were done on the same batch of seedlings in one place (Lab J.Kudla/Ulm, Germany) by coworkers from the indicated groups.
NASCARRAY142 AtGenExpress: Stress Treatments (Genotoxic stress) 2004-10-01	AtGenExpress: A multinational coordinated effort to uncover the transcriptome of the multicellular model organism Arabidopsis thaliana. The activity of genes and their encoded products can be regulated in several ways, but transcription is the primary level, since all other modes of regulation (RNA splicing, RNA and protein stability, etc.) are dependent on a gene being transcribed in the first place. The importance of transcriptional regulation has been underscored by the recent flood of global expression analyses, which have confirmed that transcriptional co-regulation of genes that act together is the norm, not the exception. Moreover, many studies suggest that evolutionary change is driven in large part by modifications of transcriptional programs. An essential first step toward deciphering the transcriptional code is to determine the expression pattern of all genes. With this goal in mind, an international effort to develop a gene expression atlas of Arabidopsis has been underway since fall 2003. This project, dubbed AtGenExpress, is funded by the DFG, and will provide the Arabidopsis community with access to a large set of Affymetrix microarray data. As part of this collaboration, we have generated expression data from 80 biologicaly different samples in triplicate. Seeds of Arabidopsis thaliana Wild Type (col-0) were sown on rafts in Magenta boxes containing MS-Agar-media. After 2 days in the cold room (4Â°C, dark) the boxes were transferred to the long day chamber. Long day conditions were 16/8 hrs light/dark, 24Â°C, 50% humidity and 150 ÂµEinstein/cm2 sec light intensity. At day 11 the rafts were transferred in Magenta boxes containing MS-liquid-media. At day 16 stress treatment started at 3 hrs of light period; samples taken at 0.5, 1, 3, 6, 12, 24 h after treatment (in selected indicated cases 0.25h and 4.0h too), control samples include 0h; roots and shoots were prepared separately; all treatments and preparations were done on the same batch of seedlings in one place (Lab J.Kudla/Ulm, Germany) by coworkers from the indicated groups.
NASCARRAY143 AtGenExpress: Stress Treatments (Oxidative stress) 2004-10-01	AtGenExpress: A multinational coordinated effort to uncover the transcriptome of the multicellular model organism Arabidopsis thaliana. The activity of genes and their encoded products can be regulated in several ways, but transcription is the primary level, since all other modes of regulation (RNA splicing, RNA and protein stability, etc.) are dependent on a gene being transcribed in the first place. The importance of transcriptional regulation has been underscored by the recent flood of global expression analyses, which have confirmed that transcriptional co-regulation of genes that act together is the norm, not the exception. Moreover, many studies suggest that evolutionary change is driven in large part by modifications of transcriptional programs. An essential first step toward deciphering the transcriptional code is to determine the expression pattern of all genes. With this goal in mind, an international effort to develop a gene expression atlas of Arabidopsis has been underway since fall 2003. This project, dubbed AtGenExpress, is funded by the DFG, and will provide the Arabidopsis community with access to a large set of Affymetrix microarray data. As part of this collaboration, we have generated expression data from 80 biologicaly different samples in triplicate. Seeds of Arabidopsis thaliana Wild Type (col-0) were sown on rafts in Magenta boxes containing MS-Agar-media. After 2 days in the cold room (4Â°C, dark) the boxes were transferred to the long day chamber. Long day conditions were 16/8 hrs light/dark, 24Â°C, 50% humidity and 150 ÂµEinstein/cm2 sec light intensity. At day 11 the rafts were transferred in Magenta boxes containing MS-liquid-media. At day 16 stress treatment started at 3 hrs of light period; samples taken at 0.5, 1, 3, 6, 12, 24 h after treatment (in selected indicated cases 0.25h and 4.0h too), control samples include 0h; roots and shoots were prepared separately; all treatments and preparations were done on the same batch of seedlings in one place (Lab J.Kudla/Ulm, Germany) by coworkers from the indicated groups.
NASCARRAY144 AtGenExpress: Stress Treatments (UV-B stress) 2004-10-01	AtGenExpress: A multinational coordinated effort to uncover the transcriptome of the multicellular model organism Arabidopsis thaliana. The activity of genes and their encoded products can be regulated in several ways, but transcription is the primary level, since all other modes of regulation (RNA splicing, RNA and protein stability, etc.) are dependent on a gene being transcribed in the first place. The importance of transcriptional regulation has been underscored by the recent flood of global expression analyses, which have confirmed that transcriptional co-regulation of genes that act together is the norm, not the exception. Moreover, many studies suggest that evolutionary change is driven in large part by modifications of transcriptional programs. An essential first step toward deciphering the transcriptional code is to determine the expression pattern of all genes. With this goal in mind, an international effort to develop a gene expression atlas of Arabidopsis has been underway since fall 2003. This project, dubbed AtGenExpress, is funded by the DFG, and will provide the Arabidopsis community with access to a large set of Affymetrix microarray data. As part of this collaboration, we have generated expression data from 80 biologicaly different samples in triplicate. Seeds of Arabidopsis thaliana Wild Type (col-0) were sown on rafts in Magenta boxes containing MS-Agar-media. After 2 days in the cold room (4Â°C, dark) the boxes were transferred to the long day chamber. Long day conditions were 16/8 hrs light/dark, 24Â°C, 50% humidity and 150 ÂµEinstein/cm2 sec light intensity. At day 11 the rafts were transferred in Magenta boxes containing MS-liquid-media. At day 16 stress treatment started at 3 hrs of light period; samples taken at 0.5, 1, 3, 6, 12, 24 h after treatment (in selected indicated cases 0.25h and 4.0h too), control samples include 0h; roots and shoots were prepared separately; all treatments and preparations were done on the same batch of seedlings in one place (Lab J.Kudla/Ulm, Germany) by coworkers from the indicated groups.
NASCARRAY145 AtGenExpress: Stress Treatments (Wounding stress) 2004-10-01	AtGenExpress: A multinational coordinated effort to uncover the transcriptome of the multicellular model organism Arabidopsis thaliana. The activity of genes and their encoded products can be regulated in several ways, but transcription is the primary level, since all other modes of regulation (RNA splicing, RNA and protein stability, etc.) are dependent on a gene being transcribed in the first place. The importance of transcriptional regulation has been underscored by the recent flood of global expression analyses, which have confirmed that transcriptional co-regulation of genes that act together is the norm, not the exception. Moreover, many studies suggest that evolutionary change is driven in large part by modifications of transcriptional programs. An essential first step toward deciphering the transcriptional code is to determine the expression pattern of all genes. With this goal in mind, an international effort to develop a gene expression atlas of Arabidopsis has been underway since fall 2003. This project, dubbed AtGenExpress, is funded by the DFG, and will provide the Arabidopsis community with access to a large set of Affymetrix microarray data. As part of this collaboration, we have generated expression data from 80 biologicaly different samples in triplicate. Seeds of Arabidopsis thaliana Wild Type (col-0) were sown on rafts in Magenta boxes containing MS-Agar-media. After 2 days in the cold room (4Â°C, dark) the boxes were transferred to the long day chamber. Long day conditions were 16/8 hrs light/dark, 24Â°C, 50% humidity and 150 ÂµEinstein/cm2 sec light intensity. At day 11 the rafts were transferred in Magenta boxes containing MS-liquid-media. At day 16 stress treatment started at 3 hrs of light period; samples taken at 0.5, 1, 3, 6, 12, 24 h after treatment (in selected indicated cases 0.25h and 4.0h too), control samples include 0h; roots and shoots were prepared separately; all treatments and preparations were done on the same batch of seedlings in one place (Lab J.Kudla/Ulm, Germany) by coworkers from the indicated groups.
NASCARRAY146 AtGenExpress: Stress Treatments (Heat stress) 2004-10-01	AtGenExpress: A multinational coordinated effort to uncover the transcriptome of the multicellular model organism Arabidopsis thaliana. The activity of genes and their encoded products can be regulated in several ways, but transcription is the primary level, since all other modes of regulation (RNA splicing, RNA and protein stability, etc.) are dependent on a gene being transcribed in the first place. The importance of transcriptional regulation has been underscored by the recent flood of global expression analyses, which have confirmed that transcriptional co-regulation of genes that act together is the norm, not the exception. Moreover, many studies suggest that evolutionary change is driven in large part by modifications of transcriptional programs. An essential first step toward deciphering the transcriptional code is to determine the expression pattern of all genes. With this goal in mind, an international effort to develop a gene expression atlas of Arabidopsis has been underway since fall 2003. This project, dubbed AtGenExpress, is funded by the DFG, and will provide the Arabidopsis community with access to a large set of Affymetrix microarray data. As part of this collaboration, we have generated expression data from 80 biologicaly different samples in triplicate. Seeds of Arabidopsis thaliana Wild Type (col-0) were sown on rafts in Magenta boxes containing MS-Agar-media. After 2 days in the cold room (4Â°C, dark) the boxes were transferred to the long day chamber. Long day conditions were 16/8 hrs light/dark, 24Â°C, 50% humidity and 150 ÂµEinstein/cm2 sec light intensity. At day 11 the rafts were transferred in Magenta boxes containing MS-liquid-media. At day 16 stress treatment started at 3 hrs of light period; samples taken at 0.5, 1, 3, 6, 12, 24 h after treatment (in selected indicated cases 0.25h and 4.0h too), control samples include 0h; roots and shoots were prepared separately; all treatments and preparations were done on the same batch of seedlings in one place (Lab J.Kudla/Ulm, Germany) by coworkers from the indicated groups.
NASCARRAY147 Transcriptomic studies to understand the circadian clock temperature buffering mechanism 2005-07-21	The circadian clock temporally orchestrates many physiological and developmental processes. The accuracy with which it co-ordinates these events with the daily light/dark cycle gives an organism a competitive advantage. For a clock to remain accurate it must be buffered against unpredictable fluctuations in temperature. We have identified the gene GI as playing a role in this buffering process. To understand the underlying temperature buffering mechanism we aim to investigate, in gi and WT changes in the transcriptome.The Arabidopsis (wt (WS) and gi11 (WS)) seeds will be sterilised and then plated on MS 3% sucrose plates. The seeds will be cold treated for 2 days and then germinated and grown under 12h light 12h dark cycles at 22Â°C for 7 days. On the seventh day plants will be transferred to constant light at a range of temperatures (12Â°C, 17Â°C, 22Â°C and 27Â°C). RNA will be isolated over one circadian cycle on the third day of adaptation and then pooled, (prior to pooling the RNA will be analysed on a denaturing gel and using real time PCR and quantified using Quant-itÃ¤ RNA assay). The pooling over one circadian cycle is essential when you are comparing WT to a mutant with an altered period. Using this approach we aim to identify temperature-regulated transcripts and transcripts that are mis-regulated in gi at specific temperatures.This transcriptomic study is funded by the BBSRC.
NASCARRAY149 AtGenExpress: Developmental series (seedlings and whole plants) 2004-07-09	AtGenExpress: A multinational coordinated effort to uncover the transcriptome of the multicellular model organism Arabidopsis thaliana. The activity of genes and their encoded products can be regulated in several ways, but transcription is the primary level, since all other modes of regulation (RNA splicing, RNA and protein stability, etc.) are dependent on a gene being transcribed in the first place. The importance of transcriptional regulation has been underscored by the recent flood of global expression analyses, which have confirmed that transcriptional co-regulation of genes that act together is the norm, not the exception. Moreover, many studies suggest that evolutionary change is driven in large part by modifications of transcriptional programs. An essential first step toward deciphering the transcriptional code is to determine the expression pattern of all genes. With this goal in mind, an international effort to develop a gene expression atlas of Arabidopsis has been underway since fall 2003. This project, dubbed AtGenExpress, is funded by the DFG, and will provide the Arabidopsis community with access to a large set of Affymetrix microarray data. As part of this collaboration, we have generated expression data from 80 biologicaly different samples in triplicate. The focus of this data set is on different tissues and different developmental stages in wild type Columbia (Col-0) and various mutants.
NASCARRAY150 AtGenExpress: Developmental series (leaves) 2004-07-09	AtGenExpress: A multinational coordinated effort to uncover the transcriptome of the multicellular model organism Arabidopsis thaliana. The activity of genes and their encoded products can be regulated in several ways, but transcription is the primary level, since all other modes of regulation (RNA splicing, RNA and protein stability, etc.) are dependent on a gene being transcribed in the first place. The importance of transcriptional regulation has been underscored by the recent flood of global expression analyses, which have confirmed that transcriptional co-regulation of genes that act together is the norm, not the exception. Moreover, many studies suggest that evolutionary change is driven in large part by modifications of transcriptional programs. An essential first step toward deciphering the transcriptional code is to determine the expression pattern of all genes. With this goal in mind, an international effort to develop a gene expression atlas of Arabidopsis has been underway since fall 2003. This project, dubbed AtGenExpress, is funded by the DFG, and will provide the Arabidopsis community with access to a large set of Affymetrix microarray data. As part of this collaboration, we have generated expression data from 80 biologicaly different samples in triplicate. The focus of this data set is on different tissues and different developmental stages in wild type Columbia (Col-0) and various mutants.
NASCARRAY151 AtGenExpress: Developmental series (roots) 2004-07-09	AtGenExpress: A multinational coordinated effort to uncover the transcriptome of the multicellular model organism Arabidopsis thaliana. The activity of genes and their encoded products can be regulated in several ways, but transcription is the primary level, since all other modes of regulation (RNA splicing, RNA and protein stability, etc.) are dependent on a gene being transcribed in the first place. The importance of transcriptional regulation has been underscored by the recent flood of global expression analyses, which have confirmed that transcriptional co-regulation of genes that act together is the norm, not the exception. Moreover, many studies suggest that evolutionary change is driven in large part by modifications of transcriptional programs. An essential first step toward deciphering the transcriptional code is to determine the expression pattern of all genes. With this goal in mind, an international effort to develop a gene expression atlas of Arabidopsis has been underway since fall 2003. This project, dubbed AtGenExpress, is funded by the DFG, and will provide the Arabidopsis community with access to a large set of Affymetrix microarray data. As part of this collaboration, we have generated expression data from 80 biologicaly different samples in triplicate. The focus of this data set is on different tissues and different developmental stages in wild type Columbia (Col-0) and various mutants.
NASCARRAY152 AtGenExpress: Developmental series (flowers and pollen) 2004-07-09	AtGenExpress: A multinational coordinated effort to uncover the transcriptome of the multicellular model organism Arabidopsis thaliana. The activity of genes and their encoded products can be regulated in several ways, but transcription is the primary level, since all other modes of regulation (RNA splicing, RNA and protein stability, etc.) are dependent on a gene being transcribed in the first place. The importance of transcriptional regulation has been underscored by the recent flood of global expression analyses, which have confirmed that transcriptional co-regulation of genes that act together is the norm, not the exception. Moreover, many studies suggest that evolutionary change is driven in large part by modifications of transcriptional programs. An essential first step toward deciphering the transcriptional code is to determine the expression pattern of all genes. With this goal in mind, an international effort to develop a gene expression atlas of Arabidopsis has been underway since fall 2003. This project, dubbed AtGenExpress, is funded by the DFG, and will provide the Arabidopsis community with access to a large set of Affymetrix microarray data. As part of this collaboration, we have generated expression data from 80 biologicaly different samples in triplicate. The focus of this data set is on different tissues and different developmental stages in wild type Columbia (Col-0) and various mutants.
NASCARRAY153 AtGenExpress: Developmental series (shoots and stems) 2004-07-09	AtGenExpress: A multinational coordinated effort to uncover the transcriptome of the multicellular model organism Arabidopsis thaliana. The activity of genes and their encoded products can be regulated in several ways, but transcription is the primary level, since all other modes of regulation (RNA splicing, RNA and protein stability, etc.) are dependent on a gene being transcribed in the first place. The importance of transcriptional regulation has been underscored by the recent flood of global expression analyses, which have confirmed that transcriptional co-regulation of genes that act together is the norm, not the exception. Moreover, many studies suggest that evolutionary change is driven in large part by modifications of transcriptional programs. An essential first step toward deciphering the transcriptional code is to determine the expression pattern of all genes. With this goal in mind, an international effort to develop a gene expression atlas of Arabidopsis has been underway since fall 2003. This project, dubbed AtGenExpress, is funded by the DFG, and will provide the Arabidopsis community with access to a large set of Affymetrix microarray data. As part of this collaboration, we have generated expression data from 80 biologicaly different samples in triplicate. The focus of this data set is on different tissues and different developmental stages in wild type Columbia (Col-0) and various mutants.
NASCARRAY154 AtGenExpress: Developmental series (siliques and seeds) 2004-07-09	AtGenExpress: A multinational coordinated effort to uncover the transcriptome of the multicellular model organism Arabidopsis thaliana. The activity of genes and their encoded products can be regulated in several ways, but transcription is the primary level, since all other modes of regulation (RNA splicing, RNA and protein stability, etc.) are dependent on a gene being transcribed in the first place. The importance of transcriptional regulation has been underscored by the recent flood of global expression analyses, which have confirmed that transcriptional co-regulation of genes that act together is the norm, not the exception. Moreover, many studies suggest that evolutionary change is driven in large part by modifications of transcriptional programs. An essential first step toward deciphering the transcriptional code is to determine the expression pattern of all genes. With this goal in mind, an international effort to develop a gene expression atlas of Arabidopsis has been underway since fall 2003. This project, dubbed AtGenExpress, is funded by the DFG, and will provide the Arabidopsis community with access to a large set of Affymetrix microarray data. As part of this collaboration, we have generated expression data from 80 biologicaly different samples in triplicate. The focus of this data set is on different tissues and different developmental stages in wild type Columbia (Col-0) and various mutants.
NASCARRAY155 AtGenExpress: Developmental series (mutants and other ecotypes) 2004-07-09	AtGenExpress: A multinational coordinated effort to uncover the transcriptome of the multicellular model organism Arabidopsis thaliana. The activity of genes and their encoded products can be regulated in several ways, but transcription is the primary level, since all other modes of regulation (RNA splicing, RNA and protein stability, etc.) are dependent on a gene being transcribed in the first place. The importance of transcriptional regulation has been underscored by the recent flood of global expression analyses, which have confirmed that transcriptional co-regulation of genes that act together is the norm, not the exception. Moreover, many studies suggest that evolutionary change is driven in large part by modifications of transcriptional programs. An essential first step toward deciphering the transcriptional code is to determine the expression pattern of all genes. With this goal in mind, an international effort to develop a gene expression atlas of Arabidopsis has been underway since fall 2003. This project, dubbed AtGenExpress, is funded by the DFG, and will provide the Arabidopsis community with access to a large set of Affymetrix microarray data. As part of this collaboration, we have generated expression data from 80 biologicaly different samples in triplicate. The focus of this data set is on different tissues and different developmental stages in wild type Columbia (Col-0) and various mutants.
NASCARRAY156 Systemic signalling of irradiance and CO2 concentration in Arabidopsis (Treatment 1: Ambient CO2 and 2004-10-11	We were awarded a BBSRC grant about a year ago to undertake some affymetrix gene chip profiling of light and CO2 systemic signalling in Arabidopsis. The design of the proposed experiment is given below and the appropriate funding has been provided by the BBSRC. The aim of the project is to identify the temporal profile of those genes that respond to light and CO2 systemic signals in developing leaves. Moreover, as thes two signals have opposing effects on leaf development to ascertain whether they involve similar or parallel signalling pathways. The experiment is to examine the effect of exposing mature leaves to high CO2 or low light or both on the gene expression profile of developing leaves. We already have data for maize that changes in gene expression profile occur within 4h and that there are a variety of temporal responses that differ between individual gene transcripts. We have also demonstrated that Arabidopsis leaf development is altered by these systemmic signals and that lesions in the jasmonate and ethylene signalling pathways block these responses. Our experimental design is shown below: We have 4 treatments and 7 timepoints. (0, 2, 4, 12, 24, 48, 96 h) We would sample from 5 individual plants that would be pooled for each RNA preparation. This would require 28 chips and this would include extra replication of the 0 time-point control (deemed by many as nessary). Experimental details: All plants were germinated for 7 days under the following conditions: Humax multi-purpose compost, ambient carbon dioxide (370 ppm) and ambient light (250 Âµmol/m/s), constant temperature of 20Â°C and a 10 h photoperiod (8 am until 6 pm). After a week the the seedlings were potted up into 104-cell plug trays for a further 2 weeks and then potted up into 10 cm pots and the bottom part of the signalling cuvette system attached (see Lake et al., Nature 10th May 2001 Vol. 411, pp 154). Twenty four, 4 week old plants, then had the top part of the signalling system attached, trapping leaf insertions 5-13. Humidified, ambient air was passed through them at 500 mls/min via an oil-free air compressor. The three target leaves (19-21) were then marked with non-toxic, acrylic paint. After a 24 h period (the plants were sealed into the cuvettes from 10 am until 10am) of adjustment, the experiment was started by harvesting the target leaves from 4 plants and immediately freezing the tissue in liquid nitrogen to give the 0 h sample before RNA extraction. The remaining 20 plants were divided into 4 groups of five and given one of the following treatments: Ambient carbon dioxide/ambient light (Control) (A) Elevated carbon dioxide (750 ppm)/ambient light (E) Ambient carbon dioxide/low light (50 Âµmol/m/s) (AS) Elevated carbon dioxide/low light (ES) - For the Elevated CO2, elevated CO2 was pumped in using a CT room next door set to same temperature but with a CO2 cylinder inside and the same pump as used in the ambient room to supply the elevated CO2 laden, humidified air into the signalling room using rubber tubing. - Shade treatment consisted of neutral density filter (Cat. 210 0.6ND, Lee Filters) that had a hole cut in the middle to allow the middle developing leaves to grow through. A timecourse of 2, 4, 12, 24, 48 and 96 h were carried out each using a batch of 24 plants. This whole process was repeated with another batch of 24 plants at the same developmental stage to give a 2, 4, 12, 24, 48 and 96 hour sample from each of the four treatments. The whole timecourse was then repeated 4 times. For the mature leaves: We had 8 chips left over so we devised this little experiment to assess the gene changes that were occurring in the enclosed, treated, mature leaves that were signalling the environment to the young developing leaves.
NASCARRAY157 Systemic signalling of irradiance and CO2 concentration in Arabidopsis (Treatment 2: Elevated CO2 an 2004-10-11	We were awarded a BBSRC grant about a year ago to undertake some affymetrix gene chip profiling of light and CO2 systemic signalling in Arabidopsis. The design of the proposed experiment is given below and the appropriate funding has been provided by the BBSRC. The aim of the project is to identify the temporal profile of those genes that respond to light and CO2 systemic signals in developing leaves. Moreover, as thes two signals have opposing effects on leaf development to ascertain whether they involve similar or parallel signalling pathways. The experiment is to examine the effect of exposing mature leaves to high CO2 or low light or both on the gene expression profile of developing leaves. We already have data for maize that changes in gene expression profile occur within 4h and that there are a variety of temporal responses that differ between individual gene transcripts. We have also demonstrated that Arabidopsis leaf development is altered by these systemmic signals and that lesions in the jasmonate and ethylene signalling pathways block these responses. Our experimental design is shown below: We have 4 treatments and 7 timepoints. (0, 2, 4, 12, 24, 48, 96 h) We would sample from 5 individual plants that would be pooled for each RNA preparation. This would require 28 chips and this would include extra replication of the 0 time-point control (deemed by many as nessary). Experimental details: All plants were germinated for 7 days under the following conditions: Humax multi-purpose compost, ambient carbon dioxide (370 ppm) and ambient light (250 Âµmol/m/s), constant temperature of 20Â°C and a 10 h photoperiod (8 am until 6 pm). After a week the the seedlings were potted up into 104-cell plug trays for a further 2 weeks and then potted up into 10 cm pots and the bottom part of the signalling cuvette system attached (see Lake et al., Nature 10th May 2001 Vol. 411, pp 154). Twenty four, 4 week old plants, then had the top part of the signalling system attached, trapping leaf insertions 5-13. Humidified, ambient air was passed through them at 500 mls/min via an oil-free air compressor. The three target leaves (19-21) were then marked with non-toxic, acrylic paint. After a 24 h period (the plants were sealed into the cuvettes from 10 am until 10am) of adjustment, the experiment was started by harvesting the target leaves from 4 plants and immediately freezing the tissue in liquid nitrogen to give the 0 h sample before RNA extraction. The remaining 20 plants were divided into 4 groups of five and given one of the following treatments: Ambient carbon dioxide/ambient light (Control) (A) Elevated carbon dioxide (750 ppm)/ambient light (E) Ambient carbon dioxide/low light (50 Âµmol/m/s) (AS) Elevated carbon dioxide/low light (ES) - For the Elevated CO2, elevated CO2 was pumped in using a CT room next door set to same temperature but with a CO2 cylinder inside and the same pump as used in the ambient room to supply the elevated CO2 laden, humidified air into the signalling room using rubber tubing. - Shade treatment consisted of neutral density filter (Cat. 210 0.6ND, Lee Filters) that had a hole cut in the middle to allow the middle developing leaves to grow through. A timecourse of 2, 4, 12, 24, 48 and 96 h were carried out each using a batch of 24 plants. This whole process was repeated with another batch of 24 plants at the same developmental stage to give a 2, 4, 12, 24, 48 and 96 hour sample from each of the four treatments. The whole timecourse was then repeated 4 times. For the mature leaves: We had 8 chips left over so we devised this little experiment to assess the gene changes that were occurring in the enclosed, treated, mature leaves that were signalling the environment to the young developing leaves.
NASCARRAY158 Systemic signalling of irradiance and CO2 concentration in Arabidopsis (Treatment 3: Ambient CO2 and 2004-10-11	We were awarded a BBSRC grant about a year ago to undertake some affymetrix gene chip profiling of light and CO2 systemic signalling in Arabidopsis. The design of the proposed experiment is given below and the appropriate funding has been provided by the BBSRC. The aim of the project is to identify the temporal profile of those genes that respond to light and CO2 systemic signals in developing leaves. Moreover, as thes two signals have opposing effects on leaf development to ascertain whether they involve similar or parallel signalling pathways. The experiment is to examine the effect of exposing mature leaves to high CO2 or low light or both on the gene expression profile of developing leaves. We already have data for maize that changes in gene expression profile occur within 4h and that there are a variety of temporal responses that differ between individual gene transcripts. We have also demonstrated that Arabidopsis leaf development is altered by these systemmic signals and that lesions in the jasmonate and ethylene signalling pathways block these responses. Our experimental design is shown below: We have 4 treatments and 7 timepoints. (0, 2, 4, 12, 24, 48, 96 h) We would sample from 5 individual plants that would be pooled for each RNA preparation. This would require 28 chips and this would include extra replication of the 0 time-point control (deemed by many as nessary). Experimental details: All plants were germinated for 7 days under the following conditions: Humax multi-purpose compost, ambient carbon dioxide (370 ppm) and ambient light (250 Âµmol/m/s), constant temperature of 20Â°C and a 10 h photoperiod (8 am until 6 pm). After a week the the seedlings were potted up into 104-cell plug trays for a further 2 weeks and then potted up into 10 cm pots and the bottom part of the signalling cuvette system attached (see Lake et al., Nature 10th May 2001 Vol. 411, pp 154). Twenty four, 4 week old plants, then had the top part of the signalling system attached, trapping leaf insertions 5-13. Humidified, ambient air was passed through them at 500 mls/min via an oil-free air compressor. The three target leaves (19-21) were then marked with non-toxic, acrylic paint. After a 24 h period (the plants were sealed into the cuvettes from 10 am until 10am) of adjustment, the experiment was started by harvesting the target leaves from 4 plants and immediately freezing the tissue in liquid nitrogen to give the 0 h sample before RNA extraction. The remaining 20 plants were divided into 4 groups of five and given one of the following treatments: Ambient carbon dioxide/ambient light (Control) (A) Elevated carbon dioxide (750 ppm)/ambient light (E) Ambient carbon dioxide/low light (50 Âµmol/m/s) (AS) Elevated carbon dioxide/low light (ES) - For the Elevated CO2, elevated CO2 was pumped in using a CT room next door set to same temperature but with a CO2 cylinder inside and the same pump as used in the ambient room to supply the elevated CO2 laden, humidified air into the signalling room using rubber tubing. - Shade treatment consisted of neutral density filter (Cat. 210 0.6ND, Lee Filters) that had a hole cut in the middle to allow the middle developing leaves to grow through. A timecourse of 2, 4, 12, 24, 48 and 96 h were carried out each using a batch of 24 plants. This whole process was repeated with another batch of 24 plants at the same developmental stage to give a 2, 4, 12, 24, 48 and 96 hour sample from each of the four treatments. The whole timecourse was then repeated 4 times. For the mature leaves: We had 8 chips left over so we devised this little experiment to assess the gene changes that were occurring in the enclosed, treated, mature leaves that were signalling the environment to the young developing leaves.
NASCARRAY159 Systemic signalling of irradiance and CO2 concentration in Arabidopsis (Treatment 4: Elevated CO2 an 2004-10-11	We were awarded a BBSRC grant about a year ago to undertake some affymetrix gene chip profiling of light and CO2 systemic signalling in Arabidopsis. The design of the proposed experiment is given below and the appropriate funding has been provided by the BBSRC. The aim of the project is to identify the temporal profile of those genes that respond to light and CO2 systemic signals in developing leaves. Moreover, as thes two signals have opposing effects on leaf development to ascertain whether they involve similar or parallel signalling pathways. The experiment is to examine the effect of exposing mature leaves to high CO2 or low light or both on the gene expression profile of developing leaves. We already have data for maize that changes in gene expression profile occur within 4h and that there are a variety of temporal responses that differ between individual gene transcripts. We have also demonstrated that Arabidopsis leaf development is altered by these systemmic signals and that lesions in the jasmonate and ethylene signalling pathways block these responses. Our experimental design is shown below: We have 4 treatments and 7 timepoints. (0, 2, 4, 12, 24, 48, 96 h) We would sample from 5 individual plants that would be pooled for each RNA preparation. This would require 28 chips and this would include extra replication of the 0 time-point control (deemed by many as nessary). Experimental details: All plants were germinated for 7 days under the following conditions: Humax multi-purpose compost, ambient carbon dioxide (370 ppm) and ambient light (250 Âµmol/m/s), constant temperature of 20Â°C and a 10 h photoperiod (8 am until 6 pm). After a week the the seedlings were potted up into 104-cell plug trays for a further 2 weeks and then potted up into 10 cm pots and the bottom part of the signalling cuvette system attached (see Lake et al., Nature 10th May 2001 Vol. 411, pp 154). Twenty four, 4 week old plants, then had the top part of the signalling system attached, trapping leaf insertions 5-13. Humidified, ambient air was passed through them at 500 mls/min via an oil-free air compressor. The three target leaves (19-21) were then marked with non-toxic, acrylic paint. After a 24 h period (the plants were sealed into the cuvettes from 10 am until 10am) of adjustment, the experiment was started by harvesting the target leaves from 4 plants and immediately freezing the tissue in liquid nitrogen to give the 0 h sample before RNA extraction. The remaining 20 plants were divided into 4 groups of five and given one of the following treatments: Ambient carbon dioxide/ambient light (Control) (A) Elevated carbon dioxide (750 ppm)/ambient light (E) Ambient carbon dioxide/low light (50 Âµmol/m/s) (AS) Elevated carbon dioxide/low light (ES) - For the Elevated CO2, elevated CO2 was pumped in using a CT room next door set to same temperature but with a CO2 cylinder inside and the same pump as used in the ambient room to supply the elevated CO2 laden, humidified air into the signalling room using rubber tubing. - Shade treatment consisted of neutral density filter (Cat. 210 0.6ND, Lee Filters) that had a hole cut in the middle to allow the middle developing leaves to grow through. A timecourse of 2, 4, 12, 24, 48 and 96 h were carried out each using a batch of 24 plants. This whole process was repeated with another batch of 24 plants at the same developmental stage to give a 2, 4, 12, 24, 48 and 96 hour sample from each of the four treatments. The whole timecourse was then repeated 4 times. For the mature leaves: We had 8 chips left over so we devised this little experiment to assess the gene changes that were occurring in the enclosed, treated, mature leaves that were signalling the environment to the young developing leaves.
NASCARRAY160 Systemic signalling of irradiance and CO2 concentration in Arabidopsis (Controls) 2004-10-11	We were awarded a BBSRC grant about a year ago to undertake some affymetrix gene chip profiling of light and CO2 systemic signalling in Arabidopsis. The design of the proposed experiment is given below and the appropriate funding has been provided by the BBSRC. The aim of the project is to identify the temporal profile of those genes that respond to light and CO2 systemic signals in developing leaves. Moreover, as thes two signals have opposing effects on leaf development to ascertain whether they involve similar or parallel signalling pathways. The experiment is to examine the effect of exposing mature leaves to high CO2 or low light or both on the gene expression profile of developing leaves. We already have data for maize that changes in gene expression profile occur within 4h and that there are a variety of temporal responses that differ between individual gene transcripts. We have also demonstrated that Arabidopsis leaf development is altered by these systemmic signals and that lesions in the jasmonate and ethylene signalling pathways block these responses. Our experimental design is shown below: We have 4 treatments and 7 timepoints. (0, 2, 4, 12, 24, 48, 96 h) We would sample from 5 individual plants that would be pooled for each RNA preparation. This would require 28 chips and this would include extra replication of the 0 time-point control (deemed by many as nessary). Experimental details: All plants were germinated for 7 days under the following conditions: Humax multi-purpose compost, ambient carbon dioxide (370 ppm) and ambient light (250 Âµmol/m/s), constant temperature of 20Â°C and a 10 h photoperiod (8 am until 6 pm). After a week the the seedlings were potted up into 104-cell plug trays for a further 2 weeks and then potted up into 10 cm pots and the bottom part of the signalling cuvette system attached (see Lake et al., Nature 10th May 2001 Vol. 411, pp 154). Twenty four, 4 week old plants, then had the top part of the signalling system attached, trapping leaf insertions 5-13. Humidified, ambient air was passed through them at 500 mls/min via an oil-free air compressor. The three target leaves (19-21) were then marked with non-toxic, acrylic paint. After a 24 h period (the plants were sealed into the cuvettes from 10 am until 10am) of adjustment, the experiment was started by harvesting the target leaves from 4 plants and immediately freezing the tissue in liquid nitrogen to give the 0 h sample before RNA extraction. The remaining 20 plants were divided into 4 groups of five and given one of the following treatments: Ambient carbon dioxide/ambient light (Control) (A) Elevated carbon dioxide (750 ppm)/ambient light (E) Ambient carbon dioxide/low light (50 Âµmol/m/s) (AS) Elevated carbon dioxide/low light (ES) - For the Elevated CO2, elevated CO2 was pumped in using a CT room next door set to same temperature but with a CO2 cylinder inside and the same pump as used in the ambient room to supply the elevated CO2 laden, humidified air into the signalling room using rubber tubing. - Shade treatment consisted of neutral density filter (Cat. 210 0.6ND, Lee Filters) that had a hole cut in the middle to allow the middle developing leaves to grow through. A timecourse of 2, 4, 12, 24, 48 and 96 h were carried out each using a batch of 24 plants. This whole process was repeated with another batch of 24 plants at the same developmental stage to give a 2, 4, 12, 24, 48 and 96 hour sample from each of the four treatments. The whole timecourse was then repeated 4 times. For the mature leaves: We had 8 chips left over so we devised this little experiment to assess the gene changes that were occurring in the enclosed, treated, mature leaves that were signalling the environment to the young developing leaves.
NASCARRAY161 Environmental Genomics of calcicole-calcifuge physiology 2011-10-20	Calcareous grasslands are among the most species-rich plant communities in Europe. Huge losses among these grasslands and their continuing vulnerability to either agricultural improvement or neglect have focused attention on the need for conservation of their biodiversity. In addition, the potential impact of climatic stresses on the structure, composition and function of calcicolous grassland ecosystems is a topic of concern. A clear understanding of the molecular mechanisms that enable calcicole species to thrive on calcareous soils is therefore essential to enable us to predict how these plant communities and their constituent species will be affected by environmental change and how the biodiversity of these ecosystems can be sustained. We are testing the hypothesis that adaptation to a calcareous environment reflects altered patterns of gene expression in Arabidopsis thaliana and related Arabis species. We have employed transcriptome analysis using the NASC Affymetrix service to identify a cohort of genes that are differentially expressed (>2-fold increase/decrease in expression) between non-calcicolous (Col-4) and the calcicolous (Cal-0) ecotypes of A. thaliana at low (1mM)and high (12.5mM)concentrations of rhizospheric calcium. We would now like to compare expression profiles of further 2 ecotypes of Arabidopsis, collected from 2 different locations in Britain, firstly from area (W1), an of low soil pH, Penicuik near Edinburgh and secondly from area (W2), an area of high soil pH, Elland near Leeds. We have collected seed from these plants and have grown them under the same laboratory conditions of low and high rhizospheric calcium described above.Plants were grown in controlled environment growth cabinets on sterile sand and were watered daily with 0.5 X Long Ashton Nutrient solution containing either 1 mM or 12.5 mM CaCl2. Plants were grown at 10 hours of light (160 PAR) and 55% humidity to growth stage 3.90 (full rossette), which occurred after approximately 30 days after the begining of treatment. RNA was extracted from 10 pooled plants for each sample using Qiagen RNEasy kits.
NASCARRAY162 Growth promotion of Arabidopsis by Trichoderma hamatum 2005-06-02	Trichoderma spp. are ubiquitous soil-borne ascomycetous fungi that have been used widely in the biological control of plant diseases. Certain strains also exhibit growth promotion of plants, but the mechanism for this phenomenon has not yet been elucidated. We have recently identified an isolate of T. hamatum that causes significant increases in the development of Arabidopsis root systems. The aim of this work is to elucidate the mechanism of root growth promotion. One approach is to analyse the transcriptome of plants grown in the presence and absence of the fungus.
NASCARRAY163 UV-B response of Arabidopsis 2004-08-23	The UV-B portion of the solar radiation acts as an environmental signal for which plants have evolved specific and sensitive UV-B perception systems. The UV-B-specific UV RESPONSE LOCUS 8 (UVR8) and the multifunctional E3 ubiquitin ligase CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) are key regulators of the UV-B response. We show here that uvr8 null mutants are deficient in UV-B-induced photomorphogenesis and hypersensitive to UV-B stress, whereas overexpression of UVR8 results in enhanced UV-B photomorphogenesis, acclimation and tolerance to UV-B stress. By using sun simulators, we provide evidence at the physiological level that UV-B acclimation mediated by the UV-B-specific photoregulatory pathway is indeed required for survival in sunlight. At the molecular level we demonstrate that the wild type but not the mutant UVR8 and COP1 proteins directly interact in a UV-B-dependent, rapid manner in planta. These data collectively suggest that UV-B-specific interaction of COP1 and UVR8 in the nucleus is a very early step in signalling and responsible for the plantâ€™s coordinated response to UV-B ensuring UV-B acclimation and protection in the natural environment.
NASCARRAY166 Peroxisomal mdh mutant 2005-07-20	The aim is to study the function of peroxisomal NAD-malate dehydrogenase in fatty acid beta-oxidation, glyoxylate cycle and photorespiration. Both peroxisomal MDH genes (At2g22780 and At5g09660) have been knocked out with T-DNA insertions and a double mutant made. Double mutant seedlings are blocked in beta-oxidation - they are 2,4DB resistant and beta-oxidation genes are repressed. They carry out glyoxylate cycle as normal. Plants grow well in soil and produce seed. Microarray analysis will tell us the extent of changes in gene expression in the mutant. For microarray analysis seeds are stratified at 4 C on agar medium with 1/2 strength M&S salts and 1% sucrose for 2 days, then seedlings grown for 2 days at 20 C in the light (100 umol/m2/s). Triplicate samples will be grown for mutant and wild type (col-0) and RNA isolated from each.
NASCARRAY167 AtGenExpress: Pathogen Series: Response to Botrytis cinerea infection 2005-02-02	AtGenExpress: A multinational coordinated effort to uncover the transcriptome of the multicellular model organism Arabidopsis thaliana The activity of genes and their encoded products can be regulated in several ways, but transcription is the primary level, since all other modes of regulation (RNA splicing, RNA and protein stability, etc.) are dependent on a gene being transcribed in the first place. The importance of transcriptional regulation has been underscored by the recent flood of global expression analyses, which have confirmed that transcriptional co-regulation of genes that act together is the norm, not the exception. Moreover, many studies suggest that evolutionary change is driven in large part by modifications of transcriptional programs. An essential first step toward deciphering the transcriptional code is to determine the expression pattern of all genes. With this goal in mind, an international effort to develop a gene expression atlas of Arabidopsis has been underway since fall 2003. This project, dubbed AtGenExpress, is funded by the DFG, and will provide the Arabidopsis community with access to a large set of Affymetrix microarray data. As part of this collaboration, we have generated expression data from 80 biologicaly different samples in triplicate. Responses to B. cinerea infection were assayed in wild-type Col-0 plants at 18 and 48 hours after inoculation of adult leaves. Leaves were inoculated by placing 4 5-ul drops of a 5x10e5 spore solution on each leaf. Control leaves were spotted with droplets of 24g L-1 potato dextrose broth medium.
NASCARRAY168 AtGenExpress: Pathogen Series: Pseudomonas half leaf injection 2005-02-02	AtGenExpress: A multinational coordinated effort to uncover the transcriptome of the multicellular model organism Arabidopsis thaliana The activity of genes and their encoded products can be regulated in several ways, but transcription is the primary level, since all other modes of regulation (RNA splicing, RNA and protein stability, etc.) are dependent on a gene being transcribed in the first place. The importance of transcriptional regulation has been underscored by the recent flood of global expression analyses, which have confirmed that transcriptional co-regulation of genes that act together is the norm, not the exception. Moreover, many studies suggest that evolutionary change is driven in large part by modifications of transcriptional programs. An essential first step toward deciphering the transcriptional code is to determine the expression pattern of all genes. With this goal in mind, an international effort to develop a gene expression atlas of Arabidopsis has been underway since fall 2003. This project, dubbed AtGenExpress, is funded by the DFG, and will provide the Arabidopsis community with access to a large set of Affymetrix microarray data. As part of this collaboration, we have generated expression data from 80 biologicaly different samples in triplicate. Half of a Columbia plant leaf was injected with Pseudomonas syringae. The other half of the leaf was collected on a time course for analysis.
NASCARRAY169 AtGenExpress: Pathogen Series: Response to Erysiphe orontii infection 2005-02-02	AtGenExpress: A multinational coordinated effort to uncover the transcriptome of the multicellular model organism Arabidopsis thaliana The activity of genes and their encoded products can be regulated in several ways, but transcription is the primary level, since all other modes of regulation (RNA splicing, RNA and protein stability, etc.) are dependent on a gene being transcribed in the first place. The importance of transcriptional regulation has been underscored by the recent flood of global expression analyses, which have confirmed that transcriptional co-regulation of genes that act together is the norm, not the exception. Moreover, many studies suggest that evolutionary change is driven in large part by modifications of transcriptional programs. An essential first step toward deciphering the transcriptional code is to determine the expression pattern of all genes. With this goal in mind, an international effort to develop a gene expression atlas of Arabidopsis has been underway since fall 2003. This project, dubbed AtGenExpress, is funded by the DFG, and will provide the Arabidopsis community with access to a large set of Affymetrix microarray data. As part of this collaboration, we have generated expression data from 80 biologicaly different samples in triplicate. Responses to E. orontii infection were assayed in wild-type Col-0 plants at 6, 12, 18, 24, 48, 72, 96, and 120 hours after inoculation of adult leaves. Inoculation was done via settling tower, using 10 day old E. orontii cultures. Leaves number 7 to 10 were selected for profiling.
NASCARRAY170 AtGenExpress: Different temperature treatment of seeds 2005-02-02	AtGenExpress: A multinational coordinated effort to uncover the transcriptome of the multicellular model organism Arabidopsis thaliana The activity of genes and their encoded products can be regulated in several ways, but transcription is the primary level, since all other modes of regulation (RNA splicing, RNA and protein stability, etc.) are dependent on a gene being transcribed in the first place. The importance of transcriptional regulation has been underscored by the recent flood of global expression analyses, which have confirmed that transcriptional co-regulation of genes that act together is the norm, not the exception. Moreover, many studies suggest that evolutionary change is driven in large part by modifications of transcriptional programs. An essential first step toward deciphering the transcriptional code is to determine the expression pattern of all genes. With this goal in mind, an international effort to develop a gene expression atlas of Arabidopsis has been underway since fall 2003. This project, dubbed AtGenExpress, is funded by the DFG, and will provide the Arabidopsis community with access to a large set of Affymetrix microarray data. As part of this collaboration, we have generated expression data from 80 biologicaly different samples in triplicate. Wild-type seeds were treated at 22 C and 4 C
NASCARRAY171 AtGenExpress: Response to sulfate limitation 2005-02-02	AtGenExpress: A multinational coordinated effort to uncover the transcriptome of the multicellular model organism Arabidopsis thaliana The activity of genes and their encoded products can be regulated in several ways, but transcription is the primary level, since all other modes of regulation (RNA splicing, RNA and protein stability, etc.) are dependent on a gene being transcribed in the first place. The importance of transcriptional regulation has been underscored by the recent flood of global expression analyses, which have confirmed that transcriptional co-regulation of genes that act together is the norm, not the exception. Moreover, many studies suggest that evolutionary change is driven in large part by modifications of transcriptional programs. An essential first step toward deciphering the transcriptional code is to determine the expression pattern of all genes. With this goal in mind, an international effort to develop a gene expression atlas of Arabidopsis has been underway since fall 2003. This project, dubbed AtGenExpress, is funded by the DFG, and will provide the Arabidopsis community with access to a large set of Affymetrix microarray data. As part of this collaboration, we have generated expression data from 80 biologicaly different samples in triplicate. This experiment studies the effect of sulfate limitation in the roots of 10-day-old Arabidopsis seedlings from Columbia-0 ecotype
NASCARRAY172 AtGenExpress: ACC time course in wildtype seedlings 2005-02-02	AtGenExpress: A multinational coordinated effort to uncover the transcriptome of the multicellular model organism Arabidopsis thaliana The activity of genes and their encoded products can be regulated in several ways, but transcription is the primary level, since all other modes of regulation (RNA splicing, RNA and protein stability, etc.) are dependent on a gene being transcribed in the first place. The importance of transcriptional regulation has been underscored by the recent flood of global expression analyses, which have confirmed that transcriptional co-regulation of genes that act together is the norm, not the exception. Moreover, many studies suggest that evolutionary change is driven in large part by modifications of transcriptional programs. An essential first step toward deciphering the transcriptional code is to determine the expression pattern of all genes. With this goal in mind, an international effort to develop a gene expression atlas of Arabidopsis has been underway since fall 2003. This project, dubbed AtGenExpress, is funded by the DFG, and will provide the Arabidopsis community with access to a large set of Affymetrix microarray data. As part of this collaboration, we have generated expression data from 80 biologicaly different samples in triplicate. Wild-type seedlings were treated with ACC for 30 min, 1 hr and 3 hr.
NASCARRAY173 AtGenExpress: Zeatin time course in wildtype seedlings 2005-02-02	AtGenExpress: A multinational coordinated effort to uncover the transcriptome of the multicellular model organism Arabidopsis thaliana The activity of genes and their encoded products can be regulated in several ways, but transcription is the primary level, since all other modes of regulation (RNA splicing, RNA and protein stability, etc.) are dependent on a gene being transcribed in the first place. The importance of transcriptional regulation has been underscored by the recent flood of global expression analyses, which have confirmed that transcriptional co-regulation of genes that act together is the norm, not the exception. Moreover, many studies suggest that evolutionary change is driven in large part by modifications of transcriptional programs. An essential first step toward deciphering the transcriptional code is to determine the expression pattern of all genes. With this goal in mind, an international effort to develop a gene expression atlas of Arabidopsis has been underway since fall 2003. This project, dubbed AtGenExpress, is funded by the DFG, and will provide the Arabidopsis community with access to a large set of Affymetrix microarray data. As part of this collaboration, we have generated expression data from 80 biologicaly different samples in triplicate. Wild-type seedlings were treated with zeatin for 30 min, 1 hr and 3 hr.
NASCARRAY174 AtGenExpress: Methyl Jasmonate time course in wildtype 2005-02-02	AtGenExpress: A multinational coordinated effort to uncover the transcriptome of the multicellular model organism Arabidopsis thaliana The activity of genes and their encoded products can be regulated in several ways, but transcription is the primary level, since all other modes of regulation (RNA splicing, RNA and protein stability, etc.) are dependent on a gene being transcribed in the first place. The importance of transcriptional regulation has been underscored by the recent flood of global expression analyses, which have confirmed that transcriptional co-regulation of genes that act together is the norm, not the exception. Moreover, many studies suggest that evolutionary change is driven in large part by modifications of transcriptional programs. An essential first step toward deciphering the transcriptional code is to determine the expression pattern of all genes. With this goal in mind, an international effort to develop a gene expression atlas of Arabidopsis has been underway since fall 2003. This project, dubbed AtGenExpress, is funded by the DFG, and will provide the Arabidopsis community with access to a large set of Affymetrix microarray data. As part of this collaboration, we have generated expression data from 80 biologicaly different samples in triplicate. Wild-type seedlings were treated with methyl jasmonate for 30 min, 1 hr and 3 hr.
NASCARRAY175 AtGenExpress: IAA time course in wildtype seedlings 2005-02-02	AtGenExpress: A multinational coordinated effort to uncover the transcriptome of the multicellular model organism Arabidopsis thaliana The activity of genes and their encoded products can be regulated in several ways, but transcription is the primary level, since all other modes of regulation (RNA splicing, RNA and protein stability, etc.) are dependent on a gene being transcribed in the first place. The importance of transcriptional regulation has been underscored by the recent flood of global expression analyses, which have confirmed that transcriptional co-regulation of genes that act together is the norm, not the exception. Moreover, many studies suggest that evolutionary change is driven in large part by modifications of transcriptional programs. An essential first step toward deciphering the transcriptional code is to determine the expression pattern of all genes. With this goal in mind, an international effort to develop a gene expression atlas of Arabidopsis has been underway since fall 2003. This project, dubbed AtGenExpress, is funded by the DFG, and will provide the Arabidopsis community with access to a large set of Affymetrix microarray data. As part of this collaboration, we have generated expression data from 80 biologicaly different samples in triplicate. Wild-type seedlings were treated with IAA for 30 min, 1 hr and 3 hr.
NASCARRAY176 AtGenExpress: ABA time course in wildtype seedlings 2005-02-02	AtGenExpress: A multinational coordinated effort to uncover the transcriptome of the multicellular model organism Arabidopsis thaliana The activity of genes and their encoded products can be regulated in several ways, but transcription is the primary level, since all other modes of regulation (RNA splicing, RNA and protein stability, etc.) are dependent on a gene being transcribed in the first place. The importance of transcriptional regulation has been underscored by the recent flood of global expression analyses, which have confirmed that transcriptional co-regulation of genes that act together is the norm, not the exception. Moreover, many studies suggest that evolutionary change is driven in large part by modifications of transcriptional programs. An essential first step toward deciphering the transcriptional code is to determine the expression pattern of all genes. With this goal in mind, an international effort to develop a gene expression atlas of Arabidopsis has been underway since fall 2003. This project, dubbed AtGenExpress, is funded by the DFG, and will provide the Arabidopsis community with access to a large set of Affymetrix microarray data. As part of this collaboration, we have generated expression data from 80 biologicaly different samples in triplicate. Wild-type seedlings were treated with ABA for 30 min, 1 hr and 3 hr.
NASCARRAY177 AtGenExpress: GA3 time course in wildtype and ga1-5 mutant seedlings 2005-02-02	AtGenExpress: A multinational coordinated effort to uncover the transcriptome of the multicellular model organism Arabidopsis thaliana The activity of genes and their encoded products can be regulated in several ways, but transcription is the primary level, since all other modes of regulation (RNA splicing, RNA and protein stability, etc.) are dependent on a gene being transcribed in the first place. The importance of transcriptional regulation has been underscored by the recent flood of global expression analyses, which have confirmed that transcriptional co-regulation of genes that act together is the norm, not the exception. Moreover, many studies suggest that evolutionary change is driven in large part by modifications of transcriptional programs. An essential first step toward deciphering the transcriptional code is to determine the expression pattern of all genes. With this goal in mind, an international effort to develop a gene expression atlas of Arabidopsis has been underway since fall 2003. This project, dubbed AtGenExpress, is funded by the DFG, and will provide the Arabidopsis community with access to a large set of Affymetrix microarray data. As part of this collaboration, we have generated expression data from 80 biologicaly different samples in triplicate. Wild-type and ga1 mutant seedlings were treated with GA3 for 30 min, 1 hr and 3 hr.
NASCARRAY178 AtGenExpress: Brassinolide time course in wildtype and det2-1 mutant seedlings 2005-02-02	AtGenExpress: A multinational coordinated effort to uncover the transcriptome of the multicellular model organism Arabidopsis thaliana The activity of genes and their encoded products can be regulated in several ways, but transcription is the primary level, since all other modes of regulation (RNA splicing, RNA and protein stability, etc.) are dependent on a gene being transcribed in the first place. The importance of transcriptional regulation has been underscored by the recent flood of global expression analyses, which have confirmed that transcriptional co-regulation of genes that act together is the norm, not the exception. Moreover, many studies suggest that evolutionary change is driven in large part by modifications of transcriptional programs. An essential first step toward deciphering the transcriptional code is to determine the expression pattern of all genes. With this goal in mind, an international effort to develop a gene expression atlas of Arabidopsis has been underway since fall 2003. This project, dubbed AtGenExpress, is funded by the DFG, and will provide the Arabidopsis community with access to a large set of Affymetrix microarray data. As part of this collaboration, we have generated expression data from 80 biologicaly different samples in triplicate. Wild-type and det2 mutant seedlings were treated with brassinolide for 30 min, 1 hr and 3 hr.
NASCARRAY179 AtGenExpress: Effect of brassinosteroids in seedlings 2005-02-02	AtGenExpress: A multinational coordinated effort to uncover the transcriptome of the multicellular model organism Arabidopsis thaliana The activity of genes and their encoded products can be regulated in several ways, but transcription is the primary level, since all other modes of regulation (RNA splicing, RNA and protein stability, etc.) are dependent on a gene being transcribed in the first place. The importance of transcriptional regulation has been underscored by the recent flood of global expression analyses, which have confirmed that transcriptional co-regulation of genes that act together is the norm, not the exception. Moreover, many studies suggest that evolutionary change is driven in large part by modifications of transcriptional programs. An essential first step toward deciphering the transcriptional code is to determine the expression pattern of all genes. With this goal in mind, an international effort to develop a gene expression atlas of Arabidopsis has been underway since fall 2003. This project, dubbed AtGenExpress, is funded by the DFG, and will provide the Arabidopsis community with access to a large set of Affymetrix microarray data. As part of this collaboration, we have generated expression data from 80 biologicaly different samples in triplicate. det2 mutant seedlings were treated with several brassinosteroid compounds.
NASCARRAY180 AtGenExpress: Comparison of plant hormone-related mutants 2005-02-02	AtGenExpress: A multinational coordinated effort to uncover the transcriptome of the multicellular model organism Arabidopsis thaliana The activity of genes and their encoded products can be regulated in several ways, but transcription is the primary level, since all other modes of regulation (RNA splicing, RNA and protein stability, etc.) are dependent on a gene being transcribed in the first place. The importance of transcriptional regulation has been underscored by the recent flood of global expression analyses, which have confirmed that transcriptional co-regulation of genes that act together is the norm, not the exception. Moreover, many studies suggest that evolutionary change is driven in large part by modifications of transcriptional programs. An essential first step toward deciphering the transcriptional code is to determine the expression pattern of all genes. With this goal in mind, an international effort to develop a gene expression atlas of Arabidopsis has been underway since fall 2003. This project, dubbed AtGenExpress, is funded by the DFG, and will provide the Arabidopsis community with access to a large set of Affymetrix microarray data. As part of this collaboration, we have generated expression data from 80 biologicaly different samples in triplicate. Comparison of plant hormone related mutants
NASCARRAY181 AtGenExpress: Cytokinin treatment of seedlings 2005-02-02	AtGenExpress: A multinational coordinated effort to uncover the transcriptome of the multicellular model organism Arabidopsis thaliana The activity of genes and their encoded products can be regulated in several ways, but transcription is the primary level, since all other modes of regulation (RNA splicing, RNA and protein stability, etc.) are dependent on a gene being transcribed in the first place. The importance of transcriptional regulation has been underscored by the recent flood of global expression analyses, which have confirmed that transcriptional co-regulation of genes that act together is the norm, not the exception. Moreover, many studies suggest that evolutionary change is driven in large part by modifications of transcriptional programs. An essential first step toward deciphering the transcriptional code is to determine the expression pattern of all genes. With this goal in mind, an international effort to develop a gene expression atlas of Arabidopsis has been underway since fall 2003. This project, dubbed AtGenExpress, is funded by the DFG, and will provide the Arabidopsis community with access to a large set of Affymetrix microarray data. As part of this collaboration, we have generated expression data from 80 biologicaly different samples in triplicate. Wild-type and ARR22 overexpressing seedlings were treated with t-zeatin for 3 hours
NASCARRAY182 AtGenExpress: ARR21C overexpression 2005-02-02	AtGenExpress: A multinational coordinated effort to uncover the transcriptome of the multicellular model organism Arabidopsis thaliana The activity of genes and their encoded products can be regulated in several ways, but transcription is the primary level, since all other modes of regulation (RNA splicing, RNA and protein stability, etc.) are dependent on a gene being transcribed in the first place. The importance of transcriptional regulation has been underscored by the recent flood of global expression analyses, which have confirmed that transcriptional co-regulation of genes that act together is the norm, not the exception. Moreover, many studies suggest that evolutionary change is driven in large part by modifications of transcriptional programs. An essential first step toward deciphering the transcriptional code is to determine the expression pattern of all genes. With this goal in mind, an international effort to develop a gene expression atlas of Arabidopsis has been underway since fall 2003. This project, dubbed AtGenExpress, is funded by the DFG, and will provide the Arabidopsis community with access to a large set of Affymetrix microarray data. As part of this collaboration, we have generated expression data from 80 biologicaly different samples in triplicate. Wild-type and ARR21C overexpressing seedlings were compared.
NASCARRAY183 AtGenExpress: Effect of ABA during seed imbibition 2005-02-02	AtGenExpress: A multinational coordinated effort to uncover the transcriptome of the multicellular model organism Arabidopsis thaliana The activity of genes and their encoded products can be regulated in several ways, but transcription is the primary level, since all other modes of regulation (RNA splicing, RNA and protein stability, etc.) are dependent on a gene being transcribed in the first place. The importance of transcriptional regulation has been underscored by the recent flood of global expression analyses, which have confirmed that transcriptional co-regulation of genes that act together is the norm, not the exception. Moreover, many studies suggest that evolutionary change is driven in large part by modifications of transcriptional programs. An essential first step toward deciphering the transcriptional code is to determine the expression pattern of all genes. With this goal in mind, an international effort to develop a gene expression atlas of Arabidopsis has been underway since fall 2003. This project, dubbed AtGenExpress, is funded by the DFG, and will provide the Arabidopsis community with access to a large set of Affymetrix microarray data. As part of this collaboration, we have generated expression data from 80 biologicaly different samples in triplicate. This experiment studies the effect of two concentrations of ABA during seed imbibition in the Col-0 ecotype.
NASCARRAY184 AtGenExpress: Basic hormone treatment of seeds 2005-02-02	AtGenExpress: A multinational coordinated effort to uncover the transcriptome of the multicellular model organism Arabidopsis thaliana The activity of genes and their encoded products can be regulated in several ways, but transcription is the primary level, since all other modes of regulation (RNA splicing, RNA and protein stability, etc.) are dependent on a gene being transcribed in the first place. The importance of transcriptional regulation has been underscored by the recent flood of global expression analyses, which have confirmed that transcriptional co-regulation of genes that act together is the norm, not the exception. Moreover, many studies suggest that evolutionary change is driven in large part by modifications of transcriptional programs. An essential first step toward deciphering the transcriptional code is to determine the expression pattern of all genes. With this goal in mind, an international effort to develop a gene expression atlas of Arabidopsis has been underway since fall 2003. This project, dubbed AtGenExpress, is funded by the DFG, and will provide the Arabidopsis community with access to a large set of Affymetrix microarray data. As part of this collaboration, we have generated expression data from 80 biologicaly different samples in triplicate. Basic hormone treatment of seeds
NASCARRAY185 AtGenExpress: Effect of gibberellic acid inhibitors on seedlings 2005-02-02	AtGenExpress: A multinational coordinated effort to uncover the transcriptome of the multicellular model organism Arabidopsis thaliana The activity of genes and their encoded products can be regulated in several ways, but transcription is the primary level, since all other modes of regulation (RNA splicing, RNA and protein stability, etc.) are dependent on a gene being transcribed in the first place. The importance of transcriptional regulation has been underscored by the recent flood of global expression analyses, which have confirmed that transcriptional co-regulation of genes that act together is the norm, not the exception. Moreover, many studies suggest that evolutionary change is driven in large part by modifications of transcriptional programs. An essential first step toward deciphering the transcriptional code is to determine the expression pattern of all genes. With this goal in mind, an international effort to develop a gene expression atlas of Arabidopsis has been underway since fall 2003. This project, dubbed AtGenExpress, is funded by the DFG, and will provide the Arabidopsis community with access to a large set of Affymetrix microarray data. As part of this collaboration, we have generated expression data from 80 biologicaly different samples in triplicate. Wild-type seedlings were treated with inhibitors of gibberellic acid biosynthesis.
NASCARRAY186 AtGenExpress: Effect of auxin inhibitors on seedlings 2005-02-02	AtGenExpress: A multinational coordinated effort to uncover the transcriptome of the multicellular model organism Arabidopsis thaliana The activity of genes and their encoded products can be regulated in several ways, but transcription is the primary level, since all other modes of regulation (RNA splicing, RNA and protein stability, etc.) are dependent on a gene being transcribed in the first place. The importance of transcriptional regulation has been underscored by the recent flood of global expression analyses, which have confirmed that transcriptional co-regulation of genes that act together is the norm, not the exception. Moreover, many studies suggest that evolutionary change is driven in large part by modifications of transcriptional programs. An essential first step toward deciphering the transcriptional code is to determine the expression pattern of all genes. With this goal in mind, an international effort to develop a gene expression atlas of Arabidopsis has been underway since fall 2003. This project, dubbed AtGenExpress, is funded by the DFG, and will provide the Arabidopsis community with access to a large set of Affymetrix microarray data. As part of this collaboration, we have generated expression data from 80 biologicaly different samples in triplicate. Wild-type seedlings were treated with auxin inhibitors.
NASCARRAY187 AtGenExpress: Effect of brassinosteroid inhibitors on seedlings 2005-02-02	AtGenExpress: A multinational coordinated effort to uncover the transcriptome of the multicellular model organism Arabidopsis thaliana The activity of genes and their encoded products can be regulated in several ways, but transcription is the primary level, since all other modes of regulation (RNA splicing, RNA and protein stability, etc.) are dependent on a gene being transcribed in the first place. The importance of transcriptional regulation has been underscored by the recent flood of global expression analyses, which have confirmed that transcriptional co-regulation of genes that act together is the norm, not the exception. Moreover, many studies suggest that evolutionary change is driven in large part by modifications of transcriptional programs. An essential first step toward deciphering the transcriptional code is to determine the expression pattern of all genes. With this goal in mind, an international effort to develop a gene expression atlas of Arabidopsis has been underway since fall 2003. This project, dubbed AtGenExpress, is funded by the DFG, and will provide the Arabidopsis community with access to a large set of Affymetrix microarray data. As part of this collaboration, we have generated expression data from 80 biologicaly different samples in triplicate. Wild-type seedlings were treated with brassinazoles.
NASCARRAY188 AtGenExpress: Effect of ethylene inhibitors on seedlings 2005-02-02	AtGenExpress: A multinational coordinated effort to uncover the transcriptome of the multicellular model organism Arabidopsis thaliana The activity of genes and their encoded products can be regulated in several ways, but transcription is the primary level, since all other modes of regulation (RNA splicing, RNA and protein stability, etc.) are dependent on a gene being transcribed in the first place. The importance of transcriptional regulation has been underscored by the recent flood of global expression analyses, which have confirmed that transcriptional co-regulation of genes that act together is the norm, not the exception. Moreover, many studies suggest that evolutionary change is driven in large part by modifications of transcriptional programs. An essential first step toward deciphering the transcriptional code is to determine the expression pattern of all genes. With this goal in mind, an international effort to develop a gene expression atlas of Arabidopsis has been underway since fall 2003. This project, dubbed AtGenExpress, is funded by the DFG, and will provide the Arabidopsis community with access to a large set of Affymetrix microarray data. As part of this collaboration, we have generated expression data from 80 biologicaly different samples in triplicate. Wild-type seedlings were treated with ethylene inhibitors.
NASCARRAY189 AtGenExpress: Effect of cycloheximide on seedlings 2005-02-02	AtGenExpress: A multinational coordinated effort to uncover the transcriptome of the multicellular model organism Arabidopsis thaliana The activity of genes and their encoded products can be regulated in several ways, but transcription is the primary level, since all other modes of regulation (RNA splicing, RNA and protein stability, etc.) are dependent on a gene being transcribed in the first place. The importance of transcriptional regulation has been underscored by the recent flood of global expression analyses, which have confirmed that transcriptional co-regulation of genes that act together is the norm, not the exception. Moreover, many studies suggest that evolutionary change is driven in large part by modifications of transcriptional programs. An essential first step toward deciphering the transcriptional code is to determine the expression pattern of all genes. With this goal in mind, an international effort to develop a gene expression atlas of Arabidopsis has been underway since fall 2003. This project, dubbed AtGenExpress, is funded by the DFG, and will provide the Arabidopsis community with access to a large set of Affymetrix microarray data. As part of this collaboration, we have generated expression data from 80 biologicaly different samples in triplicate. Wild-type seedlings were treated with cycloheximide.
NASCARRAY190 AtGenExpress: Effect of proteasome inhibitor MG13 on seedlings 2005-02-02	AtGenExpress: A multinational coordinated effort to uncover the transcriptome of the multicellular model organism Arabidopsis thaliana The activity of genes and their encoded products can be regulated in several ways, but transcription is the primary level, since all other modes of regulation (RNA splicing, RNA and protein stability, etc.) are dependent on a gene being transcribed in the first place. The importance of transcriptional regulation has been underscored by the recent flood of global expression analyses, which have confirmed that transcriptional co-regulation of genes that act together is the norm, not the exception. Moreover, many studies suggest that evolutionary change is driven in large part by modifications of transcriptional programs. An essential first step toward deciphering the transcriptional code is to determine the expression pattern of all genes. With this goal in mind, an international effort to develop a gene expression atlas of Arabidopsis has been underway since fall 2003. This project, dubbed AtGenExpress, is funded by the DFG, and will provide the Arabidopsis community with access to a large set of Affymetrix microarray data. As part of this collaboration, we have generated expression data from 80 biologicaly different samples in triplicate. Wild-type seedlings were treated with the proteasome inhibitor MG13 (carbobenzoxyl-leucinyl-leucinyl-leucinal) for 3 hours.
NASCARRAY191 AtGenExpress: Effect of photosynthesis inhibitor PNO8 on seedlings 2005-02-02	AtGenExpress: A multinational coordinated effort to uncover the transcriptome of the multicellular model organism Arabidopsis thaliana The activity of genes and their encoded products can be regulated in several ways, but transcription is the primary level, since all other modes of regulation (RNA splicing, RNA and protein stability, etc.) are dependent on a gene being transcribed in the first place. The importance of transcriptional regulation has been underscored by the recent flood of global expression analyses, which have confirmed that transcriptional co-regulation of genes that act together is the norm, not the exception. Moreover, many studies suggest that evolutionary change is driven in large part by modifications of transcriptional programs. An essential first step toward deciphering the transcriptional code is to determine the expression pattern of all genes. With this goal in mind, an international effort to develop a gene expression atlas of Arabidopsis has been underway since fall 2003. This project, dubbed AtGenExpress, is funded by the DFG, and will provide the Arabidopsis community with access to a large set of Affymetrix microarray data. As part of this collaboration, we have generated expression data from 80 biologicaly different samples in triplicate. Wild-type seedlings were treated with the photosynthesis inhibitor PNO8 (N-octyl-3-nitro-2,4,6-trihydroxybenzamide).
NASCARRAY192 AtGenExpress: Effect of ibuprofen, salicylic acid and daminozide on seedlings 2005-02-02	AtGenExpress: A multinational coordinated effort to uncover the transcriptome of the multicellular model organism Arabidopsis thaliana The activity of genes and their encoded products can be regulated in several ways, but transcription is the primary level, since all other modes of regulation (RNA splicing, RNA and protein stability, etc.) are dependent on a gene being transcribed in the first place. The importance of transcriptional regulation has been underscored by the recent flood of global expression analyses, which have confirmed that transcriptional co-regulation of genes that act together is the norm, not the exception. Moreover, many studies suggest that evolutionary change is driven in large part by modifications of transcriptional programs. An essential first step toward deciphering the transcriptional code is to determine the expression pattern of all genes. With this goal in mind, an international effort to develop a gene expression atlas of Arabidopsis has been underway since fall 2003. This project, dubbed AtGenExpress, is funded by the DFG, and will provide the Arabidopsis community with access to a large set of Affymetrix microarray data. As part of this collaboration, we have generated expression data from 80 biologicaly different samples in triplicate. Wildtype seedlings were treated with ibuprofen, salicylic acid or daminozide
NASCARRAY194 Dark-induced gene expression in sfr6 2005-01-30	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.
NASCARRAY195 AtGenExpress: Expression profiling of early germinating seeds 2005-02-02	AtGenExpress: A multinational coordinated effort to uncover the transcriptome of the multicellular model organism Arabidopsis thaliana. The activity of genes and their encoded products can be regulated in several ways, but transcription is the primary level, since all other modes of regulation (RNA splicing, RNA and protein stability, etc.) are dependent on a gene being transcribed in the first place. The importance of transcriptional regulation has been underscored by the recent flood of global expression analyses, which have confirmed that transcriptional co-regulation of genes that act together is the norm, not the exception. Moreover, many studies suggest that evolutionary change is driven in large part by modifications of transcriptional programs. An essential first step toward deciphering the transcriptional code is to determine the expression pattern of all genes. With this goal in mind, an international effort to develop a gene expression atlas of Arabidopsis has been underway since fall 2003. This project, dubbed AtGenExpress, is funded by the DFG, and will provide the Arabidopsis community with access to a large set of Affymetrix microarray data. As part of this collaboration, we have generated expression data from 80 biologicaly different samples in triplicate. Expression study of Columbia-0 seeds at 0, 1 and 3 hours post-imbibition
NASCARRAY196 Circadian gene expression under different light treatments 2005-12-02	Most eukaryotic and some prokaryotic organisms regulate their metabolic and physiological behaviour with an internal 24h 'circadian clock'. Around 6% of the Arabidopsis thaliana genome was estimated to be regulated on a transcriptional level by this clock, with various phases of peak expression throughout the daily cycle (Harmer et al., 2000). The phase and period of the internal clock is entrained by changes in light and temperature so that the clock cycles in resonance with the external environment. The temporal co-ordination provided by the clock allows organisms to anticipate and respond to the predictable environmental changes in the day/night cycle. This resonance with the external environment is believed to impart organisms with a selective advantage (Ouyang et al., 1998).Using Affymetrix micro-arrays we hope to further our knowledge of the mechanisms of the clock to help in our attempts at modelling the molecular oscillator of Arabidopsis and understanding how the clock's phase relates to fitness. Firstly we will reduce the complexity of the clock by growing seedlings under monochromatic far-red light. Under white light conditions, light signals are input to the clock via the five phytochrome and two cryptochrome photoreceptors, whereas under far-red conditions light is input solely via Phytochrome A (PHYA). Secondly, we will alter the length of the day/night cycle, away from 24 hours, by growing seedlings under different white light/dark T-cycles (T=20h and T=28h). In both sets of experiments a time course of samples will be taken over the first and/or second circadian cycle from seedlings released into constant far-red or white light, following entrainment to light/dark cycles.
NASCARRAY197 MUTANTS RESPONSE TO IAA 2005-03-11	BACKGROUND: Individual members of Auxin Response Factor (ARF) transcriptional regulators may control specific types of auxin responses. Our mutant/overexpression analysis supports this notion. Mutations in MP (IAA24/ARF5) and ARF7 (NPH4/MSG1) confer highly specific (!) auxin insensitivity. Phenotypes implicate MP and ARF7 in partially non-overlapping aspects of auxin-dependent cell elongation, but only MP in vascular differentiation, suggesting regulation of two sets of partially non-overlapping genes. RATIONALE: So far, we have identified >20 genes with highly specific auxin inducibility which is dependent on the activity of MP and/or ARF7. This is evidenced by both reduced auxin inducibility in mp and msg1 mutants and enhanced inducibility in corresponding MP and ARF7 overexpression plants. Inducibility of three of these genes is selectively affected only by MP gene activity and all three of these genes are expressed specifically in vascular tissues. Thus, large-scale differential auxin inducibility profiles could identify most auxin inducible genes in the developmental pathway of each ARF gene; e.g. for MP in vascular development. Comparison of mp versus Col-0 tissue would additionally provide a huge selective collection of vascular genes, since otherwise normal mp cotyledons nearly devoid of vascular tissues can be obtained. TREATMENT/GENOTYPE: We suggest to assess rapid (<20min) inducibility at 20ÂµM versus 0ÂµM IAA in Col-0 (1), in mp (2) and in msg1 (3) mutants (both are in Col-0). PRIORITIES/LINKS: We would suggest the sequence (1), (2), (3) as described above. This would involve 6 or 12 hybridizations, but likely (1) and possibly (3) will be suggested by others, and one should then coordinate the induction/tissue conditions towards an integrated experiment. Alternatively, we could supply RNA for all three experiments as microarray facilities are becoming available in successive rounds. TISSUE/RNA: RNA from apical seedling organs will be prepared by a senior post-doc, who has developed a highly reproducible induction protocol for immersed Col-0 seedlings. We have >20 genes with known profiles to test each RNA sample. These internal controls may also help in further microarray optimization. SIGNIFICANCE:(1) is of obvious general interest to entire auxin community. Among other goals, (2)(3) would provide the first comprehensive collection of Arabidopsis vascular genes (high general interest; later spec. microarray). PERSPECTIVE: Our long-term goal is understanding signaling in vascular development and we would concentrate on the class of specifically MP dependent gene expressions, that would be further characterized by expression pattern and reverse genetic phenotype. DOCUMENTATION: We can provide quantified Northern data to demonstrate the feasibility of the approach. RELEASE: (1)(3) immediately, (2) 3 months LIT: EMBO J 17, 1405-1411 Plant Phys 115, 419-426 Plant Phys 118, 1265.1275
NASCARRAY198 ATP CITRATE LYASE 2005-03-11	Cast as a central molecule in plant metabolism, acetyl-CoA plays a major role in many interconnected primary and secondary biochemical pathways, producing classes of compounds such as fatty acids, waxes, flavonoids and isoprenoids. Since acetyl-CoA is membrane-impermeable, synthesis is thought to occur in the subcellular compartment where it is required. Several acetyl-CoA-generating mechanisms have been proposed including one involving ATP citrate lyase (ACL). ACL catalyzes the ATP-dependent reaction between citrate and CoA to form oxaloacetate and acetyl-CoA. Our molecular characterizations of Arabidopsis ACL cDNAs indicate that the plant enzyme consists of two dissimilar subunits (A and B). Using subunit-specific antibodies and activity assays, we have shown that ACL is located in the cytosol, and is not detectable in plastids, mitochondria, or peroxisomes. ACL mRNA a accumulates preferentially in cells requiring cytosolic acetyl-CoA. Antisense ACL-A plants with reduced ACL activity have a distinct phenotype including: miniaturized organs, small cell size, aberrant plastid morphology and reduced cuticular wax. Our initial characterizations indicate that ACL may be providing acetyl-CoA for cytosolic acetyl-CoA-derived phytochemicals. We wish to compare mRNA accumulation profiles in elongating inflorescence stems of antisense ACL and wild type Arabidopsis thaliana (Columbia). Inflorescence stems have been selected for analysis in that the ACL mutants have easily comparable developmental stages, and adequate mass for the extraction of RNA and further biochemical studies. We will use the wealth of resulting data, along with other experimental data to formulate testable hypotheses to address several questions. (1) How are genes involved in cytosolic acetyl-CoA-requiring pathways effected? (2) Are genes of other acetyl-CoA generating mechanisms effected (i.e., Do alternative acetyl-CoA-generating pathways compensate for the lack of cytosolic acetyl-CoA? (3) What other metabolic pathways are involved with or linked to ACL? This analysis may also lead to the discovery of currently unknown pathways requiring ACL-derived acetyl-CoA. The use of microarray analysis would greatly enhance our current analysis of antisense ACL plants and thus our investigation into the role of ACL in acetyl-CoA generation.
NASCARRAY199 INFLORESENCE MERISTEM ARREST IN BROCCOLI 2005-03-11	The microarray analysis will be used to help identify gene regulation that leads to temperature-sensitive arrest of the broccoli meristem at the transition to floral initiation. Broccoli normally develops a ramified inflorescence, without flower bud development. After a certain period, meristems begin to make flower buds instead of more inflorescences. The meristem is arrested at this transition if the temperature is too high. This arrest is a good model for studying the regulation of inflorescence development, and is also a substantial agricultural problem. Broccoli is a good model system for studying the stages of reproductive development in crucifers because the stages exaggerated in broccoli over Arabidopsis. The homology is high enough between these species that reasonable hybridization can be expected. There are many genes that are candidates for being involved in the developmental arrest. These genes include CAL, LFY, AP1, TFL, AG, CBF1. Some have been identified as associated with the similar arrest that occurs in cauliflower. The genes actually in the regulatory sequence will be identified by high differential expression in the microarrays. This analysis will be particularly valuable in identifying additional genes that are not currently suspected. Using these results, and what is currently known about the mutual regulation among the MADS homeotic, we will propose a model for heat regulation of inflorescence genes. This model would provide the working hypothesis for an ongoing investigation using mapping and in situ hybridization to study regulation of the arrest. The RNA will be isolated from the inflorescence meristem of broccoli at the stage of development most responsive to high temperature. Control plants will be held at 22/15C (D/N) for three days before isolation, treated plants at 35/22. The genotype is a highly susceptible broccoli (Brassica oleracea var. italica) F1 hybrid, PS19590, in which we are mapping heat-tolerance QTLs
NASCARRAY200 GENES REGULATED BY AINTEGUMENTA 2005-03-11	Understanding the mechanisms involved in plant growth and development is critical to achieving increased agricultural production. Because flowers, and the fruit and seeds that develop from them, represent a major source of food, the identification of new genes involved in flower development could have enormous agricultural impact. We would like to use DNA microarrays to identify genes regulated by the transcription factor AINTEGUMENTA (ANT), which is involved in floral organ initiation and growth. ANT encodes a member of the AP2/EREBP class of transcription factors. Recently, we have used an in vitro selection procedure to identify DNA sequences bound by ANT. This will allow us to directly test whether ANT regulates target genes identified by DNA microarrays in our proposed experiment. We plan to identify ANT target genes using transgenic plants containing a steroid inducible form of ANT. The inducible ANT construct consists of full-length ANT fused at its carboxyl terminal end to the ligand binding domain of the glucocorticoid receptor. The activity of the fusion protein was tested by watering 35S::ANT-GR plants daily with the steroid dexamethasone (10mm dexamethasone in 0.1% ethanol). These plants produced flowers with larger organs that closely resembled those produced by 35S::ANT plants. No phenotypes were observed in 35S::ANT-GR mock-treated plants (watered daily with 0.1% ethanol). Inflorescences from kanamycin resistant T2 plants will be used for RNA isolation as described below. The comparison probe set will consist of RNA isolated from 35S::ANT-GR inflorescences that have been treated with dexamethasone (5mm) and cycloheximide (10mm) and RNA isolated from 35S::ANT-GR inflorescences that have been treated with cycloheximide (10mm) alone. Cycloheximide is a protein synthesis inhibitor and its inclusion should prevent the identification of more downstream genes. Inflorescences will be treated by direct pipetting of approximately 25ml of the dexamethasone/cycloheximide solution or the cycloheximide solution onto the tissue. This tissue will undergo a double induction: the first application will be followed by a second application four hours later. The tissue will be harvested approximately 6 hours after the first treatment. We plan to use two arrays for biological replicates of the same experiment.
NASCARRAY201 GENE EXPRESSION IN ANTISENSE TRANSGENIC ARABIDOPSIS 2005-03-11	Acetyl-CoA carboxylase (ACCase) catalyzes the carboxylation of acetyl-CoA to produce malonyl-CoA. Malonyl-CoA in plastids is the direct precursor for de novo fatty acid synthesis. In Arabidopsis, plastidic ACCase is a tetrameric enzyme, encoded by three nuclear and one plastidic gene. We postulate that if plastidic ACCase is a key regulatory enzyme, a decrease of plastidic ACCase activity would decrease fatty acid, and thus lipid, biosynthesis, triggering a series of physiological changes. We have generated 42 lines of transgenic Arabidopsis plants with reduced levels of the ACCase biotin carboxyl-carrier (BCC) subunit by introducing an antisense cDNA of the corresponding gene. The transgenic plants have different severities of phenotype reflecting the levels of BCC protein. A typical transgenic plant with a mild phenotype has 75-90% of BCC protein (as compared to wild type plants), grows more slowly, and has a smaller size than the wild-type plants. In these plants, the late vegetative and all cauline leaves are curled and variegated yellow. In the curled leaves, cell size, vacuolar area, and intercellular space are dramatically reduced. The subcellular structure of both chloroplasts and mitochondria is significantly altered. Although seed yield is reduced 10-100 fold, there is no significant change in fatty acid composition or content in seeds harvested from the transgenic plants. We propose to investigate changes in the mRNA profiles in the antisense Arabidopsis plants. Rosette leaves 8-12 of 30-day old Arabidopsis thaliana (ecotype Columbia) will be harvested and polyA RNA will be isolated for microarray analysis for both transgenic and wild type plants. Comparison of the results from the transgenic and wild type plants will enable us to identify the changes in global gene expression at the RNA level. Meanwhile, we are investigating the gene expression at the protein level using two-dimensional electrophoresis (2DE) and MALDI mass spectrometry. Interestingly, our preliminary results show that only the levels of very few proteins have been altered, regardless of the severe phenotype in the transgenic plants. With microarray and 2DE, we would be able to identify gene expression changes at mRNA as well as protein levels in plants under lipid stress. These data will give us a good start to investigate how the morphological changes are generated in the transgenic plants, and shed light on gene regulation networked with plastidic ACCase.
NASCARRAY202 CADMIUM AND VIRAL MOVEMENT 2005-03-11	The mechanism by which RNA molecules travel long distances through plant vasculature is unknown. We study systemic transport of two types of RNA: plant virus genomes and elicitors of post translational gene silencing (PTGS). We demonstrated that exposure of plants to non-toxic concentrations of cadmium (Cd) block viral disease. Cd-mediated viral protection was due to inhibition of the systemic movement of the virus but had no effect on viral replication and local spread. Furthermore, higher, toxic levels of Cd did not inhibit viral systemic movement (Plant J. 13, 591-602). Thus, non-toxic levels of Cd may trigger production of cellular factors which interfere with the viral systemic movement; exposure of plants to higher, toxic amounts of Cd likely represses this defensive response.Confocal immunofluorescence showed that, in the infected plants exposed to non-toxic levels of Cd, virions entered the vascular tissue but were unable to exit it into the non-inoculated, systemic organs. Thus, viral entry into and exit from the vascular cells may occur by different mechanisms. Furthermore, low Cd concentrations did not induce acquired resistance and acted independently of salicylic acid and pathogenesis related (PR) proteins (Plant J. 16, 13-20). Because viruses often adapt existing cellular machinery for their needs, they likely employ an endogenous pathway for systemic transport of RNA. Recent evidence indicates that cellular RNAs also travel through plant vasculature, revealing a novel type of systemic signaling. Specifically, PTGS, an innate plant defense mechanism, is likely elicited by such RNA signals. Our recent experiments indicate the low levels of Cd reverse PTGS, resulting in 'unsilencing' of the repressed marker genes and suggesting that viral systemic movement and PTGS may share similar transport pathways within plants. Microarray technology is especially useful to identify transcripts selectively induced or repressed under different experimental conditions. As our data indicate that Arabidopsis plants (Col-0 and Col-1) respond to low Cd concentrations by restricting viral systemic movement, we propose to perform Arabidopsis cDNA microarray experiments to identify genes induced or repressed in plants treated with non-toxic (viral movement and PTGS are restricted) but not toxic concentrations of Cd (viral movement and PTGS are not restricted). The results of these experiments may shed new light on plant factors involved in systemic traffic of RNA.
NASCARRAY203 ELEVATED CO2 2005-03-11	The ongoing rise in atmospheric CO2 concentration is likely to be the most significant and pervasive environmental factor affecting plants in the 21st century. A doubling of atmospheric CO2 will impact all plants, regardless of ecosystem or agricultural importance. Furthermore, because of the fundamental nature of CO2 in plant metabolism, it is likely that elevated CO2 will affect a very large number of metabolic pathways. Work in our lab and others has detailed many of the specific effects of elevated CO2 on the biochemistry and molecular biology of photosynthesis [for review see Moore, Cheng, Sims, & Seemann {1999} Plant Cell and Environment 22: 567-582] and it is clear that there are similar dramatic effects on many other plant processes. At the level of gene expression, we and others have begun to document the effects of elevated CO2 on the expression of a few genes in Arabidopsis, but it is likely that the number of genes whose expression is affected is exceptionally large. Microarray technology offers the ideal approach for obtaining a global overview of the effects of elevated CO2 on plant gene expression. This technology will enable us to examine the effects of elevated CO2 on the expression patterns of genes involved in a variety of plant metabolic pathways including carbon fixation, light reactions, photorespiration, respiration, glycolysis and nitrogen metabolism, as well as the associated signaling pathways. Arabidopsis [ecotype Columbia] will be grown hydroponically at optimal nutrient concentrations at either 1000 microl/L or 360 microl/L [ambient] CO2. Vegetative tissues [roots and shoots] will be harvested for RNA preparation at maturity prior to the transition to flowering. These initial experiments will allow us to isolate the effects of elevated CO2 on gene expression and minimize any effects due to developmental changes and nutritional deficiencies. Results from this experiment will identify coordinate patterns of transcriptional regulation. We will use this information to focus our future research on subsets of regulated genes. Examination of CO2 effects on the transcription of these genes will also include developmental time courses, varied nutrient regimes, preindustrial CO2 concentration, and the use of different mutant backgrounds. These studies will help unravel the complex responses of plants that will occur in the doubled atmospheric CO2 content of the 21st century.
NASCARRAY204 DAG2 TRANSCRIPTION FACTOR 2005-03-11	The Dof proteins are transcription factors widely distributed only within the plant kingdom, characterized by the Dof domain, a C2-X21-C2 zinc finger. The unusual conservation of this domain coupled with the presence of the Dof transcription factors in all plants, suggests a crucial role of these proteins in regulating different functions typical of and of universal relevance in plants. By means of a reverse genetic approach, we demonstrated that the Arabidopsis Dof protein DAG1 is involved in the control of red light-dependent seed germination (Papi et al., Genes & Development, 14, 28-33 (2000)). We subsequently identified a T-DNA insertion line in another uncharacterized gene of the Arabidopsis Dof family. A mutant line was identified and the Dof gene was denominated DAG2 for its sequence similarity with DAG1 - 57% aminoacidic identity on the whole protein. dag2 was shown to be a knock-about mutant, as the N-terminus of DAG2 is expressed in translational fusion with the GUS reporter gene. DAG2 and DAG1 might be involved in interacting pathways or even regulate with opposite roles the same gene(s) based on their overlapping expression patterns and on the opposite phenotype of the respective mutants. Purpose of the microarray screening will be to identify genes that are deregulated in the dag2 mutant as compared to the Wassilewskija wild type, with the scope of identifying the regulatory target(s) of the DAG2 transcription factor. This will allow to establish whether DAG2 and DAG1 have opposite effects in controlling the same regulatory circuit(s) in Arabidopsis. According to the DAG2 and DAG1 expression patterns, microarrays should be analyzed with RNA-derived probes from developing siliques - stages 12 to 16 - of dag2 mutant plants and from equivalent tissues of Wassilewskija wild type plants. The analysis concerning DAG2 could be coordinated with that of DAG1, just submitted from our laboratory - proposal number 957-JGE-935-JEJ.
NASCARRAY205 ZINC FINGER 2005-03-11	The zinc finger trascription factor DAG1 controls seed dormancy, in that homozygous dag1 T-DNA knock-out mutant seeds are non-dormant and do not need a red light pulse to germinate (Papi et al., Genes & Development, 14, 28-33 (2000)). The gene DAG1 is expressed in the phloem of developing siliques but not in the embryo, and this represents a first example of maternal control of seed germination, excluding mutants of the seed coat. The phytochrome?mediated pathway for seed germination is still required in dag1 mutant seeds, since a far-red light pulse inhibits dark germination of the mutant seeds. Thus DAG1 may ensure the inactivity, for dormancy initiation, of a component - transported from the mother plant to the seed - that would be activated, to trigger germination, as a consequence of red light perception. Purpose of the microarray screening will be to identify genes that are deregulated in the dag1 mutant as compared to the Wassilewskija wild type, with the scope of identifying the regulatory target(s) of the DAG1 transcription factor. This will allow to shed considerable light on the mechanism of red light?induced seed germination in Arabidopsis. Microarrays should be analyzed with RNA-derived probes from developing siliques - stages 12 to 16 - of dag1 plants and from equivalent tissues of Wassilewskija wild type plants.
NASCARRAY206 AUXIN-INDUCED LATERAL ROOT FORMATION 2005-03-11	One of the most dramatic effects of auxin is its ability to trigger the formation of lateral roots. In this manner auxin is of crucial importance for the development of the root system of plants. Nevertheless, differential screenings to identify genes that are expressed in relation to lateral root formation are scarce. For that reason we have recently started to investigate auxin-induced genes in arabidopsis roots (Neuteboom et al., Plant Mol Biol 39: 273-287, 1999; Neuteboom et al., DNA Research 6: 13-19, 1999). Despite the simplicity of our screening method, our studies showed that interesting genes with novel expression patterns can easily be found in roots. To our opinion it will be very rewarding to investigate auxin-induced changes in gene expression in roots in relation with lateral root formation with the microarray technique. To make the screening more useful for the wider scientific community interested in fundamental plant biology, thus avoiding any special tissue culturing methods, we propose the following: Seeds of the wild-type Columbia ecotype will be sown in lines on plates with half strength MS medium (containing 10 gram per liter sucrose) covered with a fine mesh nylon cloth which allows easy transfer of seedlings. The plates will be incubated at an angle of 60 degrees in a growth chamber set at 21 C under a 16 h illumination cycle. Ten days after germination the seedlings, which on average will have a 2 cm long root, will be transferred to fresh plates (control or containing 1 micromolar of the auxin 1-NAA). After 24 h the roots of the seedlings will be harvested and RNA will be isolated. We propose to pool the roots from at least 10 plates for both treatments and this in duplicate, thus resulting in two 'control RNA' samples and two 'NAA RNA' samples. Differential signals after the use of probes derived from control RNA and NAA mRNA are of interest for: A: Those interested in genes expressed in developing lateral root primordia since after 24 hours of NAA treatment almost all stages of lateral root formation can be found at much higher frequency than in the controls. B: Those interested in gene expression as a consequence of lateral root formation, which is a novel aspect of development of the root system. C: Those interested to find novel auxin-inducible genes.
NASCARRAY207 TMV INFECTION 2005-03-11	Simple RNA plant viruses such as tobacco mosaic virus (TMV) are known to alter the cellular machinery of their hosts in order to replicate their genomes and spread both locally and systemically. In addition, plants actively respond to viral infections in a variety of ways that are aimed at limiting the infection process, including host derived hypersensitive responses and gene silencing. Thus, viruses have a significant impact on the expression patterns of their host's genome. The goal of this proposal is to identify plant genes that are differentially regulated during viral infections that result in disease responses. Specifically, the interaction between TMV, the type member of the tobamovirus group, and the Arabidopsis ecotype Shahdara will be examined. This ecotype allows rapid systemic movement of TMV into upper non-inoculated leaves within five days post-inoculation. This rapid movement is accompanied by severe disease symptoms that include stunting, leaf curling, and necrosis. In comparison, ecotypes such as Columbia permit only limited systemic TMV movement after 15 days post-inoculation and display no symptoms. In addition, Northern blot analysis indicates that the host pathogen related proteins, PR-1 and PR-5, are induced in Shadhara upon infection with TMV but not in ecotype Columbia. The observed responses of Shahdara to TMV infection are similar to other virus - host interactions previously studied in tobacco, tomato, and pepper. This makes the TMV - Shahdara system ideally suited for the investigation of fundamental plant ? virus interactions on a genomic level. For the proposed array experiments, mature Shahdara leaves will be inoculated with 10 micrograms of purified TMV. RNA will be extracted from these leaves at three days post-inoculation. This is the earliest time point in which reliable PR-1 and PR-5 host gene induction can be observed. Both PR-1 and PR-5 will also serve as internal controls to determine the quality of isolated mRNA. Control RNA will be acquired in a similar fashion from mock inoculated leaves of Shahdara. Duplicate sets of RNA pooled from multiple TMV and mock inoculated leaves will be provided for the analysis. It is anticipated that information obtained from these experiments should provide new insights into how viruses regulate the gene expression of their hosts and how plants respond to the presence of a virulent viral pathogen.
NASCARRAY208 RESPONSE TO SYSTEMIC VIRUS INFECTION 2005-03-11	RNA viruses such as tobacco mosaic virus (TMV) have evolved specific interactions with their host's cellular machinery that facilitate virus replication, cell-to-cell movement and systemic spread. Ultimately, these interactions are also responsible for rendering the plant either resistant or susceptible to virus invasion. The objective of this proposal is to assess the regulation of host genes upon the establishment of a systemic virus infection that results in the display of disease symptoms. This study is an extension of an earlier microarray experiment (Proposal # 945-CJC-890-HFG-1) involving the identification of genes induced in response to TMV infection in inoculated leaf tissues. For these studies, the Arabidopsis ecotype Shahdara was chosen because it supports the rapid systemic movement of TMV throughout the plant and exhibits severe disease symptoms, such as leaf curling, stunting, late bolting and necrosis within 14 days of virus inoculation. These responses are similar to those seen in other TMV infected plant species including tobacco, tomato and pepper. Thus, the TMV-Shahdara system is ideally suited for the study of plant-virus interactions. For the current set of experiments, mature rosette leaves of Shahdara plants will be inoculated with 10ug of TMV. At 14 days post inoculation, total RNA will be extracted in duplicate sets from uninoculated upper leaves of TMV infected Shahdara plants. Previous experiments have shown that TMV moves into the uninoculated upper leaves of Shahdara within 5 - 7 days of inoculation and reaches a maximum level of accumulation within 14 days post inoculation. Control RNA will be extracted from upper leaves of mock inoculated Shahdara plants. Control and test poly A+ RNA extracted from these total RNA samples will be submitted for microarray analysis to identify the genes induced at this infection stage. The gene expression profile obtained from this microarray analysis will be useful in identifying plant genes that are altered in tissues that display virus induced symptoms. Such genes should reflect the physiological changes imparted by the virus and provide insight into the disease process. Results from this experiment can also be compared to results from our previous experiment to assess the changing status of specific host genes at different stages of infection. Together this information should provide new insights into how plants respond to the establishment of a virulent virus infection.
NASCARRAY209 T-DNA DISRUPTION OF TTR1 2005-03-11	Recently we identified an Arabidopsis gene, TTR1, that confers tolerance to tobacco ringspot virus (TRSV) (manuscript in preparation). Both sensitive (Estland) and tolerant (Columbia) plants can be infected with TRSV and accumulate virus antigen to similar levels. While TRSV-infected Columbia plants show few symptoms and produce seed, Estland plants elicit a systemic hypersensitive response (HR) to TRSV and die within two weeks of inoculation. The predicted sequence of the amino-terminal domain of TTR1 is most similar to the product of the tobacco N gene, a TIR-NBS-LRR resistance gene. As with N, the TTR1-induced HR is temperature sensitive. At 23C, the HR in TRSV-infected Estland plants follows the movement of TRSV through the plant. At 30C, Estland plants show no symptoms, but rapidly and uniformly develop a systemic HR when shifted to 23C. Unlike N, TTR1 contains a carboxy-terminal extension that is similar to nuclear-localized WRKY plant transcription factors. The predicted TTR1 promoter contains two W-boxes and a GCC-box. The purpose of these studies is to identify very early gene expression events in plant disease responses using the unique system we have developed for the coordinate induction of a HR. Most WRKY genes have been identified by differential gene expression analysis following wounding or exposure to pathogen elicitors. The proteins that sense these stimuli and induce the initial wave of gene expression are largely unknown. Because of its combination of pathogen recognition and transcription activation domains, we hypothesize that TTR1 is one of the elicitor-sensing proteins that initiates and/or controls disease responses.For these studies, TRSV-sensitive 'knockout' and tolerant wild-type Columbia plants will be grown and inoculated with TRSV at 30C. The Columbia TTR1 knockout contains an insertion of an Estland TTR1 gene within the Columbia TTR1 coding region and shows a systemic HR to TRSV similar to Estland. Plants will be maintained at 30C for a week to allow TRSV to move systemically and then shifted to 23C to coordinately induce the systemic HR in the sensitive Columbia line. For this initial test, mRNA will be purified from leaf tissue of the two groups of plants 1 hr after the shift to 23C. If these studies are successful, we will sample additional time points closer to the temperature shift to identify genes that are activated immediately following pathogen recognition.
NASCARRAY210 AGL15 OVEREXPRESSOR VS WILDTYPE 2005-03-11	Plant organs have a limited lifespan, and senescing organs have a very different profile of gene expression than immature or mature organs. We are interested in the transition between the non-senescent and senescent state. What happens at the onset of senescence? Is the transition abrupt or gradual? Does it involve large global changes, upregulation (or downregulation) of selected suites of genes, or relatively small changes initially? To address these questions, we will take advantage of the unique floral phenotype associated with overexpression of the MADS domain factor AGL15. Plants that overexpress AGL15 show delayed senescence, as well as delayed abscission, of the outermost whorls of floral organs (Plant Cell 12: 183-197, 2000). By comparing RNA populations in mature wild type flowers and mature flowers of AGL15 overexpressors, we can examine how gene expression patterns change during the transition between states and identify sequences whose expression increases (or decreases) just as the senescence programs start to unfold. To ensure that the samples we are comparing are as similar developmentally as possible, we will grow wild type plants and plants carrying a CaMV 35S: AGL15 transgene (both in WS background) side-by-side under highly controlled conditions in a growth chamber. At four days after the start of flowering, we will collect samples for RNA preparation. In wild type, the programs that drive tissue senescence appear to be activated soon after flowers open. We will collect the youngest open flower, the bud immediately above it, and the two flowers immediately below it from both sets of plants. RNA will be prepared from the pooled bud/flower samples to give us one wild type (pre-senescent) sample and one AGL15 (non-senescent) sample for microarray analysis. Most studies of senescence processes have involved comparisons between organs of different ages. The advantage of the approach we outline here is that age is no longer an experimental variable: the two samples will be developmentally equivalent in nearly every regard. Through this analysis, we expect to identify molecular events associated with the onset of senescence processes in plant tissues and learn how AGL15 overexpression affects global expression patterns during an important developmental transition.
NASCARRAY211 CO2 STRESS 2005-03-11	CO2 chamber
NASCARRAY212 CAULIFLOWER CURD 2005-03-11	The role of carotenoids in plant biology is well-established, as is their importance to human health. While much has been learned about the molecular biology of the genes encoding enzymes of carotenoid biosynthesis in plants, we still lack a clear understanding of the regulatory factors that modulate the process of carotenogenesis. Further insight into the identity of such factors should reveal strategies for modifying carotenoid content and composition in crops. This would be invaluable for reducing the incidence of human health problems such as vitamin A deficiency that are, to a significant extent, caused by a reliance on staple crops that are poor sources of carotenoids. To gain a better understanding of the regulation of carotenogenesis, we are characterizing the semi-dominant Or gene of cauliflower (Brassica oleracea). This gene causes various non-photosynthetic tissues to accumulate up to 500 times more beta-carotene than is normally found in comparable tissues of wildtype plants, thus turning them orange. We have been unable to associate the Or gene with any particular gene of either the non-mevalonate pathway or of carotenoid biosynthesis. Given that large microarrays for most plant species will not be forthcoming in the immediate future, plant biologists must seek to explore such resources in related species. We expect Arabidopsis microarrays to be useful for gene discovery in related species such as the ecomomically important Brassica species. We propose to use Arabidopsis microarrays to examine global gene expression in tissues of Or vs. wildtype cauliflower. Comparable probe RNA shall be isolated from the periphery of expanding curd tissue (the edible part), where beta-carotene deposition is particularly significant in Or plants, and virtually absent in wildtype. We will isolate RNA from our standard experimental line that is homozygous for the Or gene, and the parent cultivar from which it arose, Extra Early Snowball, or another related cultivar. We expect that this experiment will identify transcripts whose expression is significantly altered in the Or homozygous line, thus identifying candidate genes for further studies and uncovering concerted shifts in gene expression patterns that may reveal a mode of action for Or. Furthermore, this experiment will serve as a benchmark for evaluating the utility of species-specific microarrays as a tool for gene discovery in related species.
NASCARRAY213 OLEATE HYDROXYLASE OVEREXPRESSOR 2005-03-11	The fatty acid composition of plant cellular membranes is highly conserved and subject to little change due to external influences. The majority of the fatty acids in the membranes are polynsaturated and 16 or 18 carbons long. In contrast, seed storage lipids can consist of fatty acids with different chain lengths, degree of unsaturation and a variety of side chains.Overexpression of an oleate hydroxylase from Castor (Ricinus communis), a protein specifically involved in the synthesis of seed oils, results in the accumulation of ricinoleic, lesquerolic and densipolic acid in the reserve oil from transgenic Arabidopsis plants (Broun and Somerville, 1997). However, despite the use of a strong constitutive promoter, no hydroxy fatty acids accumulate in the vegetative tissue of these plants. Furthermore, the lack of hydroxy fatty acids in root tissue was shown to coincide with the absence of any detectable hydroxylase protein (Broun et al., 1998). This indicates that the plant cell is able to quickly remove any unusual fatty acids which might interfer with the hydrophobic nature of the membrane and is able to down regulate the level of the hydroxylase. This experiment is designed to identify the genes involved in this process by comparing the expression levels of genes in non-transgenic Arabidopsis seedlings to those found in seedlings of line TA-14 (Broun et al., 1998). This line contains the oleate hydroxylase gene under control of the CaMV 35S promoter.RNA was extracted from whole, 2 week old, seedlings (Columbia ecotype) grown on MS medium (supplemented with 1% Sucrose) in vertically placed plates.
NASCARRAY214 DST-MEDIATED MRNA DEGRADATION 2005-03-11	RNA was extracted from 33 day old mutant and WT plants and used for the microarray. dst1 is a mutant defective in the DST-mediated mRNA degradation pathway.
NASCARRAY216 ANTHER DEVELOPMENT AND MATURATION 2005-03-11	Jasmonic acid, synthesized in octadecanoid pathway is known to be a signal trasduction molecule involved in regulation of different cellular processes in plants. The role of methyl jasmonate in plant fertility was established by characterization of Arabidopsis male sterile mutants fad3-2 fad7-2 fad8 and opr3. The former mutant lacks hexadecatrienoic acids (16:3) and linolenic (18:3) fatty acid precursor of jasmonate while the latter lacks 12-oxophytodienoic acid reductase, one of the enzymes of jasmonate synthesis. Thus these mutants are unable to synthesize jasmonic acid. These male sterile mutants are made fertile by spraying with methyl jasmonate on flower buds especially during late stages of flower development. The Arabidopsis coi1 mutant, which is unresponsive to jasmonate and related compounds, is also male sterile but fertility is not restored by jasmonate treatment. Therefore it is suggested that jasmonate activates the set of genes during the late stages of anther development and which are probably involved in pollen maturation and fertility. The purpose of this experiment is to identify the jasmonate regulated genes in anther and subsequently assign their functional role in male fertility in Arabidopsis. The male sterile mutant mutant opr3 (ecotype Wassilewskija) will be grown in standard environmental conditions and will be used as a control to isolate a specific stage of anthers. Other plants will be sprayed with jasmonate and anthers will be isolated after 1 hr of the treatment. The RNA from both control and jasmonate treated anthers will be used to make probes for hybridization to microarrays. Ref: 1. Stintzi, A. & Browse, J. (2000) Proc. Natl. Acad. Sci. USA 97, 10625-10630. 2. McConn, M. & Browse, J.(1996) Plant cell 8, 403-416. 3. Feys, B. J. F., Benedetti, C. E., Penfold, C. N. & Turner J. G. (1994) Plant cell 6, 751-759.
NASCARRAY218 BLUE LIGHT TIME COURSE 2005-03-11	With the use of a cDNA microarray consisting of 11,500 Arabidopsis cDNA elements generated as part of the Arabidopsis Functional Genomics Consortium (AFGC; http://afgc.stanford.edu). Gene expression profiles of wild type Arabidopsis seedlings were monitored at early stages during the transition from dark to light growing conditions, including de-etiolation, cotyledon expansion and inhibition of hypocotyls elongation. Red or blue light was used as light source to investigate the effects of light quality on Arabidopsis seedling photomorphogenesis. Note: A high fluence blue light has caused elevated temperature in the growing area of the treated sample. So please expect to see some heat shock related alteration of gene expression in this set of experiments.
NASCARRAY220 CIS-JASMONE AS A NOVEL SIGNALLING MOLECULE 2005-03-11	Cis-jasmone as a novel signalling molecule in plant gene expression
NASCARRAY221 CYTOKININ TREATED LEAVES 2005-03-11	NA
NASCARRAY222 DARK RESPONSIVE DURING SEEDLING DEVELOPMENT 2005-03-11	Dark responsive gene expression during Arabidopsis seedling development
NASCARRAY224 EFFECTS OF ASCORBATE IN THE ASCORBATE-DEFICIENT VTC MUTANT 2005-03-11	NA
NASCARRAY225 RESPONSE TO IRON DEFICIENCY. 2005-03-11	Rationale: We would like to arrive at a molecular understanding of iron uptake in plants and to determine how cellular iron status may regulate the uptake, storage and utilization of iron. Understanding iron uptake in plants is important as one-third of the world's soils are iron deficient. Furthermore, iron deficiency in humans is the most prevalent nutritional problem in the world today, affecting an estimated 2.7 billion people. Iron deficiency, along with deficiencies of the micronutrients vitamin A and iodine, are often referred to as 'hidden hunger.' Clearly, we need to understand how plants acquire iron, both from the point of view of improving plant growth and crop yields and for improving human nutrition. Food-based solutions to 'hidden hunger' offer sustainable solutions to problems of malnutrition. For most of the world, plant material is the major source of iron so any progress we can make to ensure plants have higher stores of bio-available iron will go a long way towards solving an important problem in human nutrition. Experiment: We will compare the pattern of gene expression in roots of wild type plants (ecotype Columbia) grown under iron-sufficient conditions to that of roots from plants grown under iron-deficient conditions. We expect to identify 3 classes of genes: those which display up-regulation, down regulation, or those whose expression remains unchanged. We know that a number of genes are transcriptionally responsive to iron availability, including those encoding a Fe(III) chelate reductase (FRO2), a specific isoform of a H+-ATPase (AHA2), and an iron transporter (IRT1). Expression of all three genes is induced in roots under iron deficiency. We would also like to look at patterns of gene expression in roots of frd3 mutants grown with and without iron. frd3 mutants express Fe(III) reductase activity under both iron-deficient and iron-sufficient conditions, unlike wild type plants. They also show constitutive expression of IRT1. Despite having de-regulated iron deficiency responses, frd3 phenotypically resembles an iron-deficient plant i.e. it is chlorotic. Taken together, these data suggest that FRD3 may be a regulatory gene. The ability to determine whether the expression of other iron-regulated genes is also affected in a frd3 mutant should allow us to initially focus on a smaller number of genes than we anticipate will be identified in the experiment using wild type plants grown with and without iron.
NASCARRAY227 ETHYLENE TREATED LEAVES & SEEDLINGS 2005-03-11	NA
NASCARRAY228 FUNGUS INOCULATION 2005-03-11	NA
NASCARRAY229 GENES EXPRESSED BETWEEN PLANTS AND CULTURED 2005-03-11	Our final goal of this project is to identify the factors that confer the ability to cultured cells for the production of secondary metabolites. A few of the cultured cell lines have been reported to maintain the ability to produce secondary metabolites,however, it is well known that most of the cultured plant cells do not display the ability to produce secondary metabolites which are produced in plants. We are interested in analyzing why most of the cultured cells do not produce the secondary metabolites that are produced in the plant tissue and why some cultured cell lines can maintain the ability to produce secondary metabolites. Although it is most likely that the genes which are involved in the production of secondary metabolites are inactive in most of cultured cells, it is not characterized which genes are expressed between differentiated tissue cells and dedifferentiated cultured cells or between the cultured cell lines that produce a certain metabolites. To approach the key factors that involved in the production of secondary metabolites in cultured cells, as the first step, we will compare the gene expression pattern between leaves, roots and cultured cells by isolating mRNAs form Arabidopsis plants and cultured cells, and characterize the difference between cultured cells and intact plants on the gene expression level
NASCARRAY230 TRANSCRIPTIONAL PROFILING OF WT, AXR3-1 AND ARX3-1R4 2005-03-11	Microarray is a powerful tool to examine global transcriptional profiles (TF). If a mutation in a transcriptional regulator results in altered phenotypes, there should be differences in TF,implying downstream events of the mutation. Axr3 mutants are pleotropic semi-dominant mutants which encode a modified Aux/IAA protein (IAA17), a transcriptional regulator. The axr3-1 allele is a P to L change caused by a base substitution in conserved domain II and leads to a severe phenotype. The phenotype of the root of axr3-1 (Co) is shortened with loss of gravitropism, and is less sensitive to auxin by 500 fold. An intragenetic suppressor (revertant) of axr3-1, axr3-1 R4, shows the WT-like phenotype. A single base change in the axr3-1 R4 allele within conserved domain IV results in replacement of half of domain IV with 37 new amino acids.Protein-protein interaction between Aux/IAA and ARF (a transcription factor) is suggested as a regulatory mechanism in auxin-responsive plant growth and development. To answer the question, how a mutation within domain II causes a severe phenotype and how an additional mutation within domain IV of axr3-1 causes phenotypic reversion, protein-protein interactions were examined in a yeast two-hybrid system. IAA17 and axr3-1 proteins interacted with each other, ARF and other Aux/IAA proteins. However, axr3-1R4 protein did not interact with ARF or other Aux/IAA proteins. Much research in auxin signaling has been conducted on Aux/IAA proteins, but little is known about downstream events. Downstream genes of Aux/IAA proteins potentially can be identified with this mutant. Its identical genetic background as WT except for the mutation and its severe phenotype predict differences in TF between WT and axr3-1, implying genes involved in the phenotype associated possibly with events related to downstream IAA17. In addition, examination of the TF between WT and the revertant should provide mechanistic information of IAA17 protein-protein interactions. If the TF are the same between WT and revertant, the importance of domains III/IV is verified and suggests that the protein is a negative regulator; if not, the involvement of additional downstream genes in the phenotypic reversion is indicated. Identifying a downstream gene(s) will contribute to understanding of auxin biology and plant growth.RNA will be extracted from the roots of WT, axr3-1, and the revertant grown for one week under light:dark cycle.
NASCARRAY231 EPIDERMAL CELL DIFFERENTIATION COMPONENTS REGULATED BY GLABRA2 2005-03-11	GLABRA2 (GL2), a member of the HD-ZIP class of transcription factors in Arabidopsis, is involved in trichome, stomata and root-hair formation. Our work is attempting to clarify the role of the GL2 gene in epidermal cell differentiation. We followed two complementary approaches. We showed that the gl2-5 mutation can be complemented inducing the activity of GL2-G in 35S::GL2-G, gl2-5 plants. We propose the identification of putative targets of GL2 by microarray analysis. RNA samples will be prepared from10-days-old 35S::GL2-GR, gl2-5 seedlings treated (+) and untreated (-) with the DEX for 24h.
NASCARRAY232 IDENTIFICATION OF NICKEL -RESPONSIVE GENES 2005-03-11	Plants that hyperaccumulate nickel account for over three-quarters of all known species of metal hyperaccumulators, yet almost nothing is known about the molecular processes that allow these plants to accumulate this ion to concentrations exceeding 1% of their dry biomass. As with the zinc hyperaccumulator species Thlaspi caerulescens , Alyssum lesbiacum (Brassicaceae) is relatively small and slow growing. Identification and characterisation of genes involved in Ni hyperaccumulation in this species would be of use in developing plants to clean up Ni-contaminated soils (a process known as phytoremediation). It would also reveal much about the underlying mechanisms of metal ion homeostasis and trafficking in plant cells. Very little is currently known about Ni homeostasis in plants other than its requirement as a component of the enzyme urease, and no Ni-responsive genes have been identified to date in any eukaryote.Previous studies have shown that A. lesbiacum can survive in hydroponic culture with Ni concentrations in excess of 1 mM. Exposure to Ni leads to a linear increase in xylem histidine concentrations over a range of Ni concentrations. When exogenous histidine was supplied to the root medium of A. montanum (a non-hyperaccumulator), it ameliorated the toxic effect of Ni and increased Ni flux through the xylem. This suggests that histidine plays a central role in Ni detoxification (probably in the cell cytoplasm), and transport from root to shoot. However, the molecular basis of this response has still not been elucidated. The majority of Ni in the leaf tissue is water extractable, suggesting that much of it is stored in the vacuole. Yet no tonoplast (or other) membrane Ni transporters have been identified to date.We propose to identify Ni-responsive genes in A. lesbiacum by comparing 3-week-old hydroponically grown plants exposed to either 0 mM (control) or 0.3 mM NiSO4 for 6 hours prior to harvesting and RNA extraction. Arabidopsis microarrays should be an excellent means of identifying changes in the expression of conserved genes in closely related species of Brassicaceae. Data from the 13 genes cloned so far in our laboratory from A. lesbiacum indicates high sequence identity between the coding regions of homologous genes in the two species (range 87 to 93%).
NASCARRAY233 TARGET GENES OF THE TRANSCRIPTION FACTOR MYB4 2005-03-11	Identification of target genes of the transcription factor Myb4, involved in cold tolerance
NASCARRAY234 IDENTIFICATION OF GENES REGULATED BY A HISTONE DEACETYLASE 2005-03-11	Acetylation and deacetylation of histones are known to play important roles in regulating gene expression in eukaryotes. Histone acetyltransferases and histone deacetylases (HDACs) have been identified in large multisubunit complexes that target these enzymes to specific sites in nuclear DNA. In an attempt to identify auxin response mutants in Arabidopsis, we used natural and synthetic auxin response elements, linked to both the GUS reporter and a hygromycin resistance gene (HPH), in a genetic screen for plants with elevated expression of these transgenes. Five mutant lines with elevated expression of both the GUS and HPH transgenes were allelic, and the mutant gene was identified by fine mapping followed by sequence analysis. The gene encodes a HDAC, related to yeast RPD3, which we refer to as AtHDAC1. The purpose of this proposal is to identify genes that are regulated by AtHDAC1, using DNA microarrays. One of the mutant AtHDAC1 alleles contains a G to A transition in the second intron acceptor site from the 5' end of the gene. RNA gel blot analysis indicated that AtHDAC1 mRNA transcript levels are reduced in this mutant, and may be entirely absent. This mutant line has the strongest visible phenotype, characterized by increased branching and reduced fertility. Duplicate poly A+ RNA samples will be isolated from seedlings of this mutant line, and from wild-type Columbia, the ecotype from which the mutant was derived. A comparison of the microarray hybridization patterns between mutant and wild-type seedlings should indicate which genes other than the auxin regulated transgenes show altered expression in the mutant line. There have been few studies on the functions of HDACs and other chromatin remodelling enzymes in plants, and the identification of AtHDAC1 mutants in Arabidpsis provides us with an exciting opportunity to examine whether this enzyme has global or selective effects on gene expression.
NASCARRAY235 KNOCKOUT THE KDEL-PREOTEASE 2005-03-11	Senescence of higher plant is the final phase of plant organ.s development, involving the degradation of cellular constituents whose amino acids and sugars are transported for use in other organs. Vacuolar remodeling is a key feature of senescence in which the vacuole is converted from multifunctional organelles to a lytic organelle that mediates extensive autophagy. An emerging new aspect of vacuolar remodeling and senescence is the role of ER-derived protease precursor vesicles in transforming the vacuole. The protease precursor vesicles contain a unique member of the cysteine protease family that terminates in the ER-retention sequence KDEL. There are two expressed KDEL-protease genes in Arabidopsis that we have analyzed with RT-PCR. Vacuolar remodeling is an essential feature of plant development and in stress reaction occurring whenever there are large changes in tissue and cellular morphology. A knockout of KDEL-protease genes could impede vacuolar remodeling and the formation of ER-derived vesicles. We screened the Univ. Wisconsin T-DNA insertion pool with RT-PCR using primers designed to be gene specific for the two Arabidopsis KDEL-protease genes and isolated an insertion mutant for one of the genes with the insert in the first exon. The lack of the KDEL protease results in a phenotype where leaf senescence is greatly delayed with the mutant plants leaves remaining green long after the wild type plant.s leaves have died. This mutant also appears to exhibit increased drought tolerance. A complete analysis of the gene expression of this mutant using array technology will provide us with means to assess whether the mutant leaves persist in a normal vegetative state, or whether the leaves that persist because senescence gene products are not effectively utilized. We propose to use the Arabidopsis arrays to study the gene expression of the KDEL-protease knockout in comparison with the wild type plant. We will compare the gene expression of wild type green leaves (1 slide), wild type early senescence (1 slide), and mutant persistent green leaves (2 slides for replicate). These experiments will determine the whether the mutant leaves persist in a normal vegetative state or whether the mutant leaves express senescence specific genes but in the absence of vacuolar remodeling mediated by the ER-derived protease precursor vesicles the leaves retain a vegetative phenotype.
NASCARRAY236 LIGHT TREATED SEEDLINGS 2005-03-11	NA
NASCARRAY237 MECHANOSTIMULUS AND DARKNESS INDUCIBLE GENES 2005-03-11	Genes upregulated by stress provide insight into physiological changes that enable plant adaptation to diverse environments. An unusual set of genes, the Arabidopsis TCH genes, are upregulated in response to mechanical stimuli (Braam and Davis, 1990). However, darkness, temperature shocks, auxin and brassinosteroids also induce TCH expression. The characterized TCH regulatory regions lack canonical cold, heat and auxin cis-elements; thus, we hypothesize (Braam 2000) that these stimuli act directly to regulate some genes (e.g., COR, HSP, IAA/Aux) and indirectly for others (e.g., TCH) by causing mechanical perturbations. Our first proposed microarray experiments will be to determine and compare profiles of genes upregulated 30 min after touch and darkness. Perception of these stimuli is likely to occur through distinct mechanisms and yet both lead to rapid and strong TCH expression. The calmodulin-related and xyloglucan endotransglycosylase TCH proteins are proposed to function collaboratively in modifying cell walls and turgor at sites of mechanical stress (in response to touch) and cell expansion (in response to darkness) (Braam et al., 1997; Campbell and Braam 1999). A global view of the genes upregulated under these conditions will shed light on these fundamental cellular processes. The functions of the genes will be investigated using insertional mutagenesis and inducible RNAi expression methods ongoing in the lab to dissect TCH gene function.We will employ 2 slides to identify touch-inducible genes and 4 slides for darkness-inducible genes. The Davis lab has a (2-slide) duplicate experiment planned to identify touch-inducible genes; our 2-slides will provide 4-fold replication to verify our combined results. We will employ the Davis lab protocol (aerial portion of 3.5-week-old Columbia). Each replicate (untreated and treated) will be performed on identical age plants at different times of day on different days to enhance reliability of the results. We will also take advantage of available microarray data that profile genes induced by hormones (IAA and BR) and temperature shifts. We expect that genes related to the TCH genes in regulatory and functional properties will be induced by all the stimuli. The TCH genes will be positive controls if available on slides, or alternatively on northerns of RNAs to be requested from labs performing related microarrays.
NASCARRAY238 OVEREXPRESS A LAMMER KINASE 2005-03-11	NA
NASCARRAY239 RED LIGHT TIME COURSE 2005-03-11	With the use of a cDNA microarray consisting of 11,500 Arabidopsis cDNA elements generated as part of the Arabidopsis Functional Genomics Consortium (AFGC; http://afgc.stanford.edu). Gene expression profiles of wild type Arabidopsis seedlings were monitored at early stages during the transition from dark to light growing conditions, including de-etiolation, cotyledon expansion and inhibition of hypocotyls elongation. Red or blue light was used as light source to investigate the effects of light quality on Arabidopsis seedling photomorphogenesis.
NASCARRAY240 REGULATION OF AN OVERLAPPING SET OF GENES 2005-03-11	A widely accepted hypothesis is that methylation of cytosines recruits histone deacetylases which, in turn, deacetylate local histones to repress genes. The hypothesis is supported by biochemical data which show that methyl binding proteins associate with histone deacetylases in large multi-protein complexes. The hypothesis is also supported by our studies of nucleolar dominance in hybrid plants showing that repressed ribosomal RNA genes can be derepressed by either aza-deoxycytosine (aza-dC; an inhibitor of cytosine methylation) or by trichostatin A (TSA; an inhibitor of histone deacetylation). In nucleolar dominance, there is no additive or synergistic affect when both aza-dC and TSA are used, implying that cytosine methylation and histone deacetylation are partners in the same gene silencing pathway. However, other evidence in our laboratory suggests that this is not true for all genes. Specifically, studies of silenced transgenes have shown that many respond to blocking cytosine methylation with aza-dC but do not respond in similar fashion when histone deacetylation is blocked with TSA. These data suggest that only a subset of genes are regulated in a manner in which cytosine methylation and histone deacetylation are partners in a simple linear pathway. Using microarray data we hope to further test this hypothesis. Specifically, we would like to compare the genome expression profiles for Arabidopsis thaliana Col-0 seedlings germinated and grown for 17 days on sterile media containing no chromatin modifying chemicals (control), or on media containing aza-dC, TSA, or both aza-dC and TSA. This design should allow us to determine the sets of genes that respond (both positively and negatively) to cytosine methylation and to TSA-induced histone deacetylation and to determine if there are synergistic or antagonistic responses when both processes are affected.
NASCARRAY241 RESISTANCE INDUCED BY PAP 2005-03-11	Study of Plant resitance mechanisms induced by pokeweed antiviral protein (PAP)
NASCARRAY242 SALT TREATED SEEDLINGS 2005-03-11
NASCARRAY243 CORDYCEPIN TIME COURSE 2005-03-11	mRNA degradation provides a powerful means for controlling gene expression during growth, development, and many physiological transitions in plants and other systems. Rates of decay help define the steady state levels to which transcripts accumulate in the cytoplasm and determine the speed with which these levels change in response to the appropriate signals. When fast responses are to be achieved, rapid decay of mRNAs is necessary. Accordingly, genes with unstable transcripts often encode proteins that play important regulatory roles. Although detailed studies have been carried out on individual genes with unstable transcripts, there is limited knowledge regarding their nature and associations from a genomic perspective, or the physiological significance of rapid mRNA turnover in intact organisms. To address these problems, we have applied cDNA microarray analysis to identify and characterize genes with unstable transcripts in Arabidopsis thaliana (AtGUTs). Our studies showed that at least 1% of the 11,521 clones represented on Arabidopsis Functional Genomics Consortium microarrays correspond to transcripts that are rapidly degraded, with estimated half-lives of less than 60 min. AtGUTs encode proteins that are predicted to participate in a broad range of cellular processes, with transcriptional functions being over-represented relative to the whole Arabidopsis genome annotation. Analysis of public microarray expression data for these genes argues that mRNA instability is of high significance during plant responses to mechanical stimulation and is associated with specific genes controlled by the circadian clock.
NASCARRAY245 SULFATE IN PLANT DEFENSE AND GROWTH 2005-03-11	Sulfur is used to make diverse plant constituents among them the 23 different sulfur containing glucosinolates believed to be important in plant defense. We are interested in the link between sulfur nutrition, plant defense and plant growth. In earlier experiments we have looked at the influence of sulfate and sulfate starvation on activity and expression of specific proteins and sulfur containing compounds in cruciferous plants including Arabidopsis. In collaboration with Shauna Somerville we have used a small DNA microarray to analyse the effect of sulfate starvation on the transcript profile. These studies indicate that some groups of transcripts and proteins are influenced by sulfate nutrition including e.g lectin like jasmonate inducible proteins and thioglucosidases. Many potential important genes in the metabolic pathways of interest are, however, missing on this smaller array. We therefore want to use a more complete DNA microarray for a better profiling. We will use root tissue of Arabidopsis thaliana ecotype Col. from 3-4 week old plants. Sample plants will be grown on sulfate free medium where the sulfate salts have been replaced by cloride salts, while the control will be grown on medium with sulfate containing salts. At 3 weeks stage (stage 0), plants will be given a complete sulfate containg medium and roots harvested 6, 12 and 24 h thereafter to analyse transcripts regulated during recovery We would like to use 3 biological replicates at each timepoint and therefore ask for 12 arrays. If fewer arrays are available we will give priority to the stage 0 and 12 h after feeding. We are also open to use fewer time points and more replicates if this is recommended.
NASCARRAY246 SUPPRESSION OF CELL WALL ASSOCIATED KINASES 2005-03-11	There are about 1000 protein serine/threonine kinase genes identified in Arabidopsis, out of which, approximately 600 encode for RLKs (receptor like kinases). The functions of majority RLKs are largely unknown. The Arabidopsis cell wall associated kinase (WAK) gene family contains five highly related members whose products are suited for exchanging signals between the cell wall and the cytoplasmic compartment. We have recently shown that the dexamethasone (dex)-inducible WAK4 antisense expression can effectively control WAK protein levels. WAK suppression results in cell elongation inhibition and morphological alternations (Lally et al., 2001 Plant Cell, in press). We are very interested in how WAKs are specifically involved in cell expansion and Arabidopsis development. Our analyses have shown that the induced WAK suppression causes a substantial decrease of expansin transcript suggesting that WAK suppression may interact with or influence expansin. We hypothesize that there are other genes, particularly those involved in cell expansion and development, could also be affected by WAK suppression. In this proposed study, we would like to systematically compare the gene expression profile of plants in which the WAKs are suppressed by dex with that of plants of normal WAK expression (no dex treatment). Two identical groups of WAK4 antisense plants will be grown under standard conditions. At 8-leaf stage one group will be sprayed with 20 micro molar dex and the other group will be sprayed with a mock solution. 48 hours after spraying leaf tissue will be harvested from both group and used for RNA extraction. This is when phenotypes are starting to develop. We plan to request duplicate slides for each treatment. The microarray analysis of the isolated RNA will help us identify genes that are regulated in response to WAK suppression. The elucidation of these genes would provide valuable insight as to how WAKs function in cell expansion, and perhaps help us identify WAK-mediated signaling pathway components.
NASCARRAY247 TARGET GENES OF PEPPER AP2 TRANSCRIPTION FACTOR 2005-03-11	We isolated transcription factors from pepper plants inoculated bacterial pathogen. The transcription factors were induced during hyper-resistance(HR) reaction. Therefor we expect these genes could be involved in disease resistance mechanisms. As you know that it is very hard to do trnsformation in pepper plants. Therefore we generated several transgenic lines by using Arabidopsis. The sole reason to using Arabidopsis was that Arabidopsis has lots of sequences and the cDNA chip can be avaliable. Anyway our major goal is finding target genes of the transformed genes. For this purpose, we are trying to establish this system; functional analysis of pepper genes using arabidopsis. As a first step, we are trying to do a pilot experiment using one transgenic line over expression of AP2 transcription facot. We want compare col-0 as control plant and transenic line using tissues of leaves. In my oppion, this approach will be very useful both arabidopsis research and non-arabidopsis researcher. This approach give clues the fuction and/or working mechanisms of the gene in different organisms.
NASCARRAY248 TNT INDUCIBLE PROMOTERS 2005-03-11	PURPOSE. The production and use of trinitrotulene (TNT) since the early part of this century has resulted in the widespread contamination of military bases, manufacturing facilities and wastewater in these areas. We aim to engineer transgenic plants to monitor environmental contamination by TNT. This will be accomplished by identifying TNT responsive promoters and fusing these promoters with GFP. When TNT is present in the soil, the plant will produce GFP, which can be remotely sensed. RATIONALE. TNT is a xenobiotic nitoaromatic compound for which very few biological systems have evolved specialized pathways. Recently however, plants were shown to be capable of degrading TNT. The sequential reduction of the nitro groups is thought to be mediated by non specific enzymes that reduce low potential electron carriers such as glutathion reductase, cytochrome P-450, thioredoxin reductase. To date their inducibility by TNT has not been investigated, and no other TNT inducible plant gene has been reported. We propose to identify genes responsive to TNT using microarrays. EXPERIMENTAL DESIGN. To identify TNT doses affecting growth and development of Arabidopsis ecotype Col1, seeds were plated under sterile conditions on Arabidopsis MS media. After incubation at 4 degrees for 4 days, the plates were transferred to 25 degrees with a photoperiod of 16 hours. Nine days later plantlets were transferred to Magenta boxes containing 50 ml Arabidopsis MS media with TNT concentrations of 0, 1, 5, 10 and 20 uM. Toxicity was assessed as percentage of root length, rosette length and diameter relative to control 10 days after transfert to Magenta boxes. We observed that a low concentration of 1 uM TNT stimulated root and rosette growth. Higher concentrations inhibited growth, with a concentration of 10 uM reducing growth by 50%. We therefore plan to investigate the expression profile at concentrations of 1 and 10 uM TNT. With 2 technical replicates for each dose, the experiment will require the use of 4 slides. PERSPECTIVE. At 1 and 10 uM TNT we expect to find genes that are specific to the pathways affected by TNT as well as genes involved in a general stress response. A substractive comparison with existing databases will be conducted. We are also planning to investigate other nitro-substituted explosives at UNCG when we will be equipped with a scanner. Ultimately we will be able to narrow down the candidate promoters to be fused to GFP and assessed in plants.
NASCARRAY249 VIRUS-INDUCED REGULATION 2005-03-11	Simple RNA viruses such as tobacco mosaic virus (TMV)are known to alter the cellular machinery of their hosts in order replicate their genomes and spread both locally and systemically. In addition, plants actively respond to viral infections in a variety of ways that are aimed at limiting the infection process, including host derived hypersensitive responses and gene silencing. Thus, viruses have a significant impact on the expression patterns of their hosts' genomes. Our ealier microarray experiments (Proposal numbers 945-CJC-890-HFG-1 and 958-BDG-334-IBH-7) were aimed at identifying host genes that are differentially regulated during TMV infection. The Arabidopsis ecotype, Shahdara, was used for these experiments since it allows rapid systemic movement of TMV as well as displays marked disease symptoms such as stunting, necrosis, leaf curling and late bolting. Gene expression was monitored in infected whole tissues of Shahdara at 4 days (inoculated tissue) and 14 days (systemic tissue) after inoculation with TMV. Expression profiles obtained from these two time points i.e. 4dpi and 14dpi, should reflect host gene expression in response to the presence of a virulent viral pathogen and gene expression in symptom production, respectively. We now propose to verify the expression profiles of the previous two experiments using biological replicates from similarly obtained poly A+ samples. For the proposed experiments, four microarray slides will be used - the first two slides to monitor expression of host genes at 4 dpi and the second set of two slides to similarly monitor gene expression at 14 dpi. Mature Shahdara leaves will be inoculated with 10 ug of purified TMV. At 4 dpi and 14 dpi, total RNA will be extracted in duplicate sets from inoculated and systemic leaves, respectively. Control RNA will be similarly obtained from mock-inoculated Shahdara plants. Poly A+ RNA isolated from the test and control total RNA samples will be used for microarray analysis. Results thus obtained from the use of biological and technical replicates should help verify the accuracy of expression profiles from the previous two experiments. Together this information should provide a better understanding of the physiological changes occurring within the host in response to the establishment of a virulent viral pathogen.
NASCARRAY250 WHITE LIGHT TIME COURSE 2005-03-11	With the use of a cDNA microarray consisting of 11,500 Arabidopsis cDNA elements generated as part of the Arabidopsis Functional Genomics Consortium (AFGC; http://afgc.stanford.edu). Gene expression profiles of wild type Arabidopsis seedlings were monitored at early stages during the transition from dark to light growing conditions, including de-etiolation, cotyledon expansion and inhibition of hypocotyls elongation. Red or blue light was used as light source to investigate the effects of light quality on Arabidopsis seedling photomorphogenesis.
NASCARRAY252 RESPONSE TO DEOXYNIVALENOL (DON) 2005-03-11	Deoxynivalenol (DON, vomitoxin), a trichothecene mycotoxin, mainly produced by Gibberella zeae, inhibits protein synthesis via binding to the ribosome and interfering with peptidyltransferase. DON causes apoptosis (cytotoxicity) and immunotoxicity to eukaryotic organisms. In animal cells, trichothecene activates mitogen-activated protein kinases(MAPKs) (including SAPK/JNK,p38MAPK and ERK) (Yang et al, 2000). DON appears to be unique in that other classes of trichothecenes do not activate these protein kinases. In plant, DON is a virulence factor in the pathogenesis of G. zeae on wheat (Desjardins et al 1996). Our primary work on the mechanism of DON toxicity to Arabidopsis (unpublished) also showed that DON inhibits germination dramatically. However we still have little data about the molecular mechanism of DON toxicity to plants. What pathways in the plant are involved in responding to the toxin? What genes are turned on or off during cell apoptosis by the toxin? To further understand overall gene profiling of plants in response to DON, we propose to utilize the Arabidopsis Functional Genomics Consortium microarray to compare the Arabidopsis response to DON treatment. This genome-wide study will provide important clues to facilitate the improvement of plant resistance to DON. As a first step, we will prepare RNAs from young seedlings of Columbia wild type treated with DON and water. These microarray data from the treated and untreated with DON will be useful for further understanding of toxicology of DON to plants.
NASCARRAY253 AFFYMETRIX AND CDNA MICROARRAYS COMPARISON 2005-03-11	NahG A-Affy replication
NASCARRAY254 MUTANTS RESISTANT TO THAXTOMIN A 2005-03-11	Thaxtomin A is a phytotoxin produced by the gram-positive filamentous bacterium Streptomyces scabies, the causal agent of common scab disease in potato. We identified a mutant of Arabidopsis, designated txr1, which exhibited resistance to growth on media containing thaxtomin due to a difference in the rate of uptake of the toxin. The TXR1 gene was identified by map-based cloning and found to encode a 12.7 kD soluble protein with no apparent motifs or organelle targeting signals. The protein, which has homologs in all fully sequenced eukaryotic genomes, is expressed in all tissues of the plant. Microarray analysis of the effect of the mutation on the expression of 14,300 genes identified two stomatin-like genes that exhibited altered expression. We proposed that TXR1 is a component of, or regulator of, a dispensable transport mechanism.
NASCARRAY255 PROTEIN IMPORT INTO CHLOROPLASTS 2005-03-11	Purpose: We have isolated several Arabidopsis mutants which are defective in protein import into chloroplasts. The purpose of our experiments, using the microarrays, is to identify and characterize genes that are differentially expressed between the wild-type and mutants. Knowledge obtained from this study will not only help us understand the mechanism and regulation of chloroplast protein import but will also further our understanding of chloroplast biogenesis in general. Rationale: Using a transgene-based screening strategy similar to the one used to isolate yeast mitochondria protein import mutants (1), my laboratory has isolated several Arabidopsis mutants that are defective in protein import into chloroplasts. The traditional way of characterizing these mutants is to identify the defective loci by chromosome walking and then characterize the gene products by homology search, localization and identification of interacting components. All of these approaches are labor-intensive and do not always warrant a meaningful return. In contrast, the most comprehensive approach of unveiling the physiological consequences caused by the mutation, i.e. understanding the complete function of the gene product, is the one offered by the microarray technique. By hybridizing the microarrays using probes made from the mutants and the wild type, the entire spectrum of genes that are differentially expressed among the mutants and the wild type can be identified. In addition, for mutations that cause a severe reduction in the mRNA level of the encoding loci, microarrays also offer the possible advantages of identifying the mutant genes without going through the difficult and labor-intensive work of chromosome walking. However, in this proposal, we will first use a mutant for which the mutation locus has been identified and compare its gene expression pattern with its corresponding wild type. The mutant has a pale green phenotype early in development and in young tissues. The leaf color eventually darkens to a level comparable to the wild type. Plant tissue and treatment Whole seedlings grown on synthetic medium (1X MS salts and 2% sucrose) under 16 h light/8 h dark cycle at 22 degree C for 7 days. genotypes for RNA preparation Columbia Reference: 1. Maarse et al., EMBO (1992) 11, 3619-3628.
NASCARRAY256 BEAN1 MUTANT 2005-03-11	Wild Type leaf vs. leaf from mutant with deletion of disease resistance locus.
NASCARRAY257 SHOOT INDUCTION 2005-03-11	We are interested in identifying gene expression changes that occur during shoot development in Arabidopsis. We have set up a system to regenerate shoots from root explants employing a procedure originally developed by Valvekens to generate transgenic Arabidopsis plants. We have improved on the procedure by finding Arabidopsis lines that develop shoots synchronously and in great profusion. We are using this system in conjunction with mutants to study the hormonal control of organogenesis. We expect to see substantial gene expression changes during shoot development, as many differences as one might expect to find in comparing roots to shoots. In the regeneration procedure, we first incubate root explants on a callus induction medium (CIM) during which time they acquire 'competence' to respond to hormones that induce shoot formation. Explants are then transferred to cytokinin-rich shoot induction medium (SIM) where they organize meristems and undergo shoot morphogenesis. We are interested in identifying a) genes expressed during the acquisition of competence that may encode components of the hormone signaling pathway and b) genes expressed during shoot formation that may be involved in organizing meristems and organ primordia. A typical time course is 18 days total, 4 days on CIM and 14 days on SIM until shoots begin to emerge. Since this process occurs in phases and stretches out over a fairly long time course, we would like to request special permission to survey the microarrays with probes representing 3 time points: zero time derived from freshly explanted roots, a pooled 'early' set representing 2 and 4 days on CIM and 2 and 4 days on SIM, a pooled 'late' set representing 6, 8, 10 and 12 days on SIM. The pooled time points will be used to generate our own smaller arrays for more extensive time course studies and analysis of mutants with defects in shoot organogenesis.
NASCARRAY258 PROFILING GENES EXPRESSED DURING ARABIDOPSIS SHOOT DEVELOPMENT IN TISSUE CULTURE 2005-03-11	We are interested in identifying gene expression changes that occur during shoot development in Arabidopsis. We have set up a system to regenerate shoots from root explants employing a procedure originally developed by Valvekens to generate transgenic Arabidopsis plants. We have improved on the procedure by finding Arabidopsis lines that develop shoots synchronously and in great profusion. We are using this system in conjunction with mutants to study the hormonal control of organogenesis. We expect to see substantial gene expression changes during shoot development, as many differences as one might expect to find in comparing roots to shoots. In the regeneration procedure, we first incubate root explants on a callus induction medium (CIM) during which time they acquire 'competence' to respond to hormones that induce shoot formation. Explants are then transferred to cytokinin-rich shoot induction medium (SIM) where they organize meristems and undergo shoot morphogenesis. We are interested in identifying a) genes expressed during the acquisition of competence that may encode components of the hormone signaling pathway and b) genes expressed during shoot formation that may be involved in organizing meristems and organ primordia. A typical time course is 18 days total, 4 days on CIM and 14 days on SIM until shoots begin to emerge. Since this process occurs in phases and stretches out over a fairly long time course, we would like to request special permission to survey the microarrays with probes representing 3 time points: zero time derived from freshly explanted roots, a pooled 'early' set representing 2 and 4 days on CIM and 2 and 4 days on SIM, a pooled 'late' set representing 6, 8, 10 and 12 days on SIM. The pooled time points will be used to generate our own smaller arrays for more extensive time course studies and analysis of mutants with defects in shoot organogenesis.
NASCARRAY259 PBS3 MUTANT 2005-03-11	Global Gene Expression Analysis of the Arabidopsis Defense Mutant pbs3
NASCARRAY260 EDR1 MUTANT 2005-03-11	Microarray Analysis of the Arabidopsis edr1 Mutant
NASCARRAY261 IDENTIFICATION OF GENES IN CHLOROPHYLL STARVATION 2005-03-11	We have isolated a mutant, conditional chlorina (cch) that disassembles its light harvesting complex (LHC) under moderate light. The biochemical activities that underly this disassembly process have been proposed to consist of proteases that specifically degrade LHC proteins and enzymes that convert Chl b into Chl a. We propose using cch in a microarray analysis to try to define genes that are essential for LHC disassembly. When grown at low light (60 micromol photons m-2 sec-1), cch has a near normal phenotype, but when transferred to moderate light (150 micromol photons m-2 sec-1), cch appears yellow-green. The molecular basis of the cch mutant is a missense mutation (P642L) in the H subunit of Mg-chelatase, the first committed step in Chl versus heme biosynthesis. Demands on Chl synthesis increase after transfer to moderate light, and the mutant Mg-chelatase cannot synthesize Chl at an adequate rate. We hypothesize that during acclimation, the Chl that had been bound to the LHCs is recycled so that it can be used to build new photosystems in the growing cell. As Chl b can only be used for LHCs, it is likely that Chl b is reconverted into Chl a. It is unlikely that the Chl is detoxified, and then sent to the vacuole as in senescence, because the plant needs Chl. Rather, the initial steps of senescence are occuring (LHC disassembly), but the latter are not (removal of the phytol tail and the Mg ion, and tetrapyrrole ring cleavage). A rapid way to find genes involved in LHC disassembly would be a microarray experiment. We would look for mRNA species that are induced when cch plants acclimate to moderate light. These genes must be specific for LHC disassembly, and not genes whose expression is increased during faster growth rates. We propose to use two mRNA samples, WT and cch, both isolated from plants that had been in moderate light for two days. Genes whose expression is increased by higher growth rates should be found in both samples, and therefore will not be selected. Genes involved in the disassembly of the LHC and in Chl redistribution should only be expressed in cch, and therefore should be selected. The suite of genes selected by the microarray process will be analyzed using BLAST and ChloroP searches, and genes that lack chloroplast leaders will be excluded. Candidate genes that have chloroplast leaders will be overexpressed in WT and in cch, and their effects on Chl levels will be analyzed.
NASCARRAY262 LOL2 MUTANT VS WILTYPE 2005-03-11	We isolated several genes from A. thaliana encoding proteins which share a novel zinc finger motif with LSD1, a negative regulator of cell death and defense response. One of these genes was designated LOL2. Due to an alternative splice site the LOL2gene encodes two different proteins, one of which contains an additional, putative DNA binding motif. Northern analysis demonstrated that LOL2transcripts containing the additional DNA binding motif are predominantly upregulated after treatment with both virulent and avirulent Pseudomonas syringae pv maculicola strains. Two lol2 T-DNA insertion alleles (genetic background: Ws-0) have been isolated, which, surprisingly, differentially regulate LOL2 transcription. One allele, lol2-2, is a reduction of function, while lol2-1 is an overexpression allele. In these lol2 mutants expression of known defense related genes like EDS1, NDR1, PR1, PDF1.2 and Nim1 is differentially regulated as well. Interestingly, one of the mutants also shows enhanced resistance to virulent and avirulent Peronospora parasitica isolates. The Northern data obtained for the lol2 mutants so far suggest that LOL2 acts in a signal transduction pathway leading to disease resistance. Induction of defense related genes appears to be negatively regulated by LOL2. Therefore, the lol2 mutants will prove to be a unique tool to further dissect the events leading to defense in plants. As LOL2 and other defense related genes are differentially expressed in the lol2 mutants, microarrays will be an ideal tool to further investigate the dependence of defense related genes on LOL2 expression and also to identify additional, unknown genes that are regulated by LOL2. For the microarrays, we propose to compare one lol2 mutant allele to the other. Alternatively, we could compare each lol2 allele to wild-type. We will consult with AFGC as to which experiment is most logical, although we prefer the former. We will use RNA isolated from sterile grown, 2-3 week old plants of the two lol2 mutants and/or the wild-type. If desired, plants can be grown in two batches per mutant/wild-type. RNA will then be isolated separately from the two batches and after quantification will be mixed for the microarray assay.
NASCARRAY263 RBZ GENE KNOCKOUT 2005-03-11	We identified the Rbz gene by Ds tagging (Sundaresan et al. 1995 Genes Develop. 9, 1797-810) and by rbz complementation with a full cDNA (unpublished). The recessive rbz knockout is slow to green (wt. vs. rbz), similar to cue (Li et al. 1995 Plant Cell 7, 1599-1610) or yellow heart (Redei 1965 Arab. Info. Serv.) mutants, although crosses show it is not allelic to those on the same chromosome 5. rbz epithelium is normal but the leaves collapse due to retarded chloroplast and palisade cell development. The 80kD RBZ protein has Cys4 zinc fingers with a consensus specific to cytoplasmic nucleoporins which bind Ran (Nakielny et al. 1999 EMBO J. 18, 1982-1995; Haizel et al. 1997 Plant J. 11, 93-103). RBZ also contains three novel repeats, and proline-rich regions similar to SH3 interaction domains (Nguyen et al., 1998 Science 282, 2088-2092). We identified three proteins by yeast 2-hybrid screening and in vitro binding assays which specifically interact with RBZ: 1) IFAP, a coiled-coil, intermediate filament protein which may be nuclear membrane associated (AF004556; McNulty & Saunders 1992 J. Cell Sci. 103, 407-414); 2) another coiled-coil protein (MXM12.P4) similar to a yeast intermediate filament protein involved in intracellular protein transport (P25386); and 3) neoxanthin cleavage enzyme (Z93765), a chloroplast-targeted enzyme. Such RBZ and partner features, as well as the rbz phenotype, suggest that RBZ is involved in nuclear to chloroplast trafficking or signaling via novel cytoskeletal components (Jarvis et al. 1998 Science 282, 100-03). Initial northerns indicate that chalcone synthase and RBCS are over-expressed 5 fold in rbz vs. Ler wt., suggestive of an aberration in light signalling (Mayer et al. 1996 Plant Cell 8, 1951-59). Microarray analysis of rbz vs. wt. would provide a profile of global gene expression in the mutant and lead to a better understanding of RBZ function. We have already prepared 5ug mRNA from whole three week old plantlets of Ler wt. and rbz using TRIzol/Qiagen Oligotex, a protocol we have used for other mutant microarray analyses at Monsanto.
NASCARRAY264 ABI1 MUTANT 2005-03-11	Stomatal guard cells provide a well-suited model cell system to understand early signal transduction events in plants in response to endogenous and environmental stimuli. However, the molecular nature of many components in guard cell signal transduction pathways remains to be elucidated. To obtain a large number of genes involved in guard cell signal transduction, it might be an efficient way to use DNA microarray approach with probes prepared from guard cell RNA which is isolated from ABA-treated or -nontreated plants. Because we have developed a multi-step procedure to isolate Arabidopsis (ecotype, Columbia) guard cells, we could isolate many guard cell genes that are regulated by ABA using the high throughput technique.
NASCARRAY265 POTASSIUM NUTRITION 2005-03-11	Potassium (K+) is a major plant nutrient. In order to fulfil its many physiological functions K+ needs to be accumulated from soil and distributed over the whole plant. Experimental data suggest redundant pathways for root K+ uptake, and the Arabidopsis genome database contains a variety of putative K+ channels and transporters (several of which represented on the AFGC microarray). However, the physiological contributions of individual gene products are only poorly understood, and regulatory elements of K+ uptake and distribution remain to be identified. Here we propose to use microarrays as a tool for high-throughput identification of Arabidopsis genes involved in K+ nutrition, based on the hypothesis that key cDNAs are induced when K+ supplies become limiting for plant growth. Plants will be grown on 2 mM or 60 microM [K+], respectively, and mRNA isolated from 3 week old seedlings.
NASCARRAY266 PHYTOPHTHORA INFESTANS INOCULATION 2005-03-11	Phytophthora infestans, a host-specific oomycete plant pathogen, causes the late blight disease of potato and tomato. P. infestans, also known as the Irish famine fungus, remains a destructive pathogen with annual worldwide crop losses topping $3 billion. However, no sustainable source of genetic resistance is available. A number of plant species, including Arabidopsis, are fully resistant to all known strains of P. infestans. This form of resistance, known as nonhost resistance, could reveal novel defense genes and pathways, but has hardly been studied. With the sequencing of the entire genome of Arabidopsis nearing completion, this plant could develop into a key model for understanding nonhost resistance. We initiated studies aimed at exploiting nonhost resistance of Arabidopsis to Phytophthora. Following inoculation of Arabidopsis rosette leaves with zoospores of P. infestans, penetration of an epidermal cell occurred, followed by active defense responses, including the hypersensitive response. In addition, induction of BGL2 gene expression, a marker for the salicylic acid (SA) mediated defense pathway, was recorded. Inoculation of several known Arabidopsis mutant genotypes, compromised in their SA mediated defense response, did not result in a susceptible phenotype. These results suggest that novel nonhost resistance genes and defense pathways may occur in Arabidopsis and lead to the exciting prospect of utilizing Arabidopsis as a source of resistance genes to economically important pathogens, (Kamoun et al. Trends Plant Sci., 4:196, 1999). The aim of this proposal is to identify novel defense pathways associated with nonhost resistance. The micro-array approach is particularly adapted to the study of nonhost resistance, which is likely to be genetically complex and not easily dissected by mutagenesis. We propose to compare Arabidopsis plants challenged with zoospores of P. infestans to non-infected plants. Complete rosette-stage Arabidopsis (Col-0) will be inoculated by spraying either a zoospore suspension or water onto the leaves. One day after inoculation, leaves will be detached and used for RNA extractions. Gene expression data will generate an overview of activated and repressed disease pathways as well as other events associated with nonhost resistance. These results will provide a rationale for future functional assays of the identified pathways using transgenic knockouts and mutant analyses.
NASCARRAY267 GIBBERELLIN TREATMENT 2005-03-11	Searching for GA-regulated genes in ga5 mutant plants sprayed with or without 100uM GA
NASCARRAY268 XANTHOMONAS INOCULATION 2005-03-11	NA
NASCARRAY269 ALUMINUM STRESS 2005-03-11	Soluble aluminum (Al3+) is toxic to plants. Aluminum stress (Al-stress) is a major limiting factor for crop production on greater than 30% of the world's arable land. How plants respond to Al-stress is not well understood at the molecular level, while our understanding is better at the physiological and genetic levels. This is a proposal to screen for Al-inducible and repressible genes, using microarray technology, to better characterize the molecular aspects of Al-stress response in the light of physiological and genetic information. Al-stress has the ultimate effect of inhibiting root growth and function. Al-stressed plants have poorly developed roots that acquire nutrients and water inefficiently. The root apex is the most sensitive part of the root to this stress. Cell elongation and division are inhibited within minutes of the root being exposed to Al. Cellular metabolism is disturbed in several ways, as Al can displace essential cationic cofactors (e.g. Mg2+), thus inactivating proteins, and also lead to the generation of free radicals. The root apex is also the site of stress perception and response. In maize and wheat, root apices release organic acid ligands (e.g. citrate) to exclude Al within minutes of root exposure. Arabidopsis performs similarly to these plants. Citrate release is induced by Al-stress. Al-tolerance genes have been identified by QTL mapping, using the Col x Ler population. Functions for two of the four QTL have been proposed; candidate genes are being evaluated for further assessment. A confounding factor of these experiments is that Al is only soluble in acidic conditions. Thus, all experiments are carried out at pH 4.2. Arabidopsis tolerates growth at pH 4.2, but does not thrive as the acid stress also has physiological consequences. To identify changes in gene expression that are directly related to the Al-stress, we will grow both the Al-treated and control populations of Columbia seedlings (7 days old) at pH 4.2. Comparisons between existing data sets from Col roots grown under standard hydroponic conditions and both experimental treatments should clarify which genes respond to the acid stress and which respond to Al. RNA will be isolated from the roots of liquid culture grown plants eight hours after transfer to fresh acidic media with and without added Al. According to other studies, an eight-hour treatment should be sufficient to elicit most changes in gene expression
NASCARRAY270 ZINC TRANSPORT 2005-03-11	Phytoremediation of soils contaminated with toxic levels of heavy metals requires the understanding of plant metal transport and tolerance. The numerous Arabidopsis thaliana studies have given scientists the potential for dissection and elucidation of plant micronutrient/heavy metal uptake and accumulation pathways. Thlaspi caerulescens, also a crucifer, tolerates and accumulates very high concentrations of Zn (40,000 ug/g in shoot) and Cd. Our studies of this heavy metal hyperaccumulation model plant have shown enhanced Zn transport into Thlaspi roots and shoots. We also showed altered regulation of ZNT1, a Zn/Cd transporter, contributing to the high Zn uptake. Isolation and characterization of Zn/Cd hyperaccumulation genes may allow expression in higher biomass plant species for efficient contaminated soil clean up. Our laboratory is taking several approaches, including microarray analysis in A. thaliana, to identify additional Zn transport, tolerance and nutrition-related genes whose homologues in T. caerulescens could be involved in heavy metal hyperaccumulation. Additionally, Zn-binding ligands involved in Zn homeostasis or tolerance may be identified. Previously, several A. thaliana Zn transporters were identified via yeast complementation. Because Zn requirement is very low, subtle differences in transporter mRNA levels in relation to plant Zn status may exist, and require extremely sensitive analysis as is found in microarray analysis. Gene products acting in concert to effect Zn uptake, which would not have been identified in complementation experiments, including multimeric transporter proteins, could also be identified. The proposed microarray comparison set consists of probes prepared from root RNA of A. thaliana (Columbia) seedlings hydroponically grown in complete nutrient medium (control) and Zn deficient seedlings grown in -Zn nutrient medium (experimental). Sequence analysis of identified A. thaliana genes will be used to investigate similarities to known Zn and other micronutrient transporters. Homologues of the A. thaliana genes from T. caerulescens and T. arvense, a related non-accumulator, will be isolated and analyzed for differential expression and a hyperaccumulation role.
NASCARRAY271 SULFUR DEFICIENCY 2005-03-11	Sulfur is a macronutrient required by plants. It is taken up from the soil solution as in the form of sulfate anion and assimilated into Cys, GSH, Met and various metabolites. Cys residues in proteins form disulfide bonds that may affect the tertiary structures and enzyme activities. This redox balance is mediated by the disulfide/thiol interchange of thioredoxin or glutaredoxin using NADPH as an electron donor. Thus, sulfate assimilation plays important roles not only in the sulfur nutrition but also in the ubiquitous process that may regulate the biochemical reactions of various metabolic pathways. This study is aimed to identify the genes responsive to sulfur limitation stress. Sulfur limitation initially causes expression of sulfate transporter genes involved in the uptake and distribution of sulfate in plants. We have recently isolated 7 cell type specific sulfate transporter genes from Arabidopsis. In sulfate-starved plants, expression of the high-affinity transporter, AtST1-1, is induced in root epidermis and cortex for acquisition of sulfur. The low affinity transporter, AtST2-1 (AST68), accumulates in the root vascular tissue by sulfate starvation for root-to-shoot transport of sulfate. These studies have shown that the whole-plant process of sulfate transport is coordinately regulated by the expression of these 2 sulfate transporter genes under sulfur limited conditions. Recent studies have proposed that feeding of O-acetylserine, GSH and selenate may regulate the expression of AtST1-1 and AtST2-1 (AST68) in roots either positively or negatively. However, regulatory proteins that may directly control the expression of these genes have not been identified yet.The mRNA for the arrays will be prepared from the roots of Arabidopsis thaliana ecotype Columbia plants grown on the control and sulfate-less media. The quality of the RNA preparation will be strictly controlled calibrating with the expression of the 2 inducible sulfate transporter genes, AtST1-1 and AtST2-1 (AST68). Genes identified on the array are expected to encode the proteins required for acclimation to sulfur limitation stress. Further, they may have regulatory functions for the expression of AtST1-1 and AtST2-1 (AST68). Related papers/ Takahashi, H. et al. (1997) Proc Natl Acad Sci USA, 94: 11102-11107/ Leustek, T. and Saito, K. (1999) Plant Physiol, 120: 637-643.
NASCARRAY272 PHOTOTROPIC STIMULATION 2005-03-11	Our long-term goal is to understand at the molecular levelhow plants perceive changes in their light environment, transduce those signals to their site(s) of action, and ultimately respond in the proper adaptive manner. One light- dependent response we are examining is the phototropic response, which represents an ideal physiological system for the study of not only light-driven signal transduction, but also the regulation of cellular growth. We have used a mutational approach to identify a gene, NPH4, that is necessary, not only for phototropism, but several additional differential growth responses as well. Not unexpectedly, given their pleiotropic phenotypes, null nph4 mutations also disrupt auxin responsiveness, both at the level of auxin-sensitive growth and auxin-induced gene expression. We have cloned the NPH4 gene via positional cloning and found that it encodes the auxin-reulated transcriptional activator ARF7. The Cholodny-Went theory holds that auxin becomes redistributed as a result of tropic stimulation and that resultant changes in local auxin concentrations lead to differential growth that is necessary for tropic curvatures. Given the information gathered through study of the NPH4 locus, it is now reasonable to hypothesize that the growth phase of phototropism is auxin-dependent because, at least in part, proteins necessary for localized growth are transcriptionally up-regulated in response to local changes in auxin concentration. We therefore propose to use microarray technology to identify genes whose expression patterns change in response to phototropic stimulation, in a NPH4-specific manner. In particular, we propose to compare gene expression profiles of wild-type seedlings to those of an isogenic nph4 mutant, after each has been exposed to a phototropic stimulation. Our control panels will be represented by the same genotypes, except that they will be mock-irradiated (represents the basal gene expression response). All changes in expression should therefore be specific to the phototropic stimulation and dependent upon the presence of the NPH4 protein. Verification of light-dependence will be accomplished by northern blot analysis of candidate genes in similarly treated nph1 null mutant seedlings lacking a phototropic photoreceptor. These microarray experiments complement display experiments currently underway in our laboratory.
NASCARRAY273 ANTIMYCIN A TREATMENT 2005-03-11	Detached Arabidopsis leaves were incubated with 10 mM antimycin A in 1/10 strength MS medium for 30 minutes and 4 hr in the dark. Control leaves were not incubated.
NASCARRAY274 GENE EXPRESSION ANALYSIS IN AN ANEUPLOID 2005-03-11	Aneuploid lines have traditionally been important for classical and molecular genetic studies in plants. Although relatively stable aneuploid lines have been maintained for different plant species, the presence of additional chromosomes can potentially lead to physical instability of the genome and, as we have found with a transgenic tobacco aneuploid line, epigenetic changes in gene expression (Papp et al., (1996) Plant J. 10: 469-478). These observations are relevant not only for plant genetics and genome evolution, but also human health since aneuploid genomes are a hallmark of solid tumor cells. The effects of aneuploidy on gene expression are poorly understood and have been studied so far with only a small number of genes. While most of the genes tested are expressed according to the increased gene dosage (i.e. a 50% increase), some are expressed either more strongly or more weakly than expected (Matzke et al. (1999) Bioessays 21: 761-767). To begin to understand how the presence of extra chromosomes influences gene expression patterns, and to identify the features of genes that are downregulated in trisomics, we will use microarray analysis to study global changes in gene activity in Arabidopsis trisomic lines. Our first attempt will involve line T22 (Arabidopsis stock number 3234), which is trisomic for chromosome 2. Plants trisomic for chromosome 2 display a discernable phenotype and we have performed chromosome counts on individual plants selected for this phenotype to confirm the trisomic state (2n = 11). RNA will be prepared from young rosette leaves from plants exhibiting the trisomy 2 phenotype and from disomics of the same ecotype (Col-1) grown under the same conditions. For the two arrays of the experiment, RNA extracted from independent populations of plants will be provided. We hope to identify genes that are epigenetically silenced in the trisomic plants, with the ultimate aim of determining whether there are any common sequence features of these genes.
NASCARRAY275 CELL DEATH 2005-03-11	Purpose: To identify genes whose expression is altered in cell cultures of Arabidopsis during hydrogen peroxide [H2O2] and/or nitric oxide [NO]-induced programmed cell death [PCD]. Rationale: H2O2 and NO are important signalling molecules that induce key cellular responses in plants. H2O2 generation following pathogen challenge is a ubiquitous process intimately linked with the Hypersensitive Response [HR], a form of PCD that represents a central player in an orchestra of defence responses. Plants also generate NO in response to pathogen challenge, and NO and H2O2 act together to initiate a barrage of defence responses. We have shown that Arabidopsis cultures represent an excellent system for biochemical and molecular dissection of those signalling and metabolic processes that lead to H2O2 and NO synthesis and culminate in defence gene expression and PCD. Arabidopsis cells rapidly produce H2O2 following elicitation, and H2O2 induces the expression of defence genes, PCD, and activation of MAP kinases. Bacterial challenge also elicits NO synthesis, NO alone stimulates PCD, and NO and H2O2 together result in even greater levels of PCD. Inhibition of gene expression, cGMP synthesis and caspase activity prevents PCD, but as yet there is a dearth of information relating to genes whose expression is modulated during PCD. Using mRNA differential display, we have already identified several such genes; it is timely now, therefore, to analyse the global patterns of gene expression during PCD by combining our well-defined culture system with the power of microarray technology. We propose to compare gene expression patterns between control cells and those exposed to H2O2 and NO. RNA will be extracted from cells after 3h, by which time cells are already committed to PCD but not yet dead. This will identify genes whose expression is modulated by H2O2 and/or NO; further work using northern analysis will differentiate genes induced by H2O2, NO or both. The identification of such genes will then facilitate functional investigation using knock-out technology and transient expression studies. Plant tissues: Suspension cultures Treatments: (i) Control-treated cells; (ii) H2O2 and NO-treated cells Genotype: Arabidopsis thaliana var. Landsberg erecta
NASCARRAY276 SQN1 MUTANT VS WILDTYPE 2005-03-11	We are interested in using microarray technology to identify genes that are specifically expressed in juvenile and adult leaves of Arabidopsis. Despite the many documented morphological and physiological differences between juvenile and adult phases of development in woody and herbaceous plants, only two molecular differences between these phases have been defined. Therefore, the genes identified in this experiment will not only allow us to characterize the process of phase change in Arabidopsis, but should provide important clues about the regulation of phase change in other species. It is not trivial to identify differences in the pattern of gene expression in juvenile and adult leaves by any method that involves extracting RNA from immature tissue. This is because in an immature shoot leaves at different positions not only have a different identity, but also represent different stages of leaf development. For example, a shoot harvested immediately after germination will have two or more immature juvenile leaves. A shoot harvested later will have several mature juvenile leaves, as well as a series of immature adult leaves in various stages of leaf development. Furthermore, it is difficult to compare juvenile and adult leaves at the same stage of development because these types of leaves grow to be different sizes. Thus, an immature 5 mm juvenile leaf is at a different developmental stage than an immature 5 mm adult leaf. We believe that the squint mutation provides a solution to this problem. Of all the phase change mutations we have isolated, squint has the most dramatic effect on leaf identity. All of the leaves produced by squint mutants resemble adult leaves in their size, shape and pattern of trichome production. Equal important for this study is the fact that squint does not affect the timing of the production of leaves 1 and 2. squint therefore provides a way to obtain leaves of the same developmental age, but with different developmental identities. We predict that genes that are differentially expressed in 7-day old squint and wild type seedlings will very likely represent genes that are differentially expressed in juvenile and adult leaves. This hypothesis will be tested by performing a detailed analysis of the expression patterns of the genes identified in this experiment in both wild type plants, and in mutants that affect the process of phase change.
NASCARRAY277 DEMETHYLATION 2005-03-11	In plants and animals, substantial evidence has implicated DNA methylation in the differential control of gene expression (Bird and Wolffe, 1999). For individual genes, different methylation patterns can be detected when different tissues are compared. These patterns are remarkably consistent among individuals of the same species, leading to the suggestion that methylation patterns are established during gametogenesis and change consistently throughout development (Monk et al, 1987). Several DNA methyltransferases have been identified in Arabidopsis (Genger et al, 1999). Mutations that reduce methylation have a pleiotropic effect on Arabidopsis morphology, indicating that many developmentally important genes are under methylation control. Assessment of DNA methylation patterns in Arabidopsis, however, have thus far been limited to the analysis of one or a few genes. Heterochromatin, in particular, is the most highly methylated portion of the genome ( Vongs et al, 1993 ), and our recent discovery of several genes (and ESTs) located within centromeric heterochromatin (Copenhaver et al., 1999) raises the possibility that these genes, in particular, may be highly sensitive to the state of DNA methylation. We would like to use microarray technology, combined with our analysis of heterochromatic DNA in the centromere regions to address the following questions: Which genes are regulated by DNA methylation? Do genes regulated by methylation reside in particular regions of the genome? Do patterns in heterochromatic regions differ from those in the euchromatic chromosome arms? As a first step toward answering these questions, we propose a 2-slide experiment that will compare gene expression patterns in normal seedlings, and in seedlings that have been treated to remove methylation. Specifically, we have treated Arabidopsis seedlings of the Columbia ecotype with 5-Aza-Deoxycytidine (an inhibitor of methyltransferase) for 10 days. Southern blotting has confirmed that this treatment is sufficient to eliminate DNA methylation at much of the centromeric heterochromatin. We have prepared mRNA from treated plants and from untreated controls, and would like to use this mRNA to identify all of the genes that differ in their expression patterns. Subsequently, we will correlate alterations in gene expression levels with the position of the genes on the chromosome, providing insight into the correlation between methylation state and overall chromatin structure.
NASCARRAY278 SALICYLIC ACID DEPENDENT SIGNALLING IN ARABIDOPSIS 2005-03-11	NA
NASCARRAY279 POWDERY MILDEW INFECTION 2005-03-11	NA
NASCARRAY280 INHIBITION OF THE MITOCHONDRIAL ELECTRON TRANSPORT CHAIN 2005-03-11	Purpose: We will identify nuclear genes that exhibit altered expression from inhibition of the cytochrome respiratory pathway (CRP), which is one branch of the mitochondrial electron transport chain (mtETC). Rationale: One way to alter flux through metabolic pathways is to alter the levels of proteins in the pathways. Plant mitochondria contain many proteins involved in various metabolic processes, including the TCA cycle, respiration, and photorespiration. Most mtETC complexes consist of nuclearly-encoded mitochondrial proteins (NEMPs) and mitochondrially-encoded mitochondrial proteins (MEMPs). NEMPs are produced in coordination with MEMPs of the same complex and pathway and with other proteins in multi- organelle pathways. Enzymes involved in photorespiration, for example, are located in chloroplasts, mitochondria, and peroxisomes and many of the proteins are nuclearly-encoded. We know little about how plants control the expression of NEMPs, especially in coordination with expression of the other enzymes in multi-organelle pathways. We are identifying the signal transduction pathways through which plant cells control expression of NEMPs. We are beginning by exploring changes in expression of NEMP genes following inhibition of the CRP while preparing to study NEMP gene regulation more broadly. Inhibition of the CRP by antimycin A (AA) causes an accumulation of the transcript of nuclearly-encoded, mitochondrial alternative oxidase (specifically the aox1a gene), which is in the second branch of the mtETC. We are isolating Arabidopsis mutants that no longer increase AOX expression in response to AA. It will be interesting to determine if our mutants are also mutants in altered expression of other genes induced by AA treatment. We hypothesize there will be increases in expression of nuclearly-encoded subunits of complex I of the mtETC and decreases in expression of other CRP proteins. Tissue and treatment: We will use 21 day old Arabidopsis plants (Columbia, C24, stock CS906). The tissues from which we will isolate mRNA are: 1) untreated leaves; and 2) leaves treated with 10 mM antimycin A and incubated in the light for 4 hr. This AA treatment causes an accumulation of aox1a transcript, which is a positive control for testing the RNA from our treatments, and possibly, in the microarray experiment. We hope aox1a is on the microarrays because it is found only in higher plants, some fungi, Chlamydomonas and trypanosomes but not in other eukaryotes.
NASCARRAY282 SHADE AVOIDANCE 2005-03-11	Many plants respond to low R:FR ratio specifically with enhancement of cell elongation, and anticipation of flowering to the detriment of leaf production.The shade avoidance response is mediated by the phytochromes, but only recently a candidate, ATHB-2, for downstream factors participating in this response has been identified.ATHB-2 belongs to the HD-Zip class of transcription factors, peculiar to plants. ATHB-2 is rapidly and strongly induced by changes in the R:FR ratio.The phenotypes of plants overexpressing ATHB-2 are reminiscent of those displayed by wild-type plants germinated and grown in FR-rich light (longer hypocotyls and petioles, smaller and fewer leaves, and a thinner root mass).Seedlings with reduced levels of ATHB-2 show reciprocal phenotypes. Together with the tight regulation of the ATHB-2 gene by the phytochrome system, these data imply a major role for this HD-Zip protein in the regulation of the shade avoidance responses.Remarkably, anatomical studies in the hypocotyl of ATHB-2 transgenic plants indicated that the alteration of elongation growth was the result of major changes in both the orientation of cell expansion and the production of the secondary vascular tissue. Plants with reduced levels of ATHB-2 showed shorter epidermal and cortical cells while the proliferation of secondary vascular tissue was found to be strongly increased compared to wild-type plants.On the contrary, the elongated phenotype in the ATHB-2 overexpressing plants was found to be the consequence of the same two events but in opposite direction: a change in the orientation of cell expansion toward elongation in cells that do not divide and the inhibition of secondary cell proliferation. Similar changes have been observed in wild-type seedlings grown in environmental light conditions simulating canopy shade. Finally, our recent findings suggest that there is at some point a convergence between the phytochrome pathway that regulates the shade avoidance responses (through the action of ATHB-2) and auxin pathway(s) (http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?uid=10477292&form=6&db=m&Dopt=b). Here, we propose the identification of downstream components in the shade avoidance responses, some of which might be direct targets of ATHB-2. To this end, RNA samples will be prepared from 2 week-old plants grown in high R:FR ratios (-) and in high R:FR ratios followed by 4h of FR-rich light treatment (+). The RNAs will be prepared from the aerial part of the plants.
NASCARRAY283 CMV GENE IV 2005-03-11	Numerous studies have shown that gene VI of Cauliflower mosaic virus (CaMV) is an important determinant of disease severity in crucifers. For example, studies with chimeric viruses have shown that gene VI is responsible for eliciting chlorosis and mosaic symptoms. Most importantly, it has been shown that transgenic plants that express CaMV gene VI, including Arabidopsis thaliana, exhibit virus-like symptoms. Although CaMV gene VI is biologically important for pathogenesis, little is known about how this gene functions at the molecular level in the virus-host interaction. More traditional methods such as differential display have only provided limited results towards the understanding of disease development in both virus-infected and transgenic plants. The development of microarrays may prove to be a more reliable and powerful tool for an analysis of how viruses affect their hosts. We have created transgenic A. thaliana plants (ecotype Col-0) that express gene VI from two very different strains of CaMV. Plants infected with CaMV strain W260 cause chlorosis and stunting, and transgenic Arabidopsis plants that express W260 gene VI exhibit chlorosis and stunting. Plants infected with CaMV strain D4 exhibit a necrotic flecking symptom (which maps to virus genes other than VI), but no chlorosis. Transgenic lines that express D4 gene VI show no signs of any disease. Thus, these transgenic plants can serve as a good model system for understanding how CaMV gene VI contributes to the formation of specific symptoms because these transgenic lines have nearly identical genetic information, yet the two versions of CaMV gene VI elicit very different responses. We propose two experiments in this application, a total of eight slides. In the first experiment, our highest priority, we will compare two mRNA preparations from symptomatic, transgenic Arabidopsis plants that express W260 gene VI to two mRNA preparations derived from Col-0 control plants. The goal is to identify host genes whose expression is altered in response to a pathogenic form of gene VI. In the second experiment, we will compare two mRNA preparations from symptomless, transgenic Arabidopsis plants that expresses D4 gene VI to Col-0 control plants to identify host genes whose expression is altered by the presence of a nonpathogenic form of gene VI. for both experiments, we will utilize the hybrid experimental strategy, in which two biological and two technical replications are performed.
NASCARRAY284 BRASSINOSTEROID SIGNAL TRANSDUCTION 2005-03-11	Analysis of brassinosteroid (BR) biosynthesis and signal transduction, has shown that BRs are essential for normal plant development. We identified a BR-insensitive mutant in Arabidopsis, named bri1, with severe pleiotropic effects on development including dwarfism, de-etiolation, male sterility and altered leaf morphology (Clouse et al., 1996. Plant Physiol. 111:671-678). The BRI1 gene was cloned by Li and Chory (1997, Cell 90:929-938) and shows strong sequence homology to leucine-rich receptor kinases which function at the cell surface to transduce extracellular signals. We propose to identify genes that involve the BRI1 signal transduction pathway using microarray technology. Genes that require BRI1 for expression will have reduced hybridization intensity in the mutant while genes normally repressed by an active BRI1, will show elevated hybridization signal in the mutant compared to wildtype. Some of these genes may be directly involved in the BR response, while others may be secondary genes. Because bri1 is male sterile, a segregating F2 population (Col x bri1) will be used. Seeds will be surface sterilized and vernalized for 48 hours at 4 C in the dark. Seeds from mutant and wildtype will be placed on top of agar plates with half-strength MS media plus sucrose. Plates will be placed vertically in the dark for 4 days (seedlings will grow on agar surface). Col-0 seedlings will then be scraped into liquid half-strength MS media with sucrose. Mutant seedlings (easily distinguished from wildtype at this stage by their short hypocotyl) will also be rapidly placed in shaking liquid media. Col-0 and bri1 seedlings will be shaken in the light for an additional 7 days, after which seedlings will be rapidly vacuum filtered to remove culture media and frozen in liquid nitrogen. Poly A+ RNA will be isolated from mutant and wild-type tissue using the protocol suggested on the AFGC website. RNA will be used for first-strand cDNA synthesis by AFGC with reverse transcription in the presence of Cy3 (wildtype) or Cy5 (mutant) fluorescent dyes. The entire experiment will be repeated for the second array, reversing the dyes. The power of this approach has been shown in studies of yeast sporulation transcription factor Ndt80. Comparison of a strain lacking Ndt80 vs. wildtype on microarrays identified about 150 genes that required activity of this transcription factor for their expression (Chu et al., 1998, Science 282:699-705).
NASCARRAY285 ROLE OF EGS1 IN GENE EXPRESSION 2005-03-11	Sometimes transgenes are not properly expressed in plants due to gene silencing. In some of the most interesting examples, transgenes are still transcribed but the resulting RNA is rapidly degraded. How does this mechanism of post transcriptional gene silencing (PTGS) work and why does such a mechanism exist? In order to answer these questions, we propose to compare transcripts from a mutant line actively engaged in PTGS to RNA from the wild type parental line. The egs1 mutation causes consistent post transcriptional silencing of a transgene in seedlings. This is the only gene characterized to date that enhances gene silencing. We propose to compare gene expression in egs1 mutants to expression in the parental wild type genomic background to identify genes (other than the transgene target of PTGS) that are affected when the PTGS mechanism is activated. By comparing transcripts of egs1 mutant seedlings to those from age matched wild type seedlings, we expect to identify genes of several classes: 1. genes regulated by the wild type EGS1 gene product; 2. genes with products that are participants in the PTGS mechanism; and 3. endogenous genes that are targets of PTGS. This last class will answer the question of whether PTGS has a developmental function in addition to its role in controlling expression of introduced genes. PTGS has been predicted to protect plants from overactivity of transposable elements, from infection by RNA viruses and potentially to be involved in development. Because PTGS is enhanced in egs1 mutants, this comparison gives us the opportunity to discover genes that are regulated when the PTGS mechanism is activated. Since egs1 mutants resemble wild type plants when grown in the greenhouse, we cannot predict what kind of genes may be regulated by PTGS. However sensitive gene expression comparisons possible by DNA microarray comparisons are the best available method to reveal as yet unknown targets. Knowing which genes that are up or down regulated when PTGS is active will illuminate the function the PTGS mechanism normally plays in plant gene regulation.
NASCARRAY286 DOWNSTREAM GENES OF KN1 2005-03-11	Maize knotted1 (kn1)and its homologs from different plant species are essential for meristem maintenance. In order to further understand their function, we are taking several approaches to identify target genes in Arabidopsis. We have made transgenic Arabidopsis plants that express a fused KN1 - glucocorticoid receptor (GR) protein. This fused protein is cytoplasmic in the absence of an inducer, but translocates to the nucleus upon induction with dexamethasone (Dex). Consequently, the plants show no phenotype without induction, but show the typical kn1 overexpression phenotype upon Dex induction. We propose to use the Arabidopsis EST microarray to compare 2 sets of RNAs from these plants: 1. mRNA of control, uninduced kn1-GR seedlings and Dex + cyclohexamide treated kn1-GR seedlings. We will induce kn1-GR seedlings with Dex but prevent protein translation by using cyclohexamide. The use of cyclohexamide prevents the expression of genes farther downstream of the kn1 signal, which is essential if kn1 induces the expression of other transcription factors. We will collect RNA from noninduced and induced seedlings 8 hours after induction. This time point was selected because immunolocalization experiments using a KN1 antibody showed clear nuclear localization of KN1 protein 8 hours after DEX induction. We predict that the mRNA population of induced seedlings will differ from that of non-induced control seedlings only in the expression of genes that are up- or down- regulated by kn1. 2. mRNA of control uninduced kn1-GR seedlings and uninduced kn1-GR leaves. Since the KN1-GR fusion product is expressed throughout the kn1-GR plants, we expect that some of the resulting genes, differentially expressed between non induced and Dex induced kn1-GR plants, will not be relevant to meristem function. In order to eliminate those before further characterization, we propose to use the microarray for this second set of mRNA. A comparison of the uninduced whole seedlings, control to leaves only of the same uninduced seedlings, will help us enrich for mRNAs relevant for meristem function.
NASCARRAY287 PSEUDOMONAS INOCULATION 2005-03-11	We are interested in the susceptible interaction between Arabidopsis and the virulent bacterial pathogen Pseudomonas syringe pv. tomato strain DC3000 (Pst). Currently, little is known about the molecular mechanisms by which Pst subverts its plant host, although a number of genes required for bacterial virulence have been identified. The Pst hrp gene cluster is required for pathogenicity in susceptible hosts and encodes the components of a type III protein secretion system responsible for the delivery of virulence proteins to the plant host cell. We propose to utilize the Arabidopsis Functional Genomics Consortium microarray to identify Arabidopsis genes responsive to a single Pst DC3000 type III secreted virulence protein. The Pst delta CEL mutant strain lacks 6 ORFs present in the Conserved Effector Locus (CEL) (Alfano et al., PNAS 97:4856-61). The delta CEL mutant multiplies 200 to 500 fold less than wildtype Pst and fails to produce disease symptoms in Col-0 leaves. Complementation analysis of the delta CEL mutant revealed that orf3 and orf4 of the CEL are responsible for the mutant phenotype, but only Orf3 is secreted into the plant host cell (K. Nomura and S.Y. He, unpublished results). We have developed a heterologous secretion system for the analysis of the function of individual type III secreted effector proteins. We have engineered Pseudomonas fluorescens (Pf), a nonpathogenic saprophyte unable to cause disease on Arabidopsis, to contain the Pst Hrp secretion system. This Pf strain is capable of secreting virulence proteins into the plant host cell. For AFGC microarray analysis, a Pf strain with the Hrp system and a plasmid containing orf3 and orf4 of the CEL will be compared to the control Pf strain with the Hrp system and a plasmid containing only orf4. Arabidopsis leaves inoculated with 1 x 108 bacteria/mL of the treatment (Pf orf3 and orf4) and control (Pf orf4) strains will be harvested at 12 and 24 hours post inoculation and used for RNA isolation. Equal amounts of RNA from the two time-points for each treatment will be pooled and submitted for microarray analysis. Our goal is to identify Arabidopsis genes regulated by the Pst CEL orf3 virulence protein.
NASCARRAY288 CYTOKININ RESPONSE GENES 2005-03-11	Purpose: The purpose of the experiment is to identify additional primary response genes for cytokinins, which will serve as a starting point to investigate presently unknown signalling pathways of cytokinins. Rationale: Cytokinins are a class of plant hormones with pleiotropic effects. Due to lack of mutants and suitable systems to study fast responses of cytokinins the signalling pathways of the hormone are not well understood. Fast response genes would provide a starting point for biochemical and genetic studies of early events in cytokinin signalling. A great number of cytokinin-regulated genes has been identified in the past, however, most of the identified genes react slowly to cytokinins and are probably downstream targets and not primary response genes (Schmulling et al, Physiol. Plant. 100, 505-519, 1997). Only one class of rapid cytokinin response genes is currently known. This class of genes has been identified independently in maize and Arabidopsis. The proteins have significant homology to response regulators of the bacterial two-component signalling system and might function in cytokinin signalling. The strongest response shows the ARR5/IBC6 gene which is significantly up-regulated by cytokinins within 10 minutes (Ref.: Brandstatter and Kieber, Plant Cell 10, 1009-1019, 1998; Sakakibara et al., Plant J. 14, 337-344, 1998; Taniguchi et al., FEBS Lett. 429, 259-262, 1998). Material: The starting tissue of the experiments will be cell culture of Col-0. The culture is being grown in cytokinin free medium and will be induced by treatment with 0.1 microM BA. Material harvested at timepoints zero and 15 min will be used for comparison. As a positive control experiment, we have shown previously that the ARR5 gene transcript is in this culture at least six-fold more abundant 15 min after addition of cytokinin.
NASCARRAY289 VERNALIZATION 2005-03-11	Identification of vernalization-associated genes.
NASCARRAY290 VIRAL SUPPRESSION OF THE HYPERSENSITIVE RESPONSE 2005-03-11	The continuing escalation of disease and resistance has led to the development defense strategies by the host and counter- strategies by the pathogen. Inhibition of the host?s programmed cell death response is one counter-strategy exhibited by some animal viruses. To our knowledge Turnip crinkle virus (TCV) is the first plant virus which appears to share this ability. Resistant Arabidopsis Di-17 plants respond to TCV inoculation with the necrotic lesions of a classic hypersensitive response (HR), increases in salicylic acid and defense gene transcription. The virus is predominantly localized to the lesions of the HR. However, in a fraction of the plants, which can vary from 5-50% depending on growth conditions, the virus escapes the lesions. In these plants, the virus spreads to newly emerging portions of the plant, causing disease symptoms similar to those exhibited by TCV- infected, susceptible plants. Strikingly, these systemically infected portions of the plant do not exhibit the HR, though they contain the viral elicitor of the defense response. Further, systemically infected leaves will not produce a HR if directly reinoculated, though leaves of equivalent age on a disease-free inoculated plant will. However, asymptomatic leaves of a systemically infected plant contain little, if any, virus, and do evolve a HR upon direct reinoculation. Thus, the suppression of the HR appears to correlate with the presence of viral RNA. Significantly, recent literature shows that one of the TCV movement proteins, p8, is nuclearly localized. This protein has a high affinity for RNA and also binds DNA. Thus this protein may be altering gene expression patterns. We would like to use the microarray technology to discern differences in gene expression between symptomatic and asymptomatic plants. Di-17 plants will be inoculated with viral RNA. After disease symptoms become apparent, symptomatic leaves and the developmentally equivalent leaves on asymptomatic plants will be inoculated with virus. Tissue will be harvested 3 days post reinoculation, once lesions become apparent on the resisting plants. The reinoculation protocol is designed to compensate for the observed elevation in defense gene expresion in symptomatic leaves relative to uninoculated asymptomatic leaves. It is anticipated that this experiment will provide insight into virally induced differences in gene expression leading to suppression of the HR.
NASCARRAY291 KCL VS NH4CL 2005-03-11	NA
NASCARRAY292 KCL VS KNO3 2005-03-11	NA
NASCARRAY293 ANTISENSE NRT1 VS WILDTYPE 2005-03-11	Nitrate and ammonium are critical nutritional sources for higher plants, and they can also serve as signaling molecules to regulate nitrogen metabolism and plant growth. However, little is known about how signal is transmitted from external nitrate or ammonium to plant growth. Arabidopsis CHL1 is a nitrate transporter involved in uptake. Despite the fact that CHL1, a nitrate inducible gene, can contribute up to 80% of nitrate uptake activity under certain conditions, the chl1 deletion mutant plants can grow typically as do the wild type plants in all tested conditions. A CHL1 homolog AtNRT1:2, a constitutive gene, is also known to involve in nitrate uptake. However, different from CHL1, AtNRT1:2, when its expressing is suppressed by anti-sense construct, shows severe growth defect if ammonium but not nitrate is the sole nitrogen source provided. These observations of our lab indicate that CHL1 and AtNRT1:2 can apparently play different roles in regulating plant growth in response to various nitrogen conditions. We value that DNA microarray system may effectively contribute to evaluate and characterize how a signal is transmitted from external ammonium through the activity of AtNRT1:2 to disturb the cellular physiological processes critical for plant growth. The expression patterns of more than ten thousand genes can be systematically and comparatively analyzed by hybridizing to the RNA isolated from anti-AtNRT1:2 plants grown in ammonium versus the RNA isolated from the wild type. This approach will help us to identify candidates for further studies.
NASCARRAY294 ROP GTPASE SIGNALING NETWORK 2005-03-11	G proteins are universal molecular switches in eukaryotic signal transduction. Two classes of signaling G proteins are known: heterotrimeric G proteins and monomeric RAS superfamily of small GTPases. Animals possess a large number of trimeric G proteins and some RAS GTPases including Ras, Rho, Cdc42 and Rac. In contrast, plants have only one trimeric G protein and appear not to have these GTPase homologs. Instead, plants possess a unique Rac-related subfamily termed Rop. Rop is encoded by a large gene family composed of at least 11 members in Arabidopsis. Our transgenic studies have demonstrated their roles in the regulation of many important processes, including pollen tube tip growth, cell morphogenesis, leaf epidermal and vascular differentiaion, light or hormone-mediated development, and defense responses.
NASCARRAY296 POPLAR DIFFERENTIAL GENE EXPRESSION DURING SECONDARY GROWTH 2005-03-11	The goal of this experiment is to identify the genes that are specifically expressed during seconday xylem formation. Gene expression patterns between sapwood and cambial region of a mature black locust tree will be studied using Arabidopsis microarrays. The gene expression profile we obtain from this experiment will also be used in a comparative study with the gene expression profiles in Arabidopsis.
NASCARRAY297 ETR VS CTR 2005-03-11	NA
NASCARRAY298 CHITIN ELICITATION TIMECOURSE 2005-03-11	Chitin oligomers, released from fungal cell walls by endochitinase, induce defence and related cellular responses in many plants. However, little is known about chitin responses in the model plant Arabidopsis. We describe here a large-scale characterization of gene expression patterns in Arabidopsis in response to chitin treatment using an Arabidopsis microarray consisting of 2375 EST clones representing putative defence-related and regulatory genes. Transcript levels for 71 ESTs, representing 61 genes, were altered three-fold or more in chitin-treated seedlings relative to control seedlings. A number of transcripts exhibited altered accumulation as early as 10 min after exposure to chitin, representing some of the earliest changes in gene expression observed in chitin-treated plants. Included among the 61 genes were those that have been reported to be elicited by various pathogen-related stimuli in other plants. Additional genes, including genes of unknown function, were also identified, broadening our understanding of chitin-elicited responses. Among transcripts with enhanced accumulation, one cluster was enriched in genes with both the W-box promoter element and a novel regulatory element. In addition, a number of transcripts had decreased abundance, encoding several proteins involved in cell wall strengthening and wall deposition. The chalcone synthase promoter element was identified in the upstream regions of these genes, suggesting that pathogen signals may suppress the expression of some genes. These data indicate that Arabidopsis should be an excellent model to elucidate the mechanisms of chitin elicitation in plant defence.
NASCARRAY299 LEAFY SPURGE 2005-03-11	Leafy Spurge leaves vs. meristems on an Arabidopsis array
NASCARRAY300 MERISTEM VS LEAF 2005-03-11	Comparison of Arabidopsis leaves and meristems.
NASCARRAY301 POPLAR LEAF VS MERISTEM 2005-03-11	Poplar leaves vs. Poplar Meristems on an Arabidopsis array to compare gene similarities.
NASCARRAY302 LEAVES VS MERISTEMS 2005-03-11	Comparison of wild oat leaves vs. meristems on an Arabidopsis array
NASCARRAY303 AP2 MUTANT 2005-03-11	Each floral organ is composed of a unique set of tissues. To gain an understanding of how patterning of these tissues occurs during development, we study mutations which affect the final distribution of tissues in the floral organs. To further investigate the nature of these mutations, cell type specific markers are neeed. We propose using Arabidopsis floral mutants to uncover cell type and/or tissue-specific genes expressed in each floral organ. The Arabidopsis homeotic mutants agamous (ag) and apetala2(ap2) result in near reciprocal homeotic transformations of floral organs; in ag, petals and sepals replace stamens and carpels, respectively, and in strong ap2 mutants, carpels replace sepals and petals are absent due to ectopic expression of AG (reviewed in [1]). Thus, the flowers from each mutant will only have two of the four organ types normally found. We propose comparing mRNA expression patterns in the two mutants to detect genes expressed only in sepals and petals, and only in stamens and carpels. These genes may be expressed at any time during the development of the organs, and mRNA isolated from flower clusters containing all of the different developmental stages from each mutant would be used for the microarray pairs. In these comparisons, we would expect to see a lack of expression of certain mRNAs in each mutant sample that would correspond to mRNAs from the missing floral organs. There should be petal and sepal mRNAs present in ag that are not present in ap2 flowers, and carpel and stamen mRNAs in ap2 that are not in ag. Such an approach has been used successfully in a differential display experiment to isolate two carpel specific mRNAs; pi andpi ag mutants were compared [2]. The proposed experiments will not distinguish between sepal and petal specific mRNAs, and stamen and carpel mRNAs, but it will be a first step in finding floral organ/tissue specific mRNAs, and distinguishing between genes expressed in the perianth and reproductive organs. These may be target genes for regulatory organ identity genes, and researchers studying these transcription factors may follow up on the identification of these genes by analysis of their promoter regions, etc. Furthermore, their function may be critical to differentiation of specific tissues, and this may be of interest to researchers in the future. 1. Weigel, D. and Meyerowitz, E. M. (1994) Cell 78, 203-9. 2. Yung MH, Schaffer R, Putterill J (1999) Plant J. 17:203-8.
NASCARRAY304 TISSUE COMPARISON 2005-03-11	The advent of microarray technology has enabled researchers to assay thousands of genes simultaneously. This powerful capability allows for easy profiling of different plant tissues and organs which can serve as a base set of expression data for use in transcript localization and cluster analysis. The tissues tested include flowers, leaves, stems, and siliques derived from soil grown plants and root tissue from plants grown hydroponically.
NASCARRAY305 MATURE ROSETTE VS SEEDLING 2005-03-11	Test of the first CIW 11.5K arabidopsis print (CIW-2), using leaves vs. etiolated seedlings for maximal difference in gene expression. Also some doping/spiking of controls.
NASCARRAY306 ELEVATED CO2, 800 ppm 2005-03-11	Arabidopsis thaliana Col-0 grown at 800 ppm for 30 days. Harvested after bolting. Grown under long days.
NASCARRAY307 AUXIN RESPONSE PATHWAYS & VASCULAR DEVELOPMENT 2005-03-11	BACKGR: In mp leaves the vasculature is reduced, while other tissues seem unaffected. Transcript levels of vascular specific genes should therefore be specifically and dramatically reduced. Further, we have observed that a number of auxin inducible procambial genes are hyper auxin-inducible in 35S:MP plants. Differential profiling of mRNA pools from mp mutant versus 35S:MP cotyledon/young rosette leaves should provide a unique opportunity for the identification of vascular specific genes and auxin inducible genes. This approach is not covered by earlier experiments in mp mutant background, because it will not be restricted to rapid auxin responses and involves a 35S:MP background. RATION: The MP gene encodes an .auxin response. factor and a large number of auxin inducible genes are expressed at low levels in mp mutants and at excessive levels in 35S:MP plants.Northern analysis shows that this is also the case for procambial genes, reflecting the auxin inducibility of vascular differentiation. The experiment aims at identifying genes not rapidly auxin-induced in vascular differentiation. Because of the combined effects of the genotype specific anatomical differences and the genotype enhanced auxin inducibility, we expect extreme differences in transcript abundance. This could help overcoming limitations in assessing minor differences for a large number of genes, whose precise profiles can subsequently been sorted out on smaller arrays. TREATM/GENOT: We suggest assessment at 4 hour after application of 20uM IAA to 35S:MP cotyledons/young rosettes versus 0uM IAA mp mutant cotyledons/young rosettes. PRIOR/LINKS: single experiment/high priority. Open for interaction. TISSUE/RNA: RNA prepared by a senior post-doc, from immersed plants. We have >20 genes with known profiles to test each RNA sample. These internal controls may also help in further microarray optimization. SIGN: Identification of vascular genes is a worldwide enterprise, predominantly done in woody species. This approach in Arabidopsis is cost efficient and the further analysis of identified genes can take advantage of the enormous genomic resources available in Arabidopsis PERSP: Our long-term goal is understanding signaling in vascular development using mutant based profiles. DOC: We can provide quantified Northern data to demonstrate the feasibility of the approach. RELEASE: pref.3 months LIT: EMBO J 17, 1405-1411
NASCARRAY308 CIRCADIAN RHYTHM IN COL & LAN WILDTYPE & MUTANTS 2005-03-11	Five-week old wild-type (Columbia), lhy cca1 null mutants (Lansdsberg erecta) and CCA1 overexpressing (Columbia) Arabidopsis plants were entrained in a 2:12 light:dark photoperiod for four days and then released into continuous light (LL). Whole plants (excluding roots) were harvested every four hours for the second 24 hour period in LL.
NASCARRAY309 CIRCADIAN RHYTHM 2005-03-11	Plants respond to day/night cycling in a number of physiological ways. At the mRNA level, the expression of some genes changes during the 24-hr period. To identify novel genes regulated in this way, we used microarrays containing 11,521 Arabidopsis expressed sequence tags, representing an estimated 7800 unique genes, to determine gene expression levels at 6-hr intervals throughout the day. Eleven percent of the genes, encompassing genes expressed at both high and low levels, showed a diurnal expression pattern. Approximately 2% cycled with a circadian rhythm. By clustering Microarray data from 47 additional nonrelated experiments, we identified groups of genes regulated only by the circadian clock. These groups contained the already characterized clock-associated genes LHY, CCA1, and GI, suggesting that other key circadian clock genes might be found within these clusters.
NASCARRAY310 ERF DOMAIN TRANSCRIPTION FACTORS 2005-03-11	ERF domain proteins, a subfamily of ERF(EREBP)/AP2 domain proteins, are members of a novel family of transcription factors unique to plants. A highly conserved DNA binding domain known as the ERF domain is the unique feature of this family. To characterize this novel family of transcription factors in detail, we isolated five cDNAs encoding ERF proteins from Arabidopsis (AtERF1 to AtERF5) and analyzed their DNA binding specificity, transactivation ability and mRNA expression. AtERF1, AtERF2, and AtERF5 functioned as transcriptional activators and specifically bound to the GCC box (AGCCGCC), displaying a very similar in vitro DNA binding specificity. A number of stress inducible genes, such as genes for PDF1 and chitinase, have been shown to contain the GCC box in their promoter regions, and these AtERFs have been implicated to act as transcriptional regulaters for these genes. However, it is not known whether the genes target for AtERF1, AtER2 and AtERF5 are different or not. It is important to analyze the target genes for each AtERF to study the molecular mechanism how transcription factors that possess same DNA binding specificity, recognize their target genes. For this purpose we have prepared transgenic Arabidopsis plant where AtERF1, AtERF2 and AtERF5 are overexpressed and isolated mRNAs form two independent lines of each transgenic plants. We will analyze the regulated genes by AtERF1, AtERF2 and AtERF5 using DNA microarrays. Based on the results, we will study the role of ERF proteins on the regulations of stress related gene expression in more detail
NASCARRAY311 PROVASCULAR DIFFERENTIATION 2005-03-11	NA
NASCARRAY312 SALICYLIC ACID-DEPENDENT COUPLED REGULATION OF CELL GROWTH AND CELL DEATH 2005-03-11	In higher eukaryotes, the regulation of programmed cell death (pcd) and cell growth is often coupled. Using a dominant gain-of-function mutation in the Arabidopsis ACD6 gene, we recently showed that plants, like animals, have coupled regulation of cell death and cell growth responses (Rate et al., Plant Cell 11, 1695-1708, 1999). acd6-1 plants show constitutive activation of cell death, cell growth and pathogen defenses and require at least two signals that act together to achieve these phenotypes. One of these signal molecules is salicylic acid (SA). Our goal is to determine the identity of the second signal and determine how it acts with SA to regulate cell fate. SA is absolutely required for the regulation of defenses in plants and it is sometimes required for the regulation of cell death. Until now, SA was not known to play a clear role in cell growth responses. Removal of SA from acd6 plants using a nahG transgene, whose product metabolizes SA, completely suppresses all the acd6 phenotypes. The acd6-conferred phenotypes are restored and hyperactivated by treatment of the acd6nahG plants with low levels a synthetic analogue of SA. Activation of the SA pathway does not induce cell death or cell growth in wild type plants. This led us to propose that SA acts with a second signal to regulate these cell fate changes. Because reactivation of the SA pathway hyperactivates the acd6 phenotypes, SA likely negatively regulates the second signal.s level or activity. Using the microarray technology could point us to candidate genes and pathways that are regulated by the second signal. In acd6nahG plants, the second signal should be building up so that when we reactivate the SA pathway, we get a hyperactivated response. By comparing the gene expression patterns from nahG and nahGacd6 plants, we will identify SA-independent genes regulated by the second pathway. If the regulatory signal is already known for these genes, then we will be in a position to directly test the interaction between it and SA in inducing cell growth and cell death. We believe that to interpret this experiment, it needs to be accompanied by a comparison between nahG and wild type, to establish what pathways that are acd6-independent might be affected by the SA pathway. This is basic information that would be useful for the community of researchers interested in SA-dependent responses. Our experiments require RNA prepared from the following leaf samples: acd6nahG, nahG, wild type (Columbia)
NASCARRAY313 Gentle Mechano-stimulation 2005-03-11
NASCARRAY314 ECOTYPE COMPARISON(DNA) 2005-03-11	Ecotype Comparison(DNA)
NASCARRAY315 SUCROSE REGULATION OF GENE EXPRESSION 2005-03-11	Synopsis. Sucrose (suc) concentration in the guard-cell (gc) apoplast depends on environmental and physiological parameters, suggesting that it may be a signaling molecule that regulates gc gene expression to balance transpiration and photosynthesis. In collaboration with the lab of Dr. George W. Bates (Bio, FSU), our initial study will examine effects of suc on gene expression in leaf slices, and will be followed by gc studies at a later date.Rationale and Purpose. In apoplastic phloem loaders such as Arabidopsis, suc is excreted from the mesophyll into the apoplast. Then, suc diffuses to the phloem, where it is taken up by H+-suc co-transport. These facts imply that the apoplastic suc concentration is heterogeneous and depends on the rates of photosynthesis and translocation. Additionally, suc reaches 200 mM in the gc apoplast because of evaporation from this site. Therefore, the osmotic effect of external suc decreases stomatal aperture size when transpiration is high and CO2 is not limiting.Suc is a known regulator of gene expression. We propose that leaves adapt to a particular environment through suc (and ABA) regulation of gc gene expression. As an example, high external suc should depress gc Calvin-Cycle activity, avoiding an elevation of P-glycerate that would stimulate starch biosynthesis. In this way, gc starch degradation proceeds in light, releasing the H+ (via malate synthesis) that balance the H+ extruded during stomatal opening. As implied above, suc would affect gc gene expression in an environment conducive to high rates of photosynthesis and transpiration. However, gc are interspersed with other cells and the ordinary means of cell isolation (protoplasts, 'isolated' gc on epidermal strips) would confound an interpretation of transpiration-linked gc gene expression. As a first step, we therefore propose to study suc control of gene expression in leaf strips. These data will provide a baseline for future studies of gc specific gene expression.Plant Tissues. Ecotype Columbia will be grown at 20ÂºC under a 16-h photoperiod to promote large leaf size. Low light (150 ÂµmolÂ·m-2Â·s-1) and high humidity (80% RH) during leaf development will be maintained to minimize leaf apoplastic suc. Leaf strips, 2-mm wide, will be vacuum infiltrated with 5 mM PO42- (pH 6.1), 0.1 mM Ca2+ and Â± 150 mM sucrose. After incubation for 4 h with aerated solution, leaf strips will be harvested and RNA will be isolated.
NASCARRAY316 XANTHOPHYLL-INDEPENDENT PHOTOPROTECTION 2005-03-11	When exposed to excess light, plants sustain damage to their chloroplasts. This photodamage is caused largely by the formation of free radicals and reactive oxygen species. These radicals interact with, and destroy proteins of the photosynthetic apparatus and cause lipid peroxidation in the thylakoid membranes. One intensively studied photoprotection mechanism is the dissipation of excess excitation energy, observed as non-photochemical quenching (NPQ) of chlorophyll fluorescence. The xanthophyll cycle, leading to accumulation of zeaxanthin, plays a major role in NPQ. The xanthophylls were also shown to have anti-oxidant function in thylakoid membranes.Characterization of NPQ-deficient mutants of Arabidopsis has shown that acclimation to prolonged high light conditions can occur in the absence of the xanthophyll cycle. Recent studies also show that in young leaves, xanthophyll cycling is not necessary for photoprotection. These finding strongly suggest that other mechanisms of photoprotection are able to compensate for the lack of the xanthophyll-dependent NPQ and anti-oxidant functions. Over-accumulation of tocopherols, changes in the composition of the light-harvesting complexes and thylakoid membrane structure, and chloroplast movement may be involved in acclimation of xanthophyll- and NPQ-deficient mutants to provide the observed compensation in photoprotection.Here we propose to compare the expression pattern of high light-exposed wild-type plants with that of the triple mutant npq1npq4lut2. This triple mutant is deficient in zeaxanthin, PsbS (a PSII subunit that is necessary for NPQ), and lutein (a carotenoid with a structural role in the light harvesting complexes, which may also have NPQ and anti-oxidant activities). We will grow triple mutant and wild-type plants in short days under low light conditions (150 Âµmol photons m-2 s-1) for 5-6 weeks. Plants will then be exposed to high light conditions (1500 Âµmol photons m-2 s-1) for 4 hours, after which leaf tissue will be harvested for RNA extraction. This experiment will be repeated a second time. Physiological and biochemical analyses will be performed in parallel. We predict that compensation for the NPQ and anti-oxidant deficiencies of the triple mutant will be evident in the gene expression pattern, compared with that of the wild-type. Genes that show differences in expression will be used for further study of photoprotection in Arabidopsis.
NASCARRAY317 glutamine sensing 2005-03-11	Plants co-regulate carbon and nitrogen assimilation, metabolism, transport and storage by sensing the concentration of certain metabolites. In the presence of methylamine (MA), an ammonium analog, the growth of Arabidopsis thaliana is markedly reduced because methyl-glutamine accumulates. This metabolite signals N sufficiency to plant cells and metabolism is altered to favour C assimilation, transport and storage. A hypersensitive mutant was isolated by selection on media containing methylamine. The mutants segregated for green:chlorosis (3:1) and for sensitivity to exogenous ammonium chloride (1:2:1). The mutation altered the enzyme activity of GS, NR and GDH and was dominant, overdominant and codominant in effect respectively. Transcription of several genes was altered, the lesion eliminated the ability of GDH NR ASN and GS to respond to exogenous C and N sources, glutamine and sucrose. Therefore, we conclude the underlying gene participates in glutamine sensing and regulation. The gene underlying the mutation has been isolated in from a BAC library and will be identified by complementation. Gene expression networks in all cases appear to confer hypersensitivity but global analysis of their expression patterns has not been possible until recently. Understanding the nature of gene expression waves of Arabidopsis will allow comparison with the networks underlying glutamine sensing. The identification of common gene homologues between this model system and crop plants, particularly soybean, will also be of import. We will generate probe cDNA from mRNA extracted from wild type Arabidopsis thaliana cv. Columbia seedlings at 14-DAP. Alterations in gene expression encompassing this period must generate the ability to survive glutamine miss-sensing. The analysis will allow the classification of genes within Arabidopsis relative to glutamine sensing. Plants are grown to maturity under conditions of light, temperature and humidity that promote normal development (22 C, 20 hr photoperiod). Synchronously growing plants were selected for tissue collection. Sufficient whole seedlings to purify ten micrograms of mRNA at 14-DAP were collected. Aliquots of seedlings were reserved for probe duplication, quantitative PCR confirmation of expression profiles and proteomics (2D gels, enzyme assay, Western hybridization).
NASCARRAY318 Potassium nitrate treatment 2005-03-11	unknown
NASCARRAY319 Analysis of gene expression in temperature sensitive mutants 2005-06-03	The samples are derived from experiments aimed to describe changes in gene expression under different temperature conditions by using wild type and virescent mutants.
NASCARRAY321 Comparative ABA-treatment of Col-O vector control and transgenic plants 2006-01-03	The aim of this experiment is to understand the impact of overexpression of ERD15 on the transcriptome of Arabidopsis thaliana. ERD15 was isolated from a screen for genes rapidly induced after pathogen treatment from Arabidopsis. This gene was originally found as early responsive to drought (Kiyosue et al., 1994, Plant Physiol 106, 1707). ABA is central phytohormone in drought response, but increasing information is pointing to its significant role in pathogen responses as well. We are interested to see the effect of this hormone on plants overexpressing ERD15 compared to control plants. The samples (rosette leaves)will be harvested from 3-week old soil grown plants 90 min after spraying with 100 micromolar ABA. Comparison will be made with non-treated plant samples.
NASCARRAY324 Low chronic exposure of Arabidopsis thaliana to Caesium-137 2006-01-03	Arabidopsis thaliana plants are grown for one week in a hydroponic growth system and transferred to new plant medium containing low levels of Caesium-137 (control is transferred to new medium with no radioactivity) and left for further two weeks. Levels of Caesium-137 are chosen according to research and are reflecting occurring levels found in radioactive contaminated soil. The plants are then harvested and the samples divided into shoot and root samples.
NASCARRAY326 Targets of AtWRKY27 in Arabidopsis leaves 2006-06-29	The Arabidopsis transcription factor WRKY27 was found to be involved in plant defense towards Ralstonia solanacearum GMI1000. To identify target genes of WRKY27, we introduced a functional tagged version of WRKY27 into two independent Arabidopsis wrky27 knockout lines (ecotype Columbia) under the control of the estrogen receptor-based chemical-inducible system. 18 days old plants grown on soil (Metro Mix 200) at 22oC under a 10/14 h light/dark cycle were treated for 6h with 10 microM beta-estradiol after which RNA was immediately isolated. The two independent knockout lines transformed with the empty vectors (pMD::XVE-SALK and pMD::XVE-ETL) served as controls and were grown under the same conditions and treated identically as the experimental plants.
NASCARRAY327 Novel Target Genes Regulated By LEUNIG 2006-10-27	Regulation of homeotic gene expression is critical for proper developmental patterns in plants. The Arabidopsis thaliana floral homeotic gene AGAMOUS is regulated by LEUNIG (LUG). Mutations in LUG result in homeotic transformations of floral organ identity, lug mutants also exhibit other defects that are independent of AG, including abnormal carpel and ovule development, reduced female and male fertility, and narrower leaves and floral organs suggesting LUG has a wider role in development. LUG is structurally similar to the transcription co-repressors Tup1 (S. cerevisiae) and Groucho (Drosophila) suggesting developmental pathways regulated by LUG may be controlled by a conserved eukaryotic repression mechanism. We aim to determine novel target genes regulated by LUG using transcriptome analysis. A mutant lug-3 line has been obtained from Dr. Zhongchi Liu. This line was used in the initial studies of LUG in the Meyerowitz lab. Lug-3 is a strong allele in the Lansberg erecta background, caused by a nonsense mutation that results in early termination of the protein and is likely null. Lug-3 mutant exhibit narrow floral organs with staminoid petals and carpelloid sepals, abnormal carpel and ovule development, reduced plant height, increased lateral branching and narrow and smaller leaves. We proposed to use the lug-3 line in elucidating the role of LUG as a potential general regulator of transcription in Arabidopsis. mRNA populations will be isolated from wild type (Ler) and lug-3 plants that have produced inflorescence. This stage of development has been selected for several reasons. Firstly mRNA populations from widest range of tissue types from a single plant can be collected. Secondly, as AGAMOUS, which is involved in the regulation of floral development, is present in the Affymetrix gene chip it will provided an excellent internal control to monitor between chips. Results will demonstrate wheter LUG is likely to have a more global role in regulating transcription, and will identify pathways under the regulation of thisco-repressor like protein.
NASCARRAY328 Environmental Genomics of Calcicole-calcifuge physiology 2005-11-04	The purpose of this set of arrays is to provide a third replicate to use in data analysis addition to the 2 previous replicates for all samples already generated on this project. Our hypothesis is that adaptation to a calcareous environment will be reflected in altered gene expression including genes encoding transporters, ion channels, transcription factors, etc. To test this hypothesis we grew a laboratory non-calcicole (Col-4) and a laboratory calcicole (Cal-0) ecotypes of A. thaliana at low (1 mM) and high (12.5 mM) rhizospheric Ca2+ and compare the patterns of gene expression by microarray analysis. We then collected from the wild, a putative calcicole ecotype (Elland)and a putative non-calcicole (Penicuik) and grew both at low (1 mM) and high (12.5 mM) rhizospheric Ca2+ and compared expression profiles to the laboratory ecotypes.
NASCARRAY329 Gene Expression During Recovery from Freezing 2005-11-04	Freezing causes physiological changes even in a hardy, cold-acclimated wild type. During recovery from freezing, gene expression will reflect the induction of damage-repair processes distinct from the damage-prevention associated with cold acclimation. This will be detected by observing the wild-type transcriptome at two time points during recovery from a freezing episode. The appropriate control is the unfrozen, cold-acclimated wild type.
NASCARRAY330 Transcriptome changes of Arabidopsis during pathogen and insect attack 2005-07-25	Plant defenses against pathogens and insects are regulated differentially by cross-communicating signaling pathways in which salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) play key roles. To understand how plants integrate pathogen- and insect-induced signals into specific defense responses, we monitored the dynamics of SA, JA, and ET signaling in Arabidopsis after attack by a set of microbial pathogens and herbivorous insects with different modes of attack. Arabidopsis plants were exposed to a pathogenic leaf bacterium (Pseudomonas syringae pv. tomato), a pathogenic leaf fungus (Alternaria brassicicola), tissue-chewing caterpillars (Pieris rapae), cell-content-feeding thrips (Frankliniella occidentalis), or phloem-feeding aphids (Myzus persicae). Monitoring the signal signature in each plant-attacker combination showed that the kinetics of SA, JA, and ET production varies greatly in both quantity and timing. Analysis of global gene expression profiles demonstrated that the signal signature characteristic of each Arabidopsis-attacker combination is orchestrated into a surprisingly complex set of transcriptional alterations in which, in all cases, stress-related genes are overrepresented. Comparison of the transcript profiles revealed that consistent changes induced by pathogens and insects with very different modes of attack can show considerable overlap. Of all consistent changes induced by A. brassicicola, P. rapae, and F. occidentalis, more than 50% were also induced consistently by P. syringae. Notably, although these four attackers all stimulated JA biosynthesis, the majority of the changes in JA-responsive gene expression were attacker-specific. All together our study shows that SA, JA, and ET play a primary role in the orchestration of the plant's defense response, but other regulatory mechanisms, such as pathway cross-talk or additional attacker-induced signals, eventually shape the highly complex attacker-specific defense response. In the experimental set up we intended to select for genes that showed a more than 2-fold change in the same direction(up or down)at two time points after pathogen or insect attack. The results are described in the following paper: De Vos, M., Van Oosten, V.R., Van Poecke, R.M.P., Van Pelt, J.A., Pozo, M.J., Mueller, M.J., Buchala, A.J., Metraux, J.-P., Van Loon, L.C., Dicke, M. and Pieterse, C.M.J. (2005). Signal signature and transcriptome changes in Arabidopsis upon pathogen and insect attack. Molecular Plant-Microbe Interactions, in press.
NASCARRAY331 Transcriptional Programs of Early Reproductive Stages in Arabidopsis 2005-08-08	The life cycle of flowering plants alternates between a diploid sporophytic and a haploid gametophytic generation. After fertilization of each the egg and central cells by one male gamete, the development of both fertilization products occurs coordinated with the maternally derived seed coat and carpel tissues forming the fruit. The reproduction program is likely to involve the concerted activity of many genes. To identify genes with specific functions during reproduction, we have analyzed the expression profile of more than 22,000 genes present on the Arabidopsis ATH1 microarray during three stages of flower and fruit development. We found 1,886 genes regulated during reproductive development and 1,043 genes that were specifically expressed during reproduction. When compared to cells from an Arabidopsis suspension culture, S-phase genes were underrepresented and G2 and M-phase genes were strongly enriched in the set of specific genes, indicating that important functions during reproduction are exerted in the G2 and M phases of the cell cycle. Many potential signaling components, such as receptor-like protein kinases, phosphatases, and transcription factors, were present in both groups of genes. Members of the YABBY, MADS box, and Myb transcription factor families were significantly overrepresented in the group of specific genes, revealing an important role of these families during reproduction. Furthermore, we found a significant enrichment of predicted secreted proteins smaller than 15 kD that could function directly as signaling molecules or as precursors for peptide hormones. Our study provides a basis for targeted reverse-genetic approaches aimed to identify key genes of reproductive development in plants. During evolution, both animals and plants developed distinct pathways of sexual reproduction. The haploid life cycle starts with the formation of the meiotic products, for instance arrested primary oocytes and spermatocytes in mammals or micro- and megaspores in plants. The haploid phase ends when a sperm cell fuses with an egg cell to form the diploid zygote, which initiates embryo development. In animals, gametes are derived from a population of germ cells committed early during development to their reproductive fate and which usually rest until the onset of sexual maturity. By contrast, stem cells in shoot apical meristems of flowering plants divide continuously during postembryonic development. Only after formation of vegetative organs, the meristem produces specialized reproductive organs in which meiosis occurs, the stamens and carpel-borne ovules of the flower (for review, see Bowman and Eshed, 2000; Carles and Fletcher, 2003). As early as 1790, Goethe suggested that floral organs are derived from leaves and adapted to their specialized tasks (Goethe, 1790). Animal gametes differentiate directly from the products of meiosis, whereas in plants the meiotic products (spores) typically undergo two or three mitotic divisions to give rise to the male or female gametophytes, respectively (Drews et al., 1998; Grossniklaus and Schneitz, 1998). In several plant species, including Arabidopsis, the female gametophyte consists of seven cells: three antipodal cells, two synergid cells, one egg cell, and one central cell that contains two polar nuclei (Drews et al., 1998; Grossniklaus and Schneitz, 1998). The mature male gametophyte comprises two sperm cells contained within a vegetative cell (McCormick, 1993; da Costa-Nunes and Grossniklaus, 2003). During fertilization one sperm cell nucleus fuses with the egg cell nucleus, giving rise to the diploid embryo, whereas the other sperm cell fuses with the homodiploid central cell nucleus, generating the triploid endosperm (Drews and Yadegari, 2002). Subsequent seed and fruit development are highly coordinated processes between the embryo and endosperm, as well as the maternally derived testa (seed coat) and carpels. Because of the significant economical importance of flowers, seeds, and fruits, the complex processes involved in plant reproduction are not only of academic interest but also of high economic relevance. Many genes have been identified genetically that have a function in plant reproduction. The establishment of floral organ identity and initiation of seed development have been studied extensively, and many regulators of these processes have been identified (McElver et al., 2001; Zik and Irish, 2003; for review, see Chaudhury et al., 2001). High-throughput RNA profiling technologies can now complement the genetic and molecular approaches to provide new insights into plant reproduction. In particular, oligonucleotide-based microarrays can produce reliable, high-quality data (Lockhart et al., 1996; Hennig et al., 2003) to establish new biological knowledge on the transcriptional programs that are active during developmental processes (Spellman et al., 1998; Harmer et al., 2000; Becker et al., 2003; Honys and Twell, 2003). Here, we report a comprehensive Affymetrix GeneChip analysis of the Arabidopsis transcriptome at key steps of reproductive development. We identified 1,043 genes that were specifically expressed during reproduction. Among those genes were many potential signaling components, such as receptor-like protein kinases, phosphatases, and transcription factors. In addition, we found a significant enrichment of predicted secreted proteins smaller than 15 kD that could function directly as signaling molecules or as precursors for peptide hormones. Our study provides the basis for targeted reverse-genetic approaches aimed to identify important regulators of reproductive development in plants.
NASCARRAY334 Identifying targets of FLC at 27oC 2005-11-04	FLOWERING LOCUS C (FLC) is a MADS box transcription factor that plays a well characterised role in repressing the vegetative to floral transition of Arabidopsis thaliana. FLC has also been shown to affect the Arabidopsis circadian clock, with mutant seedlings showing short circadian periods. In a previous study, we identified the temperature-dependent circadian period QTL PerCv5b near the FLC locus on the top arm of Chromosome 5. PerCv5b caused a significant period effect at 27oC but not at 12oC or 22oC. Temperature-dependent circadian period phenotypes and a known polymorphism in the Ler allele made FLC a strong candidate gene for PerCv5b. The period effect of FLC was enhanced by combination with alleles of FRIGIDA (FRI), a gene shown to up-regulate FLC's expression. We were interested in identifying how FLC affects the circadian clock, so we decided to identify its target genes. Greatest phenotypic difference was observed between fri; flc and FRI; FLC genotype seedlings at 27oC, so expression was compared between these lines (previously described in Michaels and Amasino1999 and 2001) on the Affymetrix ATH1 microarray. Seedlings were grown on media (MS 1.5% Agar containing 3% Sucrose) for 6 days under constant cool white fluorescent light (55-60 micro EINSTEINS) at 22oC then entrained for 4 days under (12h , 12h) light , dark cycles at 22oC. At dawn on the fourth day of entrainment they were transferred to constant light (25-30 micro EINSTEINS) at 27oC. Four samples were taken at 6 hour intervals starting 24h after the transfer to continuous conditions at the times 24h, 30h, 36h and 42h. Equal amounts of total RNA were pooled from the three time points to produce one sample per genotype. The pooling strategy was employed to reduce the effect of circadian regulation on genes expression. This was particularly important in our case as some interesting genes would likely be regulated by the circadian clock and may only show expression differences at particular phases that could easily be missed if using just one time point.
NASCARRAY335 Over-expression of MBF1c enhances stress tolerance 2005-08-24	Multiprotein bridging factor 1c MBF1c (At3g24500) is a stress-response transcription co-activator. To test the function of MBF1c, we over-expressed it in transgenic Arabidopsis plants using the 35S-CaMV promoter. T4 seeds form 3 independent lines were tested for their tolerance to biotic and abiotic stress conditions. Constitutive expression of MBF1c in Arabidopsis enhanced the tolerance of transgenic plants to bacterial infection, salinity, heat and osmotic stress. Moreover, the enhanced tolerance of transgenic plants to osmotic and heat stress was maintained even when these two stresses were combined. The expression of MBF1c in transgenic plants augmented the accumulation of a number of sugars and defense transcrtipts in response to heat stress. Transcriptome profiling and inhibitor studies suggest that MBF1c expression enhances the tolerance of transgenic plants to heat and osmotic stress by partially activating, or perturbing, the ethylene-response signal transduction pathway. MBF1 proteins could be used to enhance the tolerance of plants to different abiotic stresses. Suzuki et al., 2005 Plant Physiology, submitted.
NASCARRAY336 Determination of the transcriptome of gun1, a mutant defective in plastid-to-nucleus signalling, in 2007-05-30	Background The photosynthetic apparatus in chloroplasts of higher plants is encoded by genes distributed between the plastid and nuclear genomes. Chloroplast biogenesis and the assembly of a functional photosynthetic apparatus are therefore dependent on co-ordinated expression of genes in the plastid and nuclear genomes. There is evidence to suggest that this co-ordination is achieved by retrograde signalling from the plastid to the nucleus. Treatment with norflurazon, an inhibitor of plastid carotenoid biosynthesis, or lincomycin, an inhibitor of plastid translation results in reduced nuclear photosynthetic gene transcription. gun (genomes uncoupled) mutants express nuclear photosynthetic genes in the presence of norflurazon. Five gun mutants have been identified from screens of EMS-mutated Arabidopsis thaliana seed harbouring an Lhcb1 promoter-uidA fusion (Susek et al., 1993). The gun mutants fall into two groups; gun2-gun5 have defects in genes involved in tetrapyrrole biosynthesis (Mochizuki et al., 2001; Larkin et al., 2003) and gun1 has an uncharacterised mutation but is distinct from the gun2-gun5 group (Vinti et al., 2000; Mochizuki et al., 2001). gun1 has been shown to express nuclear photosynthetic genes in the presence of lincomycin, whereas gun2-gun5 do not. Experiment Description RNA will be prepared in triplicate. Two Arabidopsis thaliana lines, Col-0 and the mutant gun1, will be subjected to four experimental conditions as follows: To determine differences between the transcriptomes of Col-0 and gun1 in light the two lines will be grown on 1x Murashige and Skoog basal salts solidified with agar (MS) and containing 2% sucrose. Seedlings will be subjected to a regime of 5 days darkness and two days continuous 200 ?mole m-2 s-1 white light. To determine differences between the transcriptomes of Col-0 and gun1 in the absence of light the two lines will be grown on 1x MS containing 2% sucrose. These seedlings will be subjected to a regime of 7 days darkness. The effect of lincomycin, at a concentration of 0.5mM, on the transcriptomes of both Col-0 and gun1, grown under both light and dark regimes, will be determined. Comparisons between the transcriptomes of Col-0 grown with and without lincomycin, under both dark and light regimes, will reveal which nuclear transcripts are affected by loss of chloroplast translation. Comparisons between the transcriptomes of Col-0 and gun1 grown with lincomycin, under both dark and light regimes, will reveal the transcripts affected by the gun1 mutation. References Larkin, R.M., Alonso, J.M., Ecker, J.R. and Chory, J. 2003. GUN4, a regulator of chlorophyll synthesis and intracellular signaling. Science 299: 902-906.Mochizuki, N., Brusslan, J.A., Larkin, R., Nagatani, A. and Chory, J. 2001. Arabidopsis genomes uncoupled 5 (GUN5) mutant reveals the involvement of Mg-chelatase H subunit in plastid-to-nucleus signal transduction. Proc Natl Acad Sci U S A 98: 2053-2058. Susek, R.E., Ausubel, F.M.and Chory, J. 1993. Signal transduction mutants of Arabidopsis uncouple nuclear CAB and RBCS gene expression from chloroplast development. Cell 74: 787-799.Vinti, G., Hills, A., Campbell, S., Bowyer, J.R., Mochizuki, N., Chory, J. and Lopez-Juez, E. 2000. .Interactions between hy1 and gun mutants of Arabidopsis, and their implications for plastid/nuclear signalling. Plant J. 24: 883-894.
NASCARRAY337 Arabidopsis E2F target genes 2005-08-18	Entry into the S phase of the cell cycle is controlled by the E2F transcitption factors that induce the transcription of genes required for cell cycle progression and DNA replication. To identify E2F target genes of Arabidopsis on a genome-wide scale, the transcriptome of wild-type plants was compared with that one of plants ectopically expressing the E2Fa-DPa genes. In four independent experiments, E2Fa-DPa overexpressing plants were grown side-to-side with wild type (Col-0) plants. RNA was extracted from 6-day-old seedlings. Each biological sample was harvested and processed independently, and finally all probed individually to Affymetrix ATH1 microarrays, resulting into 8 hybridization signals for each probeset.
NASCARRAY338 Hydrogen peroxide stress and Zat12 over-expression in Arabidopsis. 2005-08-22	In this experiment we tested the transcriptome of transgenic Arabidopsis seedlings (5-day-old) constitutively expressing the zinc-finger protein Zat12 (At5g59820) under the control of the 35S-CaMV promoter (Zat12). The transcriptome of these seedlings was compared to that of wild type seedlings grown under the same conditions (WT) and to that of wild type seedlings grown under the same conditions and subjected to a hydrogen peroxide stress (WT+H2O2). Hydrogen peroxide treatment was performed by applying 20 mM hydrogen peroxide for 1 hour. In parallel to these experiments transgenic plants expressing Zat12 were subjected to a similar hydrogen peroxide stress (Zat12+H2O2). All treatments were performed with similar size and age seedlings grown in liquid culture (MS) and sampled at the same time as described by Davletova et al., 2005.
NASCARRAY339 Using transcriptome analysis to define the developmental state of fn10, a mutant in Arabidopsis thal 2005-09-20	The fn10 mutant was identified from the same high-dormancy mutant screen as the cts-1 (comatose) mutant (Russell et al. 2000; Footitt et al. 2002). As well as showing reduced germination potential, the fn10 mutant is hypersensitive to abscisic acid (ABA) and fails to establish on sucrose. In these experiments the transcriptome profile of fn10 seed that has been stored for 1 month will be compared with after-ripened fn10 seed that has been stored for 6 months. Under optimal conditions the non-after-ripened fn10 seed will not germinate, whereas the after-ripened seed will. We will also look at after-ripened fn10 seeds imbibed on 100 nM ABA, a concentration that will prevent germination of the seed. Control samples will be after-ripened wild-type Landsberg erecta (Ler) seeds, and Ler imbibed on 10 ?M ABA, a concentration that will prevent germination. From these experiments, and from experiments previously performed by the group, we hope to be able to define the developmental state of non-germinating fn10 as either dormant or non-dormant. We hope this will aid us in the understanding of this mutant, and the role the protein plays in the control of dormancy and germination. Reference List 1. Footitt S. et al. (2002) Control of germination and lipid mobilization by COMATOSE, the Arabidopsis homologue of human ALDP. Embo Journal 21, 2912-2922. 2. Russell L. et al. (2000) The Arabidopsis COMATOSE locus regulates germination potential. Development 127, 3759-3767.
NASCARRAY340 Genome-wide transcriptional analysis of the compatible Arabidopsis thaliana-Pseudomonas syringae pv. 2005-10-06	Pseudomonas syringae pv. tomato DC3000 (Pst) is a virulent pathogen, which causes disease on tomato and Arabidopsis. The type III secretion system (TTSS) plays a key role in pathogenesis by translocating virulence effectors from the bacteria into the plant host cell, while the phytotoxin coronatine (COR) contributes to virulence and disease symptom development. Recent studies suggest that both the TTSS and and COR are involved in the suppression of host basal defenses. However, little is known about the interplay between the host gene expression associated with basal defenses and the virulence activities of the TTSS and COR during infection. The global effects of the TTSS and COR on host gene expression associated with other host cellular processes during bacterial infection are also not well characterized. In this study, we used the Affymetrix full genome chip to determine the Arabidopsis transcriptome associated with basal defense to Pst DC3000 hrp mutants and the human pathogenic bacterium Escherichia coli O157:H7. We then used Pst DC3000 virulence mutants to characterize Arabidopsis transcriptional responses to the action of hrp-regulated virulence factors (e.g., TTSS and COR) during bacterial infection. Additionally, we used bacterial fliC mutants to assess the role of the PAMP flagellin in induction of basal defense-associated transcriptional responses. In total, our global gene expression analysis identified more than 5000 Arabidopsis genes that are reproducibly regulated more than 2-fold in three independent biological replicates of at least one type of comparison. Regulation of these genes provides a molecular signature for Arabidopsis basal defense to plant and human pathogenic bacteria, and illustrates both common and distinct global virulence effects of the TTSS, COR, and possibly other hrp-regulated virulence factors during Pst DC3000 infection.
NASCARRAY341 Nuclear gene expression in response to chloroplast division inhibition 2006-10-26	The aim of this investigation is to analyse the affect on nuclear gene expression of inhibition of chloroplast division. Transgenic Arabidopsis lines (Ler) were generated that contained a chemically inducible promoter (XVE)upstream of AtMinD1. AtMinD1 encodes a product involved in chloroplast division, overexpression of AtMinD1 leads to chloroplast division inhibition. Three treatments were performed: Treatment 1; Empty vector control seedlings (transformed with PER-10 only)were sown onto lehle and sprayed at 13 days old with 17-B-estradiol and were harvested 24 hours later. Treatment 2; seeds were sown on lehle containing inducer and sprayed every 24 hours until harvested at 14 days old. Treatment 3; seeds were sown on lehle plates and sprayed at 13 days old and harvested 24 hours later. For all three treatments seedlings were grown in constant light conditions, tissue was harvested from 14 day old seedlings (4 rosette leaves) and snap frozen in liquid nitrogen betore RNA preparation.
NASCARRAY342 Transcriptional profiling in gnc mutant and wild type plants 2005-10-21	The Arabidopsis GATA transcription factor family has 30 members and the biological function of most of them is poorly understood. Homozygous T-DNA insertion lines for 23 of the 30 members were identified and analyzed. Genetic screening of the insertion lines on a defined growth condition revealed one line with an altered phenotype, while the rest showed no obvious change. This line, SALK_001778, has a T-DNA insertion in the second exon of At5g56860 which prevents the expression of the GATA domain. Genetic analysis of the mutant demonstrated that the phenotypic change is caused by a single gene effect and is recessive to the wild type allele. In wild type plants, the expression of At5g56860 is shoot-specific, transcribed at an early stage of development and is inducible by nitrate. Loss of its expression in the loss-of-function mutant plants resulted in reduced chlorophyll levels. A transcript profiling experiment revealed that a considerable proportion of genes down-regulated in the loss-of-function mutants are involved in carbon metabolism and At5g56860 is thus designated GNC (GATA, Nitrate-inducible, Carbon metabolism-involved). gnc mutants with no GNC expression are more sensitive to exogenous glucose and two hexose transporter genes, with a possible connection to glucose signaling, are significantly down-regulated, while GNC over-expressing transgenic plants up-regulate their expression and are less sensitive to exogenous glucose. These observations suggest a function for GNC in regulating carbon and nitrogen metabolism.reference: Yong-Mei Bi et al, Plant J. in press
NASCARRAY343 Global Transcript Profiling of the Green Sectors of the immutans Variegation mutant of Arabidopsis 2005-10-25	Green and white variegation in the Arabidopsis immutans (im) mutant is caused by a nuclear recessive gene. The green sectors contain cells with normal-appearing chloroplasts, while cells in the white sectors have photooxidized plastids lacking organized lamellae. The white and green cells of im have the same genotype (im im), i.e., the tissues are not genetic chiamaeras of wild-type and mutant tissue. The green im sectors have altered leaf morphology, higher than normal rates of carbon assimilation (monitored by 14CO2 uptake) and corresponding increases in the activities of Rubisco and SPS, elevated starch and sucrose pool sizes, and an altered pattern of carbohydrate partitioning that favors sucrose over starch. To gain insight into the morphological and biochemical changes observed in the immutans green sectors, we conducted microarray studies to ask whether or not the immutans green sectors and wild-type Arabidopsis leaves had similar transcriptomes. For these experiments, we used the Affymetrix oligorrays (~22,500 genes) and compared the population of RNAs from dissected green sectors of mature im leaves to the population of RNAs from similarly-aged Columbia leaves growing under the same continuous light (100 Âµmoles m-2 s-1) conditions. Total RNA was isolated using the TRIzol Reagent (GIBCO BRL, Rockville, MD) according to the manufacturers protocol. RNA samples were prepared from wild-type Arabidopsis leaves and im green leaf sectors randomly collected from multiple 3-4 wk old plants; three independent collections were performed and six microarray slides (3 replicates) were used to analyze mRNA abundances in each sample. cDNAs were prepared from 10 Âµg of total RNA following instructions in the Affymetrix GeneChip Expression Analysis manual, and the double-stranded cDNAs were biotinylated by in vitro transcription using the BioArray High Yield RNA Transcript Labeling Kit (ENZO Diagnostics Inc. Farmingdale, NY). The labeled probe (cRNA) was then purified using an RNeasy column (Qiagen. Valencia, CA). Approximately 20 Âµg of fragmented (940C for 25 min) cRNA was sent to the University of Iowa DNA Facility for hybridization, staining and scanning of the Affymetrix Arabidopsis oligoarrays. Signal intensities and detection calls were generated by Affymetrix Microarray Suite v5.0 software (Affymetrix Inc., Santa Clara, CA) and exported to Microsoft EXCEL. The data were then loaded into GeneSpringR v5.1 for detailed statistical analysis.
NASCARRAY344 Transcriptome response to change in ploidy level in Arabidopsis 2007-01-15	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: DIP diploid TET tetraploid TFE triploid female excess TME triplod male excess
NASCARRAY345 AvrPtoB effector function 2009-05-18	In planta expression of the bacterial effector AvrPtoB induces susceptibility to bacterial invasion. We have used stable transgenic lines of ecotype Nd expressing avrPtoB under the control of a dexamethosone inducible promoter.Expressing lines given no treatment or Dex induction are left for 6h before challenge with suspensions of Pseudomonas syringae pv tomato strain DC3000 hrpA mutant or a mock inoculation with MgCl2. Tissues were sampled 0, 2, 4 and 12h after bacterial inoculation. Preliminary northern analyses indicated a specific suppression of defence-related transcripts by AvrPtoB. The array will provide a global picture.In a related experiment we examine the effect of AvrptoB when delivered by bacteria into arabidopsis. Triplicate samples are studied at each time point. Leaves from half the plants were treated with a solution of dexamethasone (6ÂµM in water containing 0.02% Silwet). Six hours later (our start time in this time course), Dex treated and untreated leaves were either harvested (t=0) or infiltrated with either 10 mM MgCl2 or a suspension of Pseudomonas syringae pv. tomato hrpA mutant (DC3000; 1.9x108 colony forming units/ml) in 10mM MgCl2.
NASCARRAY346 Responses to overexpression of wild type and constitutively active Arabidopsis MAP Kinase Kinase 2 2005-11-14	This experiment has been annotated by TAIR (http://arabidopsis.org). We examined transcript profiles triggered by three different arabidopsis R genes that recognize distinct Peronospora parasitica isolates.
NASCARRAY348 Tissue Type Arrays of Columbia-0 2006-03-20	This experiment has been annotated by TAIR (http://arabidopsis.org). In this experiment, different tissue preparations of wild type Columbia-0 Arabidopsis thaliana plants were hybridized and run on the ATH1 Affymetrix platform.
NASCARRAY349 Response to cold, plate grown plants 2006-03-20	This experiment was annotated by TAIR (http://arabidopsis.org). This experiment looks at changes in gene expression in Col-0 plants grown in plates in response to low temperature over time.
NASCARRAY350 Response to cold, soil grown plants 2006-03-21	This experiment was annotated by TAIR (http://arabidopsis.org). This experiment looks at changes in gene expression in Col-0 plants grown in soil in response to low temperature over time.
NASCARRAY351 Response to CBF2 expression 2006-03-22	This experiment was annotated by TAIR (http://arabidopsis.org). This experiment looks at changes in gene expression in response to constitutive expression of the transcription factor CBF2.
NASCARRAY352 Whole genome expression in response to herbicidal levels of 2,4-D application 2006-03-22	This experiment was annotated by TAIR (http://www.arabidopsis.org). In this experiment we examined whole genome response to herbicidal levels of 2,4-D application. Arabidopsis plants (14 days) grown in vitro on MS medium in petri dishes were flooded with 1 mL stock of 2,4-D to enable root uptake. Plants were treated with 1.0 mM 2,4-D for a period of 1 hour immediately after which RNA was extracted and used for the microarray based experiments. the goal of this study was to explore the herbicidal mode of action of auxinic herbicide 2,4-D.
NASCARRAY353 Response to ZAT12 expression 2006-03-24	This experiment was annotated by TAIR (http://arabidopsis.org). This experiment looks at changes in gene expression in response to constitutive expression of the transcription factor ZAT12.
NASCARRAY354 Response to potassium starvation in roots 2006-04-28	This experiment was annotated by TAIR (http://arabidopsis.org). This experiment studies the response of gene expression in roots of 25-35 day old plants grown on hydroponics after 6, 48 and 96 hours of potassium starvation. RNA from roots was extracted after transfer to control (control) or potassium free nutrient solution respectively (starvation).
NASCARRAY355 Mutant array 2006-04-20	This data was annotated by TAIR (http://arabidopsis.org). Two week-old Arabidopsis aerial tissues from Columbia-0 and cpr5, cpr5npr1, cpr5scv1, cpr5npr1svi1, and npr1 lines were collected for analysis.
NASCARRAY356 FRI FLC combos 2006-04-20	This experiment was annotated by TAIR (http://arabidopsis.org).FRI FLC time series
NASCARRAY357 Floral transition and early flower development 2006-04-19	This experiment was annotated by TAIR (http://arabidopsis.org). Floral transition and flower development are regulated by numerous environmental and endogenous signals, which are integrated at a relatively small number of floral integrators, such as FLOWERING LOCUS T (FT) and SUPPRESSOR OF CONSTANS OVEREXPRESSION 1 (SOC1). Of the environmental factors, photoperiod is regarded the most important one in promoting floral transition in Arabidopsis thaliana and most labstrains will flower earlier under long day (LD) conditions than under short day (SD) conditions. Arabidopsis is therefore considered a facultative LD plant. To monitor gene expression changes during floral transition and early flower development plants were grown under SD (9 hr light, 15 hr dark) for 30 days. Plants were then shifted to LD (16 hr light, 8 hr dark) conditions to induce flowering. RNA was isolated from micro-dissected apical tissue harvested 0, 3, 5, and 7 days after the shift to LD and double-stranded cDNA was synthesized. Biotinylated cRNA probes were prepared and hybridized to the Affymetrix ATH1 array in duplicate (biological replicates). To study floral transition, we not only analyzed response of wildtype Landsberg erecta (Ler) plants, but also the effect of mutants in the flowering time genes CONSTANS (CO; co-2) and FT (ft-2). Early flower development was analyzed by comparing Col-0 wildtype plants with the meristem identity mutant lfy-12 (Col-0).
NASCARRAY360 Genome-wide cell cycle studies 2006-06-20	This experiment was provided by TAIR (http://arabidopsis.org). Effective analysis of gene expression during the cell cycle depends on achieving a good level of synchronisation. Until recently, analysis of cell cycle processes in plants has been hampered by the lack of synchronizable cell suspensions for Arabidopsis. We have recently developed a cell synchrony system for Arabidopsis cell suspensions MM1 and MM2d, and have developed two methods of synchronization. The first synchronizes cycling cells by blocking cells at the G1/S boundary using aphidicolin. The second uses sucrose removal and resupply to synchronize cells during re-entry into the cell cycle. Cell cycle synchrony in suspension cultured cells: cells can be reproducibly synchronized by blocking at the G1/S boundary or in early S phase using aphidicolin for 24 hr and then reversing the block by washing (Menges and Murray, 2002). On aphidicolin removal, the synchronous resumption of S phase and progression through the cell cycle occur and sequential RNA samples were taken at 2-3 hourly intervals over a 19 hour period. We have carried out a transcriptional profiling analysis with the aim to study gene expression during cell cycle progression after aphidicolin treatment of suspension-cultured cells using the near full genome ATH1 arrays (Menges et al., 2003).
NASCARRAY361 In vitro tracheary element transdifferentiation of Col-0 suspension cells. 2006-04-20	This experiment was annotated by TAIR (http://arabidopsis.org). Col-0 suspension cells provided by Dr. C. Koncz and Dr. M. Umeda. 15 ml of Arabidopsis Col-0 suspension cells (Mathur et al. 1998) was transferred to 35 ml of a fresh modified Murashige and Skoog (MS) medium supplemented with 1 ug /ml 2,4-dichlorphenoxyacetic acid and 3% sucrose every 7 day, and cultured on a rotary shaker at 120 rpm in the dark at 22 C for subculture. For xylem vessel element induction, a 7.5 ml aliquot of 7-day-old subcultured cells was transferred into 42.5 ml of fresh medium that included 1 uM brassinolide and 10 mM H3BO3, and cultured as described above. The frequency of xylem vessel element formation was calculated as the proportion of xylem vessel elements to the number of living cells and the vessel elements.
NASCARRAY363 Identification of candidate Arabidillo target genes in Arabidopsis 2007-01-11	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. We aim to identify candidate Arabidillo target genes by comparing the transcriptomes of wild type, arabidillo-1/2 mutant and Arabidillo-1 overexpressing lines. The experiment will compare Col-0 (3 slides) with a 35S::Arabidillo-1-YFP overexpressing line (3 slides) and Col-3 with the arabidillo-1/2 mutant (3 slides). Each RNA prep will be from plate-grown 7-day old seedlings. Reference: Coates, JC (2003) Armadillo repeat proteins: beyond the animal kingdom? Trends in Cell Biology 13 p.463-471
NASCARRAY364 Transcript profiling of auxin/cytokinin crosstalk in the Arabidopsis primary root apex 2007-02-03	AtIPT8/pga22 seedlings (gain of function mutant in Ws background; Sun et al. 2003, Plant Physiology 131, pp167-176) were grown on vertical plates for 7 days in LD. The seedlings were then incubated directly on the plate with medium containing 5 mM 17-beta-estradiol (for induction of the IPT8 gene) or 5 mM trans-zeatin for 12 and 24 h. 5 mm primary root tips were harvested from the seedlings and pooled for microarray analysis. Total RNA was isolated from the samples with the RNeasy Plant Mini Kit from Qiagen.
NASCARRAY365 Expression Level Polymorphism Project (ELP) - Col-0 2006-02-24	This experiment was donated by The ELP Project website at elp.ucdavis.edu that was supported in part by the Arabidopsis 2010 project, NSF Division of Molecular and Cellular Biosciences, award 0115109. The study of natural genetic variation for plant disease resistance responses is a complementary approach to utilizing mutants to elucidate genetic pathways. While some key genes involved in pathways controlling disease resistance, and signaling intermediates such as salicylic acid and jasmonic acid, have been identified through mutational analyses, the use of genetic variation in natural populations permits the identification of change-of-function alleles, which likely act in a quantitative manner. Whole genome microarrays, such as Affymetrix GeneChips, allow for molecular characterization of the disease response at a genomics level and characterization of differences in gene expression due to natural variation. Differences in the level of gene expression, or expression level polymorphisms (ELPs), can be mapped in a segregating population to identify regulatory quantitative trait loci (expression QTLs) affecting host resistance responses. In order to identify an appropriate RIL population to map QTL controlling disease resistance responses, we performed a parental survey of 7 different Arabidopsis accessions. We treated vegetatively grown plants with either salicylic acid or a control solution, and harvested the plants at 3 different time points after chemical treatment. We present Affymetrix GeneChip microarray expression data for 3 biological replications of this parental survey.
NASCARRAY367 A gene expression map of the Arabidopsis root 2006-02-28	This experiment was donated by Philip N. Benfey's lab through ArexDB (http://www.arexdb.org). A global map of gene expression within an organ can identify genes with coordinated expression in localized domains, thereby relating gene activity to cell fate and tissue specialization. Here, we present localization of expression of more than 22,000 genes in the Arabidopsis root. Gene expression was mapped to 15 different zones of the root that correspond to cell types and tissues at progressive developmental stages. Patterns of gene expression traverse traditional anatomical boundaries and show cassettes of hormonal response. Chromosomal clustering defined some coregulated genes. This expression map correlates groups of genes to specific cell fates and should serve to guide reverse genetics.
NASCARRAY368 Cellulose synthase mutants 2007-02-12	We compared the transcriptome of homozygous mutants for AtCes4 and AtCesA8 (cellulose synthase genes) to their heterozygous counterparts that have a wild type phenotype.All plants were 4 week old and grown under short day conditions.
NASCARRAY370 Identification of AtATM dependent X-ray inducible transcript 2007-07-11	ATM plays an important role in the response of plants to forms of DNA damage that cause DNA double strand breaks. Arabidopsis ATM has been shown to be important for the transcriptional induction of several DNA repair genes including AtRAD51 (Garcia et al 2003 Plant Cell 15, 119-132). In our experiment we compare the transcript profiles of wild type and mutant plants exposed to 10Gy X-rays (dose rate ~0.8 Gy/min). Plants will be grown on 0.5*MS + 1%sucrose at 22degC under short (9h) day conditions and treated after 5h light. We will irradiate wild type (Col-0) and atatm-3 mutant (N589805) seedlings at growth stage 1.06 and harvest tissue 1h post irradiation. RNA will be isolated using the SV RNA isolation kit (Promega). Comparison of the transcript profiles will then allow AtATM-dependent X-ray inducible transcripts to be identified.
NASCARRAY373 Expression Level Polymorphism Project (ELP) - Cvi-1 2006-02-24	This experiment was donated by The ELP Project website at elp.ucdavis.edu that was supported in part by the Arabidopsis 2010 project, NSF Division of Molecular and Cellular Biosciences, award 0115109. The study of natural genetic variation for plant disease resistance responses is a complementary approach to utilizing mutants to elucidate genetic pathways. While some key genes involved in pathways controlling disease resistance, and signaling intermediates such as salicylic acid and jasmonic acid, have been identified through mutational analyses, the use of genetic variation in natural populations permits the identification of change-of-function alleles, which likely act in a quantitative manner. Whole genome microarrays, such as Affymetrix GeneChips, allow for molecular characterization of the disease response at a genomics level and characterization of differences in gene expression due to natural variation. Differences in the level of gene expression, or expression level polymorphisms (ELPs), can be mapped in a segregating population to identify regulatory quantitative trait loci (expression QTLs) affecting host resistance responses. In order to identify an appropriate RIL population to map QTL controlling disease resistance responses, we performed a parental survey of 7 different Arabidopsis accessions. We treated vegetatively grown plants with either salicylic acid or a control solution, and harvested the plants at 3 different time points after chemical treatment. We present Affymetrix GeneChip microarray expression data for 3 biological replications of this parental survey.
NASCARRAY374 Expression Level Polymorphism Project (ELP) - Est 2006-02-24	This experiment was donated by The ELP Project website at elp.ucdavis.edu that was supported in part by the Arabidopsis 2010 project, NSF Division of Molecular and Cellular Biosciences, award 0115109. The study of natural genetic variation for plant disease resistance responses is a complementary approach to utilizing mutants to elucidate genetic pathways. While some key genes involved in pathways controlling disease resistance, and signaling intermediates such as salicylic acid and jasmonic acid, have been identified through mutational analyses, the use of genetic variation in natural populations permits the identification of change-of-function alleles, which likely act in a quantitative manner. Whole genome microarrays, such as Affymetrix GeneChips, allow for molecular characterization of the disease response at a genomics level and characterization of differences in gene expression due to natural variation. Differences in the level of gene expression, or expression level polymorphisms (ELPs), can be mapped in a segregating population to identify regulatory quantitative trait loci (expression QTLs) affecting host resistance responses. In order to identify an appropriate RIL population to map QTL controlling disease resistance responses, we performed a parental survey of 7 different Arabidopsis accessions. We treated vegetatively grown plants with either salicylic acid or a control solution, and harvested the plants at 3 different time points after chemical treatment. We present Affymetrix GeneChip microarray expression data for 3 biological replications of this parental survey.
NASCARRAY375 Expression Level Polymorphism Project (ELP) - Tsu-1 2006-02-24	This experiment was donated by The ELP Project website at elp.ucdavis.edu that was supported in part by the Arabidopsis 2010 project, NSF Division of Molecular and Cellular Biosciences, award 0115109. The study of natural genetic variation for plant disease resistance responses is a complementary approach to utilizing mutants to elucidate genetic pathways. While some key genes involved in pathways controlling disease resistance, and signaling intermediates such as salicylic acid and jasmonic acid, have been identified through mutational analyses, the use of genetic variation in natural populations permits the identification of change-of-function alleles, which likely act in a quantitative manner. Whole genome microarrays, such as Affymetrix GeneChips, allow for molecular characterization of the disease response at a genomics level and characterization of differences in gene expression due to natural variation. Differences in the level of gene expression, or expression level polymorphisms (ELPs), can be mapped in a segregating population to identify regulatory quantitative trait loci (expression QTLs) affecting host resistance responses. In order to identify an appropriate RIL population to map QTL controlling disease resistance responses, we performed a parental survey of 7 different Arabidopsis accessions. We treated vegetatively grown plants with either salicylic acid or a control solution, and harvested the plants at 3 different time points after chemical treatment. We present Affymetrix GeneChip microarray expression data for 3 biological replications of this parental survey.
NASCARRAY376 Expression Level Polymorphism Project (ELP) - Van-0 2006-02-24	This experiment was donated by The ELP Project website at elp.ucdavis.edu that was supported in part by the Arabidopsis 2010 project, NSF Division of Molecular and Cellular Biosciences, award 0115109. The study of natural genetic variation for plant disease resistance responses is a complementary approach to utilizing mutants to elucidate genetic pathways. While some key genes involved in pathways controlling disease resistance, and signaling intermediates such as salicylic acid and jasmonic acid, have been identified through mutational analyses, the use of genetic variation in natural populations permits the identification of change-of-function alleles, which likely act in a quantitative manner. Whole genome microarrays, such as Affymetrix GeneChips, allow for molecular characterization of the disease response at a genomics level and characterization of differences in gene expression due to natural variation. Differences in the level of gene expression, or expression level polymorphisms (ELPs), can be mapped in a segregating population to identify regulatory quantitative trait loci (expression QTLs) affecting host resistance responses. In order to identify an appropriate RIL population to map QTL controlling disease resistance responses, we performed a parental survey of 7 different Arabidopsis accessions. We treated vegetatively grown plants with either salicylic acid or a control solution, and harvested the plants at 3 different time points after chemical treatment. We present Affymetrix GeneChip microarray expression data for 3 biological replications of this parental survey.
NASCARRAY377 Expression Level Polymorphism Project (ELP) - Kin-0 2006-02-24	This experiment was donated by The ELP Project website at elp.ucdavis.edu that was supported in part by the Arabidopsis 2010 project, NSF Division of Molecular and Cellular Biosciences, award 0115109. The study of natural genetic variation for plant disease resistance responses is a complementary approach to utilizing mutants to elucidate genetic pathways. While some key genes involved in pathways controlling disease resistance, and signaling intermediates such as salicylic acid and jasmonic acid, have been identified through mutational analyses, the use of genetic variation in natural populations permits the identification of change-of-function alleles, which likely act in a quantitative manner. Whole genome microarrays, such as Affymetrix GeneChips, allow for molecular characterization of the disease response at a genomics level and characterization of differences in gene expression due to natural variation. Differences in the level of gene expression, or expression level polymorphisms (ELPs), can be mapped in a segregating population to identify regulatory quantitative trait loci (expression QTLs) affecting host resistance responses. In order to identify an appropriate RIL population to map QTL controlling disease resistance responses, we performed a parental survey of 7 different Arabidopsis accessions. We treated vegetatively grown plants with either salicylic acid or a control solution, and harvested the plants at 3 different time points after chemical treatment. We present Affymetrix GeneChip microarray expression data for 3 biological replications of this parental survey.
NASCARRAY378 Expression Level Polymorphism Project (ELP) - Mt-0 2006-02-24	This experiment was donated by The ELP Project website at elp.ucdavis.edu that was supported in part by the Arabidopsis 2010 project, NSF Division of Molecular and Cellular Biosciences, award 0115109. The study of natural genetic variation for plant disease resistance responses is a complementary approach to utilizing mutants to elucidate genetic pathways. While some key genes involved in pathways controlling disease resistance, and signaling intermediates such as salicylic acid and jasmonic acid, have been identified through mutational analyses, the use of genetic variation in natural populations permits the identification of change-of-function alleles, which likely act in a quantitative manner. Whole genome microarrays, such as Affymetrix GeneChips, allow for molecular characterization of the disease response at a genomics level and characterization of differences in gene expression due to natural variation. Differences in the level of gene expression, or expression level polymorphisms (ELPs), can be mapped in a segregating population to identify regulatory quantitative trait loci (expression QTLs) affecting host resistance responses. In order to identify an appropriate RIL population to map QTL controlling disease resistance responses, we performed a parental survey of 7 different Arabidopsis accessions. We treated vegetatively grown plants with either salicylic acid or a control solution, and harvested the plants at 3 different time points after chemical treatment. We present Affymetrix GeneChip microarray expression data for 3 biological replications of this parental survey.
NASCARRAY379 Identification of genes regulated by NMD in Arabidopsis 2007-07-11	The nonsense-mediated mRNA decay pathway (NMD) is a quality control mechanism that detects and degrades mRNAs that contain premature translation termination codons (PTCs), thus preventing the production of potentially harmful truncated proteins. NMD has been studied in a number of organisms including humans, yeast, Drosophila, C. elegans and recently Arabidopsis. Recently the NMD process has been shown to have a wider significance in regulating the levels of mRNA expression for a wide range of genes in yeast and Drosophila. Several genes have been found to be required for NMD in a variety of organisms, including UPF1 and UPF3. We have identified and characterised a number of alleles of UPF1 and UPF3 in Arabidopsis that are defective in NMD. By doing a transcriptomic analysis of two NMD mutants, upf1-1 and upf3-1, and wild type plants we aim to identify the subset of genes that are co-regulated by UPF1 and UPF3 and, therefore, by NMD in Arabidopsis. RNA will be extracted from 17 day-old mutant and wild type seedlings grown at 22-24 C under constant light.
NASCARRAY381 Growth of suspension-cultured cells 2006-06-20	The growth of Arabidopsis cell cultures following their sub-culture into fresh media follows standard growth kinetics of a period of exponential increase associated with high rate of cell division, followed by a slowing of the rate of increase as cells approach stationary phase. For the analysis described here, MM2d cells were subcultured into fresh MSS-medium and samples were taken at day 1, day3, day 5 and day7. We have carried out transcriptional profiling analysis with the aim to follow growth stage specific gene expression during unperturbed growth using the near full genome ATH1 arrays (Menges et al., 2003). Journal Absract: (Plant Molecular Biology: 53, 2003) Plant cell suspension cultures are invaluable models for the study of cellular processes. Here we develop the recently described Arabidopsis suspension culture MM2d as a transcript profiling platform by means of Affymetrix ATH1 microarrays. Analysis of gene expression profiles during normal culture growth, during synchronous cell cycle re-entry and during synchronous cell cycle progression provides a unique integrated view of gene expression responses in a higher-plant system. Particularly striking is that expression of over 14 000 genes belonging to all defined categories can be reliably detected, suggesting that integrated and comparative analysis of data sets derived from transcript profiling of cultures is a powerful approach to identify candidate components involved in a wide range of biological processes. Combinatorial analysis of independent cell cycle synchrony methods allows the identification of genes that are apparently cell-cycle-regulated but are most likely responding to the induction of synchrony. We thus present an integrated genome-wide view of the transcriptional profile of a plant suspension culture and identify a refined set of 1082 cell cycle regulated genes largely independent of synchrony method.
NASCARRAY383 Cold acclimation in the sfr3-1 mutant 2008-04-30	The sfr3-1 mutation causes freezing-sensitivity in Arabidopsis thaliana. The mutated gene has been identified by positional cloning and is currently being characterised. The mutant appears normal when grown in the warm (no phenotype has been identified associated with such growth). However, following cold acclimation and subsequent freezing mutant plants are severely damaged whilst wild type plants are not. This suggests that sfr3 is deficient in the cold acclimation process. Micro-array analysis will enable the identification of any transcriptional changes during the cold acclimation process. This information will then be used, together with information obtained by gene characterisation, in order to more fully understand the nature of the sfr3 mutation.
NASCARRAY384 Cesium Toxicity in Arabidopsis 2009-03-17	Caesium (Cs+) is a potentially toxic mineral element that is released into the environment and taken up by plants. Although Cs+ is chemically similar to potassium (K+), and much is known about K+ transport mechanisms, it is not clear through which K+ transport mechanisms Cs+ is taken up by plant roots. In this study, the role of AtHAK5 in high affinity K+ and Cs+ uptake was characterized. It is demonstrated that AtHAK5 is localized to the plasma membrane under conditions of K+ deprivation, when it is expressed.
NASCARRAY385 GA Treatment of Arabidopsis Root Tips 2006-05-24	The plant hormone gibberellin represents an important regulator of growth and development. Early transcriptional events controlled by GA are not well characterised. Previous microarray studies have identified genes responsive to GA treatment in the whole seedling. The whole seedling represents many tissues where subtle effects of GA treatment in specific tissues may be masked. When treated with GA an effect on the growth rate of roots was observed. More specifically the shorter root of a GA deficient plant can be rescued to wild type length by the application of GA. This experiment was designed to identify GA regulated genes in the root tips of Arabidopsis. The use of a GA deficient mutant provides a greater potential to identify genes responding to GA treatment. Root tips are ideally suited for the quick uptake of the hormone treatment. There will be two biological replicates which will each consist of a control treatment at 0 minutes and 2 hours as well as the experimental GA treated 2 hour time point. This system provides an opportunity to compare gene expression between treated and non-treated root tips and allow the identification of early GA responsive genes.
NASCARRAY386 The early post-germinative embryo and endosperm transcriptomes in Arabidopsis 2006-06-27	Arabidopsis seed germination is coordinated with the strong induction of metabolic pathways required for the mobilisation and utilization of seed storage reserves. These are essential to support the seedling before the establishment of photoauxotrophic growth. The activity of genes encoding enzymes required for lipid mobilisation is regulated largely at the level of transcription, but our knowledge of how this regulation occurs is extremely limited. After germination the rate of lipid reserve mobilisation is determined by the carbohydrate status of the seedling and by the osmotic potential of the growth substrate. The plant response to both of these requires the action of the hormone abscisic acid (ABA). We have shown that this regulation is tissue specific (Penfield et al., 2004 Plant Cell 16, 2705-2718), and that although lipid breakdown in the embryo is inhibited by ABA, lipid breakdown in the endosperm tissues is not. Furthermore, in many species the action of the endosperm is central to the processes controlling seed germination, yet very little is known about gene expression in this tissue, or of the function of the endosperm in mature Arabidopsis seeds. Mature Arabidopsis seeds can be dissected into embryo and endosperm/seed coat fractions, with the latter fraction containing RNA only from the endosperm as the seed coat cells undergo programmed cell death during the latter stages of seed development. In this experiment we divide seeds into embryo and endosperm tissues and transcript profile both shortly after germination, or when germination and lipid reserve mobilisation are inhibited by ABA or by the gibberellin biosynthesis inhibitor paclobutrazol. In this way we can discover the endosperm transcriptome and uncover candidate regulators of lipid mobilisation by searching for genes showing differential expression between embryo and endosperm before and after ABA treatment.
NASCARRAY387 Microarray analysis of fdh1-3(avrB) 2008-01-10	To track gene expression profile in atgsnor1-3 mutant in response to Pst(avrB)
NASCARRAY388 Genes Involved in Meristemoid Cell Fate 2008-01-11	The objective of this experiment is to identify genes that are regulated by the LRR-receptor-like protein TOO MANY MOUTHS (TMM) to control stomatal precursor (meristemoid) cell fate and behavior. An interesting aspect of the tmm phenotype is that mutant plants overproduce stomata on leaves, but lack stomata entirely on inflorescence stems and in other locations on the plant. Despite the failure to produce mature stomata, mutant stems produce presumptive stomatal precursors through asymmetric divisions. This suggests that normal TMM signaling is required to maintain meristemoid cell fate but not to execute formative asymmetric divisions. Because stem stomata develop in a morphogenetic wave from tip to base, it is possible to harvest tissues that contain meristemoids but not guard mother cells or mature stomata. This provides an opportunity to compare the gene expression profiles of tissues with meristemoids that maintain their cellular identity and ultimately produce stomata (Col gl1), to tissues in which meristemoids form but rapidly lose their cellular identity due to a failure in TMM signaling (tmm-1). Because guard mother cells and stomata are not present in the tissue harvested, genes that change in abundance during these developmental stages will be filtered out. Ultimately we hope to reveal genes that operate downstream of TMM to confer meristemoid cell fate or to provide 'niche factors' suitable for their development into stomata. For analysis, approximately 90 developing inflorescence stem tips were collected and flower buds and meristems removed prior to flash freezing for each replicate sample. The region harvested had been previously determined to contain developing meristemoids but not stomata or guard mother cells by microscopic observation. An identical region was harvested from tmm-1 mutant plants grown at the same time, in the same flat.
NASCARRAY389 Comparison of transcriptional profiles between sni1 and wild type 2006-09-03	Microarray experiment was performed using 4-week old plants to compare transcriptional profiles between sni1 (suppressor of npr1) and wild type (Col-0). Three biological replicates were included.
NASCARRAY390 Transciptome differences between two vitamin C-deficient mutants and the wild type 2007-11-19	The Arabidopsis mutant vtc2 is vitamin C-deficient and high light sensitive ((MÃ¼ller-MoulÃ© et al., 2002. Plant Physiol 128: 970-977; MÃ¼ller-MoulÃ© et al., 2003. Plant Physiol 133: 748-760). Even though the VTC2 gene has been cloned (Jander et al., 2002. Plant Physiol 129: 440-450), its function is still unclear. The goal of this experiment is to learn more about the function of the VTC2 gene by determining differences in the transcriptome of the wild type and the vtc2-1 mutant. In order to differentiate between VTC2 gene specific differences and vitamin C-induced differences, a second vitamin C-deficient mutant vtc1-1 (Conklin et al., 1996. PNAS 93: 9970-9974) is included in the experiment. The plants will be grown on agar plates in a growth chamber with a 16 hour light (about 40 Âµmol photons mâ»Â²sâ»Â¹)/8 hour dark regime at 20Â°C for 14 days to stage 1.04. Whole plants will be collected and total RNA will be isolated using the Qiagen Rneasy plant mini kit (Qiagen, Hilden, Germany).
NASCARRAY392 BTH time course 2006-09-19	Chemical induction of Systemic Acquired Resistance (SAR). Plants were sprayed with 60uM BTH (Benzothiadiazole S-methylester). Tissue was collected 0, 8, and 24 hours after treatment. Three genotypes were included -- wild type, npr1-1, and wrky18 (Salk_093916) -- to identify BTH-induced transcriptome changes, and NPR1- and WRKY18-dependent genes. Three biological replicates were included.
NASCARRAY393 Response of the transcription factor mutants wrky11and wrky17 to virulent Pseudomonas(Pst DC3000) 2006-09-19	WRKY11 and WRKY17 act in a partially redundant manner as negative regulators of basal resistance to Pseudomonas. In order to identify candidate target genes that are either directly or indirectly regulated by them, a genome-wide expression analysis in mutant plants was undertaken.Three independent experiments were performed in which leaves of 4 week-old wild-type Col-0, wrky11.1 (ZIGIA En-insertion line 5AAH57) or wrky17.1 plants (T-DNA insertion lines SALK_176337) were inoculated with Pst DC3000 (107 cfu per mL) and harvested 0, 2, 5 and 12 hours after inoculation. After verifying the induction of marker genes of the defence response by Q-RT-PCR, each individual sample from each of the three biologically independent experiments was used for the hybridisation of one GeneChip.
NASCARRAY394 Temperature-controlled inhibition of phyB action 2008-01-10	It has been established that the frequently reported early flowering phenotype of the phyB mutant is very sensitive to small changes in ambient temperature (Halliday et al., 2003). A 6oC drop in temperature to 16oC is sufficient to abolish the pronounced early flowering phyB phenotype observed at 22oC. Using Affymetrix technology we will identify genes that are differentially regulated at 16oC and 22oC in a phyB-specific manner using phyB mutant and wild type seedlings.
NASCARRAY395 Arabidopsis pvip mutants 2008-01-15	Transcriptional comparison of Arabidopsis thaliana pvip1 ; pvip2 RNAi double mutants against Col-0 wildtype;Tissue used: whole rosettes of 4-week old plants grown under short-day conditions (8h light; 20oC); 3 biological replicates for each, control = Col-0 and mutant = pvip1 pvip2;PVIP = Potyvirus-VPg-interacting protein (Dunoyer et al. 2004 J. Virol. 78: 2301-2309).
NASCARRAY397 Group II-A WRKY transcription factors and early leaf senescence 2007-01-09	In our laboratory we are interested in studying the functions of WRKY zink finger type transcription factors. There are 74 members of this gene family in Arabidopsis. WRKY factors are key regulators of distinct plant defense responses and are involved in certain developmental programs e.g. plant senescence. We would like to determine the functions of a small sub-group (group II-a) of WRKY factors. Our aim to compare and contrast the gene expression profiles of 35 days-old untreated wild type and WRKY T-DNA knockout plants grown in a growth chamber under long day growth conditions. All plants chosen at this stage showed slight yellowing of the first two to four leaves.
NASCARRAY398 Group II-A WRKY transcription factors and early leaf senescence 2007-01-09	In our laboratory we are interested in studying the functions of WRKY zinc finger type transcription factors. There are 74 members of this gene family in Arabidopsis. WRKY factors are key regulators of distinct plant defense responses and are involved in certain developmental programs e.g. trichome development and plant leaf senescence. We would like to determine the functions of a small sub-group (group II-a) of WRKY factors in response to bacterial infection. Our aim is to compare and contrast the gene expression profiles of 3-week-old wild type and WRKY T-DNA knockout mutants grown in a growth chamber under long day growth conditions and subsequently challenged for 6 hours with the virulent bacterial pathogen /Pseudomonas syringae/ DC3000.
NASCARRAY399 Seed development 2009-11-24	Reciprocal crosses of a diploid Arabidopsis with different ploidies results in different endosperm development patterns and phenotype. Typically if the ploidy is higher on the maternal side there are fewer endosperm cells which cellularize early and conversely if the paternal ploidy is higher, there is more number of endosperm cells which cellularize late. Crosses involving diploid and tetraploid Arabidopsis (C24) are viable whereas crosses involving the diploid and hexaploid even though exhibit the above mentioned directional trend in endosperm development, abort (Scott et al 1998). The 'maternalised' and 'paternalised' development of endosperm is also observed in crosses involving some Arabidopsis mutants. Mutants in the fis class of genes e.g. fis1-medea when crossed with a diploid Arabidopsis (pollen parent) show endosperm development-seed development similar to a diploid (seed parent) crossed with hexaploid pollen parent. Reciprocal crosses of homozygous met1 and diploid Arabidopsis also exhibit reciprocal trends in endosperm development where a homozygous met1 mutant (seed parent) crossed with diploid is similar in phenotype to a diploid (seed parent) - tetraploid cross. Endosperm development in the reciprocal cross has phenotypic similarity to the tetraploid (seed parent)-diploid cross (Adams et al 2000).We are interested in understanding gene profiles and trends in expression underlying the endosperm development in the interploidy crosses as well as the fis and met1 mutant.
NASCARRAY400 Incompatible interaction Plasmodiophora-Arabidopsis 2012-02-15	A monogenetic inherited resistance phenotype to Plasmodiophora brassicae has been found in Arabidopsis ecotype Tsu-0. The dominantly inherited RPB1 gene mediates a hypersensitive-like response reaction. Inoculated roots of the resistant line will be compared to control roots at different time points after inoculation in order to get insights into the incompatible interaction. The corresponding dataset has been published (Siemens et al. 2006, MPMI). Furthermore, crossing experiments of the line Tsu-0 with mutant lines of salicylic acid, jasmonic acid and ethylene revealed no influence of these hormones in the resistance reaction. Furthermore, mutations of sgt1b and rar1 showed no influence on RPB1 mediated resistance, but a mutation of sgt1a (At4g23570) reveals to be epistatic to RPB1, indicating to protein ubiquitination and protein degradation as important part of clubroot resistance mechanism. The RPB1 locus has been localized to a region on chromosome 1 with 14 coding sequences according to the sequence of ecotype Columbia. One of these genes is polymorphic between resistant and susceptible ecotypes and encodes for a protein interacting with a SCF-complex. Therefor this gene is probably identical to RPB1.
NASCARRAY403 Systemic response to avirulent bacterial infection 2007-01-15	In the absence of adaptive immunity displayed by animals, plants respond locally to biotic challenge via inducible basal defense networks activated through recognition and response toconserved pathogen associated molecular patterns (PAMPs). In addition, immunity can be induced in tissues remote from infection sites via systemic acquired resistance (SAR), initiated following gene-for-gene recognition between plant resistance proteins and microbial effectors.The nature of the mobile signal and remotely activated networks responsible for establishing SAR remain unclear. Here we show that despite the absence of PAMP contact, systemically responding leaves rapidly activate a SAR transcriptional signature with strong similarity to local basal defense. Jasmonates have previously been implicated in systemic signalling in response to wounding and plant herbivory but not SAR. We present several lines of evidence that suggest jasmonates may also be central to SAR. Jasmonic acid (JA) rapidly accumulates in phloem exudates of leaves challenged with an avirulent strain of Pseudomonas syringae. In systemically responding leaves transcripts associated with jasmonate biosynthesis are upregulated and JA increases transiently. SAR can be mimicked by foliar JA application and is abrogated in mutants impaired in jasmonate synthesis or response. We conclude that, jasmonate signalling appears to mediate long-distance information transmission. Moreover, the systemic transcriptional response shares extraordinary overlap with local herbivory and wounding responses, indicating that jasmonates may be central to an evolutionarily conserved signalling network, which decodes multiple abiotic and biotic stress signals.
NASCARRAY404 Cold Acclimation Time Course 2006-10-25	OBJECTIVE: To determine expression profile of Arabidopsis (Columbia) under temperature stress and to identify genes that may be involved in short-term and long-term stress tolerance.Temperature stress treatments consist of three sub experiments each beginning at 2 hr into the light period: 1- heat shock at 40C over 4 hr with same light intensity as control 2- cold shock at 4C over 96 hr with same light intensity as control 3- deacclimation after cold acclimation for 96 hr at 20C for 24 hr with same light/dark conditions. Note: In these experiments circadian clock regulation is taken in to account as much as possible with 2 possible controls taken. 1- Control (20C) was taken 2 hr after the lights turned on and this was the time temperatures stress samples were taken 2- control (20C) 6 hr after the lights turned on and this was the time that 4 hr heat stress and 4 hr cold stress samples were taken Plant Growth Conditions: Light: Photosynthetically active radiation is 100-150 micromol/m2/s. Light regime is 15/9 hr light/dark cycles. Cool white fluorescent and incandescent lamp are used as light sources. Light quality was very uniform throughout the growth area at all temperature conditions. Soil: Plants were grown in Fafard 2 Mix (Canadian Sphagnum Peat (55%), Perlite, and Vermiculite). Soil is autoclaved. Temperature: Plants were grown at 20C for about 3 weeks (8.5 leaf stage) at 15/9 D/L with irradiance as stated above prior to initiation of temperature treatments. All aerial tissue above the soil line was harvested for RNA an sample. Each sample was composed of 16 plants. Kaplan et al., Plant Physiol. 136, 4159-4168.
NASCARRAY405 Cytokinin treatment on aerial parts of seedlings 2006-12-06	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.
NASCARRAY406 Cytokinin treatment on roots of seedlings 2006-12-06	According to the well-documented scenario with regard to the cytokinin-mediated phosphorelay signal transduction in Arabidopsis thaliana, certain members of the type-B ARR family are crucially implicated in the regulatory networks that are primarily propagated by the cytokinin-receptors (AHKs) in response to cytokinin. Nevertheless, clarification of the biological impact of these type-B ARR transcription factors is at a very early stage. Here we focused on a pair of highly homologous ARR10 and ARR12 genes by constructing an arr10 and 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 showed remarkable phenotypes with special reference to the cytokinin-action in roots (e.g., inhibition of root elongation, green callus formation from explants). Furthermore, we demonstrated that ARR10 and ARR12 are involved in the AHK-dependent signaling pathway that modulates the differentiation of root-vascular tissues (i.e., protoxylem-specification), suggesting that ARR10 and ARR12 are the prominent players that act redundantly in the AHK-dependent cytokinin signaling in roots. Keeping this in mind, we then collected the root-specific and combinatorial DNA microarray datasets with regard to the cytokinin-responsible genes by employing both the wild-type and arr10 arr12 double mutant plants. In this study, wild type and the arr10 arr12 mutant grown vertically on MS agar plates for 2 weeks were treated with 20 microM trans-zeatin or 0.02% DMSO (solvent for trans-zeatin solution) for 1h. These treated plant samples were divided into three portions, from which RNA samples were prepared separately from roots 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 roots of seedlings in Arabidopsis thaliana.
NASCARRAY408 Transcriptional analysis of Arabidopsis plants grown under defined nitrogen conditions 2007-07-17	Nitrogen (N) is the most important inorganic nutrient to plants. The production of high-yielding crops is associated with the application of a large quantity of N fertilizers at a substantial cost, and N pollution is becoming a threat to our global ecosystems. To understand more about the N response and regulation, we developed a growth system where N is the growth-limiting factor, and used an Arabidopsis whole genome array for a global evaluation of gene expression under different N conditions. Significantly differentially expressed genes under different degree of N stress or after different time frame of N induction were identified. They provided additional insights into candidate regulator genes of their coordinating the complex N responses of plants and the components of the N response mechanism. Putative N regulatory elements were also identified, and multiple transcription factor binding sites revealed a complex N regulatory network.
NASCARRAY409 Oligogalacturonide treatment of Arabidopsis seedlings 2007-01-15	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.
NASCARRAY410 Expression Profile of ASL9 Overexpressor 2007-02-15	In Arabidopsis thaliana, each member of a large family of AS2/LOB (ASYMMETRIC LEAVES 2 / LATERAL ORGAN BOUNDARIES) genes encodes a highly homologous plant specific protein. A mutational lesion of the representative AS2 gene results in the development of anomalous asymmetric leaves, implying that these family members might commonly play some roles in plant development. Here, we found that an ectopic and over-expression of ASL9 (ASYMMETRIC LEAVES 2 LIKE 9) in transgenic plants displayed a markedly anomalous architecture during development of adult plants. The expression pattern of ASL9 was quite characteristic in that it was expressed at the restricted bases of lateral organs formed from vegetative, inflorescence, and floral meristems. It was also demonstrated that, among the AS2/LOB family members, ASL9 is uniquely distinct from others in that this one is exclusively, markedly, and specifically regulated by the plant hormone cytokinin in a manner dependent on the His-Asp phosphorelay signal transduction. To gain further insight into the function of ASL9, we carried out transcriptome analysis of transgenic lines that ectopically overexpress ASL9 (ASL9-ox). Wild-type and ASL9-ox plants were grown on agar plates containing MS salt and 1% sucrose under continuous fluorescent light for 2 weeks. For each strain these 2-week-old 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). The resultant data set will be of help to discuss and hypothesize that ASL9 might be implicated in the regulation of differentiation of lateral organs with a linkage to the actions of cytokinin that is primarily propagated through the His-Asp phosphorelay signal transduction.
NASCARRAY411 Transcript inhibition from limited starch degradation 2008-07-21	In starchless mutants of Arabidopsis, pgm, and wild type, Col0, there are small differences in transcript levels at the end of the day, but large differences at the end of the night. Many of the transcripts of genes involved in biosynthesis and growth are repressed in the pgm mutant (Thimm et al., 2004). It is hypothesized that a transient depletion in sugars at the end of the night inhibits carbon utilization at the beginning of the day (Gibon et al., 2004). In order to test if the same inhibition of transcripts involved in biosynthesis and growth also occurs in starch excess mutants of Arabidopsis Affymetrix arrays will be generated for WT (Col0), and the following mutants: sex4-3 (Salk 102567), ptpkis2 (Salk 053285), and sex1-3 (Yu et al., 2001). WT, sex4-3, and ptpkis2 plants were grown for 30 days and sex1-3 plants were grown for 36 days. All plants were grown in a growth chamber with a 12 hour photoperiod, quantum flux 100 mol m-2s-1, relative humidity was at least 75%, and a constant temperature of 20oC. Three leaves from 8 different plants of each genotype were harvested using a green safe lamp 0.5 hours before the lights came on in the morning and immediately frozen in LN2. Leaf material was powered using a mortar and pestle and RNA was extracted using a Purescript RNA isolation kit (Gentra Systems, MN, USA). Samples were then treated with DNaseI (Invitrogen, CA, USA).
NASCARRAY412 Gametophytic transcription factors 2008-07-28	Following our initial transcriptomic analyses of the male gametophyte development (Honys and Twell, Genome Biol 5:R85, 2004), we identified several candidate genes for the function of transcriptional regulators of the male gametophyte development. Two transcription factors with verified function (bZIP and C2H2 ZF) were selected for the identification of their regulons. Mature pollen was isolated as published previously (Honys and Twell Plant Physiol 132:640-652, 2003) from Col-0 wild type plants and T-DNA insertion mutants (SAIL) homozygous for tested genes.
NASCARRAY413 Cis-jasmone as a novel signalling molecule in plant gene expression 2008-07-28	(Z)-Jasmone (cis-jasmone) is a highly volatile product of jasmonic acid (JA) catabolism and is not only a constitutive compound of flower volatiles but also induced by insect damage of vegetative tissue. It has been speculated that cis-jasmone might be a biologically inactive sink for the JA pathway but recently it was demonstrated that cis-jasmone acts as an insect semiochemical. In addition, we have shown by differential display that cis-jasmone induces plant gene expression qualitatively distinct from that elicited by methyl jasmonic acid (Birkett et al, PNAS 97: 9329-9334). Thus it is likely that cis-jasmone is a new lipid-derived signalling compound as opposed to the previously proposed inert sink. We are therefore interested in using the Arabidopsis DNA microarray to search for genes whose expression is altered by cis-jasmone treatment. As it is not known yet what role cis-jasmone might play in oxylipin signalling, we hope that the microarray identification of such genes will give an indication as to whether cis-jasmone is involved solely in defence signalling or whether it has additional functions. Arabidopsis thaliana ecotype Columbia will be exposed to cis-jasmone for 1 or 3 hours respectively. For each treatment (and the mock treated control plants) 8 rosettes will be harvested, pooled and used for RNA extraction. Three biological replicates will be generated per treatment.
NASCARRAY414 Arabidopsis thaliana leaves treated with Pseudomonas syringae ES4326 2007-02-16	In order to better understand the transcriptional networks triggered by pathogen inoculation, we monitored gene expression in Arabidopsis leaves inoculated with Pseudomonas syringae ES4326. Individual leaves of Col-0 plants were injected in the morning using a needle-less syringe with 10E5 cfu cm-2 PsmES4326 (suspended in 5mM MgSO4) or a MgSo4 control and were harvested 9, 24 or 32 hours later. Plants were grown in pots with BM-2 soil (Berger Peat Moss Ltd, Quebec, Canada) at a density of 9 plants per pot and kept at 22 degrees Celsius and 75% humidity with a 12 hour day length.
NASCARRAY415 Effects of the cucumber mosaic virus 2b silencing suppressor protein on inducible plant defence path 2008-07-30	In this study, we have examined the effects of the cucumber mosaic virus 2b protein on the salicylic acid (SA) and methyl jasmonate (meJa) induced defence pathways in A. thaliana. We have also investigated the effects of the 2b protein on antimycin A-induced defence, which represents a virus-specific branch of SA-induced defence. Transgenic A. thaliana (ecotype Col 0) expressing the 2b gene from CMV (strain Fny) were treated independently with SA, meJa and AA, as were wild-type controls. After 25 hours, RNA was extracted from aerial tissue for comparison of gene expression profiles in the presence and absence of the 2b protein. The 2b protein is known to suppress SA-induced resistance to plant viruses as well as the siRNA and miRNA-mediated gene silencing pathways. It is able to suppress SA-mediated induction of alternative oxidase, a gene that functions in the AA-induced virus-specific branch of SA-induced defence (Ji and Ding, 2001, Mol. Plant Microbe Interactions 14, 715-724). Furthermore, the 2b protein is the major determinant of viral symptoms in CMV and causes symptom-like alterations in the phenotype of Arabidopsis thaliana plants expressing 2b transgenes (Lewsey et al., 2007, Plant J., in press). However, its effects on the meJa-induced defence pathway, which has crosstalk with the SA-induced defence pathway, are not known.
NASCARRAY416 Microarray analysis of HopAO1 transgenic Arabidopsis plants 2007-02-28	This experiment was performed to identify gene expression changes resulting from the expression of the Pseudomonas syringae pv. tomato (Pst) strain DC3000 type III effector tyrosine phosphatase HopAO1 in stable Arabidopsis transgenic lines. Wild-type (Col gl1) plants and transgenic plants expressing HopAO1 or a mutant derivative, HopAO1 C378S, lacking phosphatase catalytic activity were vacuum-infiltrated with either a mock inoculum of sterile water or a suspension of the Pst DC3000 hrpA mutant and changes in gene expression in infiltrated leaf tissues were investigated 7 hours after inoculation using Affymetrix ATH1 genechips.
NASCARRAY417 Microarray analysis of Arabidopsis plants inoculated with HopAO1 mutants of Pst DC3000 2007-02-28	This experiment was performed to identify gene expression changes in wild type (Col-0) Arabidopsis plants after inoculation with Pseudomonas syringae pv. tomato (Pst) strain DC3000 or a mutant derivative lacking the type III effector tyrosine phosphatase HopAO1. Col-0 Arabidopsis plants were inoculated by vacuum-infiltration with suspensions of Pst DC3000 or the deltaHopAO1 mutant at 10e8 cfu/ml or with a mock inoculum of sterile water. Inoculated leaf tissues were harvested 7 hours after infiltration and used for RNA isolation and subsequent microarray analysis using Affymetrix ATH1 genechips.
NASCARRAY418 Genes co-regulated by the NMD factors UPF2 and SMG5 2011-10-05	Eukaryotic cells have evolved a mechanism called nonsense mediated mRNA decay (NMD) that detects and degrades aberrant mRNAs that contain premature termination codons (PTCs). NMD has recently acquired broader importance as it has been found to regulate not only aberrant mRNAs but also a great diversity of transcripts, including wild type genes in mammals, Drosophila and yeast. Seven proteins are the core of the NMD complex: SMG1, SMG2 (UPF1), SMG3 (UPF2), SMG4 (UPF3), SMG5, SMG6 and SMG7. Plants have orthologues of most of these proteins. We have identified and characterized a number of alleles of UPF1, UPF3 and SMG5 and made 35S:UPF2 RNAi (upf2i) transgenic plants, all of which share some phenotypic characteristics. Transcriptome analysis of upf1 and upf3 mutants has identified a subset of genes coregulated by UPF1 and UPF3 and, therefore, by NMD (unpublished data). By doing further transcriptome analysis on smg5 mutants and upf2i plants we aim to build a more robust subset of NMD targets in Arabidopsis. RNA will be extracted from 17 day-old mutant, transgenic and wild type seedlings grown at 22-24 C under constant light.
NASCARRAY419 Signaling from an Altered Cell Wall to the Nucleus Mediates Sugar-responsive Growth and Development 2007-08-31	Sugars such as glucose function as signal molecules that regulate gene expression, growth and development in plants, animals and yeast. To understand the molecular mechanisms of sugar responses, we isolated and characterized an Arabidopsis thaliana mutant, high sugar- response 8 (hsr8), which enhances sugar responsive growth and gene expression. Light- grown hsr8 plants exhibited increased starch and anthocyanin and reduced chlorophyll content in response to glucose treatment. Dark- grown hsr8 seedlings show glucose- hypersensitive hypocotyl elongation and development. The HSR8 gene, isolated using map-based cloning, is allelic to the MUR4 gene involved in arabinose synthesis. Dark- grown mur1 and mur3 seedlings also exhibit similar sugar responses to hsr8/mur4. The sugar- hypersensitive phenotypes of hsr8/mur4, mur1 and mur3 were restored to those of WT by boric acid, suggesting that alterations in the cell wall cause hypersensitive sugar- responsive phenotypes. Genetic analysis showed that sugar- hypersensitive responses in hsr8 mutants were suppressed by prl1, indicating that nuclear-localised PRL1 is required for enhanced sugar-responses in hsr8 mutant plants. Microarray analysis revealed that expression of many cell wall-related and sugar-responsive genes was altered in mur4-1, and expression of a significant proportion of these genes was restored to wild type levels in the mur4-1prl1 double mutant. These findings reveal a pathway that signals changes in the cell wall through PRL1 to altered gene expression and sugar- responsive metabolic, growth and developmental changes. The above experiments are conducted on 6d-old seedlings grown on medium containning 1% glucose in dark.
NASCARRAY420 prr mutants and 35S::PRR transgenics under constant light conditions 2008-02-18	Previous microarray analyses had revealed that myriad of genes are regulated under the circadian clock. These transcripts appear peak abundances at all phases of the subjective day and night, indicating that a complex network of transcriptional factors is used to regulate these transcripts throughout the various phases. The clock core to generate circadian rhythmic gene expression relies on the interaction of genetic feedback loops. The PSEUDO RESPONSE REGULATOR (PRR) gene family including TIMING OF CAB EXPRESSION 1 (TOC1) is involved in the mechanism. Indeed, prr9-10 prr7-11 prr5-11 (d975) triple mutant showed the arrhythmicity of circadian-associated genes expression. Prr9-10 is the SALK_007557 T-DNA insertion line, whose insertion is located the second exon of PRR9 coding sequence. Prr7-11 is the SALK_030430 T-DNA insertion line, whose insertion is located the first exon of PRR7 coding sequence. Prr5-11 is the KAZUSA_KG24599 T-DNA insertion line, whose insertion is located the fourth exon of PRR5 coding sequence. In this study, we use microarray analyses employing the d975, prr9-10 prr7-11 (d97), prr9-10 prr5-11 (d95), prr7-11 prr5-11 (d75), 35S::PRR5 (5ox), 35S::PRR1 (=35S::TOC1) (1ox), phyB-9 (phyB), and wild type (Col-0) to describe the global CCG regulatory network. These plants were grown on MS plate containing 2% sucrose for 18 days under constant light conditions. Under the conditions, the circadian phases of the plants in the same sample were different from each other because the germination timing differs between plants. Therefore, the gene expression level of CCGs should be a diurnal average (or mixed) level. Three independent biological samples of plants were harvested in 8h intervals. The RNA samples were prepared from the plants with use of RNeasy Plant Mini Kit (Qiagen, Valencia, CA). The quality of RNA 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 global CCG regulatory network by PRR9, PRR7, and PRR5.
NASCARRAY421 prr9prr7prr5 time course during late daytime 2007-05-01	Previous microarray analyses had revealed that myriad of genes are regulated under the circadian clock. These transcripts appear peak abundances at all phases of the subjective day and night, indicating that a complex network of transcriptional factors is used to regulate these transcripts throughout the various phases. The clock core to generate circadian rhythmic gene expression relies on the interaction of genetic feedback loops. The PSEUDO RESPONSE REGULATOR (PRR) gene family including TIMING OF CAB EXPRESSION 1 (TOC1) is involved in the mechanism. Indeed, prr9-10 prr7-11 prr5-11 (d975) triple mutant showed the arrhythmicity of circadian-associated genes expression. Prr9-10 is the SALK_007557 T-DNA insertion line, whose insertion is located the second exon of PRR9 coding sequence. Prr7-11 is the SALK_030430 T-DNA insertion line, whose insertion is located the first exon of PRR7 coding sequence. Prr5-11 is the KAZUSA_KG24599 T-DNA insertion line, whose insertion is located the fourth exon of PRR5 coding sequence. In this study, we use microarray analyses employing the d975 mutant to describe the global CCG regulatory network. The wild type (Col-0) and d975 were grown on MS plate containing 2% sucrose for 18 days under 12h light / 12h dark conditions. Then, three independent biological samples of plants were harvested at ZT8 (Zeitgeber time 8; ZT0 means the time light on), ZT10, ZT12, and ZT14. The RNA samples were prepared from the plants with use of Rneasy Plant Mini Kit (Qiagen, Valencia, CA). The quality of RNA 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 global CCG regulatory network by PRR9, PRR7, and PRR5.
NASCARRAY423 Gibberellin responsive genes in Arabidopsis 2013-01-03	The aim of the experiment was to identify early gibberellin (GA) responsive genes in the roots of an Arabidopsis GA deficient mutant. The GA deficient mutant used in this study is a transgenic line overexpressing the PcGA2ox1 gene. This mutant has an identical phenotype to ga1-3, but it does not require exogenous GA treatment for germination. Seeds were germinated on 1xMS + 1% (w/v) sucrose plates containing 0.7% gelrite, and grown under continous light. The plates were orientated vertically. After six days growth the plates were transferred to acclimatisation baths containing 1 x MS liquid media so that the roots were submerged. After 24 hours acclimatisation plates were treated with or without 2uM GA4 for 0, 30, 60 and 180 minutes. Root tips (up to the end of the elongation zone) were harvested from these plants (approx 150 per sample), giving a total of 7 experimental samples (1: untreated, 2: 30 mins GA4, 3: 30 mins untreated, 4: 60 mins GA4, 5: 60 mins untreated, 6: 180 mins GA4, 7: 180 mins untreated. Three biological replicates were performed giving a total of 21 root samples. Total RNA was isolated from the roots using the QIAGEN RNeasy method.
NASCARRAY425 Polycomb mutant seedlings 2008-09-02	We have identified a histone methyltransferase gene CURLY LEAF (CLF), which is required to repress a floral homeotic gene AGAMOUS (AG). CLF acts redundantly with a related gene SWINGER (SWN), so that clf swn double mutants have extreme, near lethal phenotypes in which expression of many target genes is disrupted. To understand better when and where these genes act, we propose to use a system for generating mosaic plants, in which marked sectors of cells are created that gain or lose CLF (or other Pc-G) gene function in SWN+ and swn- mutant backgrounds. This will allow us to address questions such as whether the Pc-G genes are needed throughout development to silence their targets, whether they are needed once cell division has ceased, whether they can silence their targets once the targets have been activated, and whether genes that are epigenetically silenced by CLF are impervious to activating factors. Source: Justin Goodrich staff homepage Aim is to compare gene expression in wild type and polycomb mutant seedlings. Comparison will be of bulked seedlings of four genotypes: Columbia, clf-28, swn-7, clf-28 swn-7. Tissue will be grown in tissue culture.
NASCARRAY426 Photoreceptor control of shoot meristem activity and leaf initiation 2007-07-31	When Arabidopsis seedlings are grown in the dark, their shoot meristem remains in a repressed state and does not develop leaves. The tight control of leaf development by light provides the opportunity to analyse a fundamental plant developmental process in its very early stages. We have observed that activation of photoreceptors in dark-grown seedlings leads to a rapid and dramatic increase in mitotic activity in and around the shoot meristem. Cotyledons also exhibit an induction of cell cycle activity by light, but this consists of endoreduplication and occurs later. Many other processes take place upon induction by light, including the development of the photosynthetic apparatus and the synthesis of sunscreens, but these processes are expected to take place in both the leaf primordia and the cotyledons. We have used this information to carry out a whole transcriptome analysis of shoot meristem activation and early stages of leaf development by light. For this we dissected the shoot apical regions from seedlings grown in the dark or 1, 2, 6, 24, 48 or 72 h after transfer to light. We also dissected cotyledons from equivalent seedlings grown in the dark or 1 or 6 h after transfer to light. From these samples we obtained RNA used for hybridisations against the ATH1 GenChip.
NASCARRAY428 NPA-induced vascular overgrowth in Arabidopsis 2007-08-23	We used NPA-induced vascular overgrowth in Arabidopsis leaves to look for differential up-regulation of genes in NPA-treated tissues that may be involved in vascular differentiation. Arabidopsis thaliana Col-0 plants were grown for approximately 2 weeks on solid ATS medium (1) containing a final concentration of 10 um NPA (dissolved in DMSO) or an equivalent volume of DMSO (control). At this stage plants had approximately 6 rosette leaves. RNA was prepared from entire shoot tissues of control (DMSO) or NPA-treated plants.(1) Lincoln et al., 1990. Plant Cell 2: 1071-1080.
NASCARRAY429 Identification of target genes of the NMD factor SMG7b 2011-10-05	Nonsense-mediated mRNA decay (NMD) is a conserved mRNA quality control mechanism that identifies and destroys aberrant mRNAs that contain premature termination codons (PTCs). The NMD core comprises seven proteins, from SMG1 to SMG7. Arabidopsis has orthologues of most of these proteins. Studies on yeast, Drosophila, Humans and Arabidopsis reveal that NMD not only functions to target PTC-containing mRNAs but also acts as a global regulator of gene expression. It has also been reported that the NMD pathway branches with some target genes being dependent on specific NMD factors. In order to determine the extent to which different NMD factors co-regulate or independently regulate Arabidopsis genes, transcriptome analysis of smg7b-1 mutants will be carried out and added to existent data of upf1, upf3 and smg5 NMD mutants for comparison. RNA will be extracted from 17 day-old mutant and wild type seedlings grown at 22-24 C under constant light.
NASCARRAY430 Identifying Y candidate genes 2009-11-11	The circadian clock generates biological rhythms with a period of approximately 24 hours. Using microarray experiments, we have previously shown that approximately 16% of the Arabidopsis genome is regulated in a circadian manner (Edwards et al., 2006). Previous work from our lab in modelling the molecular oscillator of Arabidopsis introduced a hypothetical component Y into an evening loop of the clock gene network (Locke et al., 2005). GIGANTEA (GI) was suggested as a strong candidate for Y based on genetic evidence and its close matching of the expression profile predicted by the mathematical modelling. Recent experimental evidence suggests that GI may only partially account for the function of Y. Thus, we are undertaking a genomics approach to identify other candidate genes that match the predicted expression profile of Y. Samples were taken from wild type and lhy cca1 double mutant seedlings from 4 timepoints around the night to day transition (-15mins, +15mins, +30mins and +60mins) after 9 days of growth in 18:6 light dark cycles. We aim to identify genes showing the light induction response predicted for Y around dawn.
NASCARRAY435 Etiolated seedling of the exo70A1 mutant versus wild type 2011-08-23	The EXO70A1 protein is supposed to be a subunit of putative plant exocyst complex. We characterized two independent T-DNA insertional mutants of the AtEXO70A1 gene (exo70A1-1 and exo70A1-2). Heterozygous EXO70A1/exo70A1 plants appear normal and segregate in a 1:2:1 ratio, suggesting that neither male nor female gametophytes are affected by the EXO70A1 disruption. However, both exo70A1-1 and exo70A1-2 homozygotes exhibit a suite of phenotypic defects. Polar growth of root hairs and stigmatic papillae is disturbed. Hypocotyls of etiolated seedlings are shorter. Organs are generally smaller, plants show loss of apical dominance and an indeterminate growth where instead of floral meristems new lateral inflorescences are initiated in a reiterative manner. Both exo70A1 mutants have dramatically reduced fertility. These results suggest that the putative exocyst subunit EXO70A1 is involved in cell and organ morphogenesis.
NASCARRAY436 Investigating the function of the circadian clock at high temperatures 2009-06-15	A significant proportion of the Arabidopsis transcriptome is under endogenous circadian regulation, that is entrained by light-dark cycles (Reviewed in: Hall and McWatters 2005). These genes are associated with photosynthesis, metabolism, growth and photo-protection, amongst others. Hence, it is not surprising that the circadian clock has been shown as a key factor for plants fitness (Dodd et al. 2005), such that mutants which lack this endogenous control show a rather poor performance. In this experiment, we aim to investigate how important is the clock for plants growing at high temperatures, using WT (Col) and plants with no functional clock (cca1-ox line 038). Seeds were sown on Araflat (Arasystem, Betatech bvba, Gent, Belgium) filled with a 3:1 mixture of compost and perlite (John Innes compost No.2, KS Horticultural Products, Seaview Nurseries, Egremont, Cumbria) maintained at field capacity and containing 0.2g per l insecticide Intercept 70WS (The Scotts Company Ltd, UK), in 12L:12D cycles at 22ÂºC in growth chambers until two real leaves were developed (12 days) . They were then transplanted to 20 hole multicell half trays (MC20, Desch-Plantpak) containing the same soil and at the same temperature for three days to recover from the transplant stress. Finally, plants were transferred to either 12L:12D cycles (160 umolm-2s-1) and 17ÂºC or 27ÂºC. Plants, were grown for a further 7days at these two temperatures, until six to eight leaves were developed (developmental stage 1,6-1,8). At this point none of the plants had flowered.Plant material was collected one hour before dawn and one hour after dusk. A green safety light was used to harvest plants prior to dawn. We collected a composite sample of 10 plants for each treatment combination in total eight samples: 2 genotypes x 2 temperatures x 2 sample times.
NASCARRAY437 Systemic responses to Pseudomonas syringae infection 2009-05-15	We used three different strains of Pseudomonas syringae pv tomato DC3000 to investigate systemic responses to infection in Arabidopsis and the development of SAR. Wildtype DC3000, the hrpA mutant and DC3000 carrying the avirulence gene avrRpm1 were syringe infiltrated into 4 week old plants at a concentration of 10e8 cfu/ml. At least 5 leaves per plant were infiltrated and at least 10 plants were pooled for each sample. Systemic, uninfected tissue was then harvested at 8, 12 and 21h after inoculation. Three independent experiments were carried out to give three biological replicates for each timepoint.
NASCARRAY438 Determining the downstream genetic targets of the transcription factor AtGLK1 2009-11-11	AimTo discover the target genes of Golden2-like transcription factors in Arabidopsis.BackgroundThe Golden2-like (GLK) family of transcription factors is essential for proper chloroplast development in several plant species. In Arabidopsis, there are two largely redundant GLK genes, GLK1 and GLK2. Double mutant plants are pale green and reduced in stature. Their most notable chloroplast-related phenotype is impaired thylakoid membrane appression and reduction in steady-state levels of Photosystem II components. GLK proteins group within the GARP family of myb transcription factors; however, the genes they regulate remain unknown.Experimental DesignWe have employed a two-component dexamethasone-inducible expression system [1]. An overexpressed fusion protein comprising the glucocorticoid receptor (GR), the lac repressor and the Gal4 activation domain resides in the plant cytoplasm. This protein, termed LHGR-N, enters the nucleus once dexamethasone binds to the GR domain. It then activates transcription from the lac operator promoter sequences present on separate transgenes which carry the cDNA to be induced. Since overexpression of either GLK1 or GLK2 is sufficient to complement the mutant phenotype, we reasoned that induction of expression of GLK1 and GLK2 cDNAs in the glk1;glk2 double mutant background should allow determination of their downstream genetic targets. In the first case, we decided to focus on GLK1 only.Genetic linesPlants carrying a single copy of the LHGR-N transgene, conferring kanamycin resistance, were crossed with glk1;glk2 double mutant plants. A single homozygous F3 line was selected on the basis of pale green mutant phenotype and kanamycin resistance. This line was transformed with a construct carrying the GLK1 cDNA downstream of six repeats of the lac operator (pOp6) and a single CaMV minimal promoter. This construct confers hygromycin resistance. Transformants were confirmed by PCR and southern blot, before being selected on the basis of transcript accumulation following induction by dexamethasone. The line with the strongest induction response was chosen for microarray experiments.Tissues: Approx. 100 10-day old seedlings were grown on MS plates and transferred to 100 ml liquid MS for two further days prior to induction. Whole seedlings were harvested.Treatment: either a) 10 uM dexamethasone dissolved in DMSO (INDUCED samples) or b) 0.1% v/v DMSO (CONTROL samples). Samples were harvested 4 and 24 hours post-treatment. Separate vessels were used for each time point and treatment. Two complete replicates were performed.Reference1. Craft, J. et al. (2005) Plant J. 41 899-918.
NASCARRAY439 DUO1 ectopic expression 2009-05-15	The experiment is designed to examine changes in gene expression upon ectopic expression of the germ line transcription factor DUO1 in Arabidopsis. Plants were transformed with the vector pMDC7DUO1, allowing induction of DUO1 by treatment with estradiol. T2 seedlings were selected on agar plates containing hygromycin for 12 days before seedlings were transferred to plates containing either 2 ÂµM 17ÃŸ-estradiol (induced), or control plates containing DMSO (non-induced). Plants were grown for 6, 12 or 24 hours before harvesting. RNA was extracted using the RNAeasy kit from Qiagen. Each sample consists of 20 seedlings combined and all treatments have been conducted in triplicate.
NASCARRAY440 HAP5 Triple Mutant vs Constans Mutant 2009-04-21	Aims:To compare the transcriptional profiles of a a AtHAP5 (AtNF-YC, AtCBF-C) triple mutant vs. Constans mutant. Background Information on Mutants:AtHAP5 Triple mutant created by cross between the following SALK TDNA inserts; SALK_034838, SALK_058903, SALK_032163 these correspond to At1G54830, At1G08970, At5G63470, respectively. co-SAIL TDNA insertion SAIL_24_H04 corresponds to AT5G15840 Tissue:Above ground whole plants grown for 10 days, under 16 hr light 8 hrs dark at constant 22ÂºC. Tissue collected 14 hrs after lights on. RT-PCR analysis on test RNA confirmed FT levels reduced in mutants lines compared to wild-type.
NASCARRAY442 Analysis of anther development by identifiying downstream genes controlled by MS35/MYB26 2008-07-21	MALE STERILITY 35 (MS35/ MYB26) is a R2R3-type MYB transcription factor (Steiner-Large et al., 2003). MS35 is suggested to be a key regulator in the process of lignified secondary thickening in the anther endothecium which provides mechanical force for anther dehiscence (Dawson et al., 1999). The ms35 mutant bears non-dehiscent anthers and thus exhibits a male sterile phenotype. This microarray experiment aims to identify genes controlled by MS35 and thus establish the precise role MS35 plays during anther development.We have demonstrated that MS35 is expressed in the anther during pollen mitotic divisions. Comparisons of gene expression will be carried out between anthers from the Ler.gl (wild-type control) and ms35.gl (mutant), to identify genes with altered expression due to the ms35 mutation. The use of the gl marker will enable a prompt identification of the ms35 mutant amongst the segregating population. The wild-type control line was chosen to be Ler.gl which ensures that the changes of gene expression are not due to the gl mutation.Plants were grown in growth chambers at 22h of daylight (167 umol.m2.sec-1) at 24 C and 2h of night at 24 C. Developmental stages of anthers were defined by bud position and DAPI staining. As soon as the plants showed three fertile or sterile siliques, anther and filaments were collected from both lines at Pollen Mitosis I (PM I), bicellular, and PM II stage, separately. RNA was extracted using RNeasy Mini Kit (Qiagen, USA) and digested with DNase to eliminate genomic DNA contamination. The identified genes will be further analysed using bioinformatics and genomic (knockout) resources that are available for Arabidopsis.
NASCARRAY444 ces transcript analysis 2009-11-10	ces transcript analysis
NASCARRAY445 Dissecting the genetic basis of nonhost resistance to Phytophthora infestans in Arabidopsis 2009-10-21	Causal agent of the notorious late blight disease of potato is the oomycete Phytophthora infestans. Research on late blight resistance has focussed on the identification and genetic mapping of qualitative (R-genes) and quantitative (QTL) resistance, mainly in host plants like potato and its wild Solanum relatives. However, relatively little has been done to identify genes that are involved in nonhost resistance, presumably the most durable type of resistance. To gain insight into the genetic basis of nonhost resistance to P. infestans in Arabidopsis thaliana, we generated a gamma radiated deletion population (40 Krad, 0.0125-0.250 deletion frequency, 9 kb deletion (70%)) in both Landsberg erecta (Ler) and Colombia (Col) ecotypes, and screened 10.000 M2â€™s for reduced resistance to P. infestans. A total of 62 â€˜susceptibleâ€™ mutants were identified, all in the Ler background, 44 of which were crossed with Col for genetic analysis. In one of the mutants(1784) that harboured a mutation that segregated as a single locus, we mapped the mutation to an intergenic region of 8.6 kb on chromosome 2, between the annotated genes AT2g31270 and AT2g31280. In this region many small RNAâ€™s have been predicted (ASRP database). Small RNAâ€™s are known to act as regulatory factors during growth and development and function in several epigenetic and post-transcriptional silencing systems, including resistance mechanisms (Navaro et al, Science 312, 436-439, 2006). The nature of the mutation causing the â€˜susceptibleâ€™ phenotype will be presented as well as the results of an Affymetrix array based gene expression study.
NASCARRAY447 Interaction Arabidopsis thaliana vs. Ralstonia solanacearum 2008-06-06	Herein, whole genome expression analysis was conducted on rosettes of resistant and susceptible Arabidopsis thaliana accessions challenged with Ralstonia solanacearum. Two disease situations were considered: Col-5 plants inoculated with the virulent GMI1000 strain, as well as Nd-1 plants challenged with the same bacterial strain deleted of the avr PopP2 gene (DeltaPopP2), in both cases developing wilt disease symptoms. In contrast, Nd-1 plants challenged with GMI1000 are fully resistant to the pathogen and served as a control.Samples were harvested at the time of inoculation, as well as at 6, 12, 24 hours -at these times no symptoms were visible-, 5 days after inoculation when the first wilt symptoms became visible (25% of wilted leaves: disease index 1, D1), and finally 8 days after inoculation when 75 % of leaves were completely wilted (disease index 3, D3).
NASCARRAY448 DEK1 calpain overexpression 2009-11-02	AIM: To understand the role of DEK1 in Arabidopsis development. BACKGROUND: DEK1 has an essential role during embryogenesis and is involved in the maintenance of the epidermis tissues: Comparison of wild-type and ADEK1 calpain overexpressing line
NASCARRAY450 Epigenetic mutant gene expression analysis 2009-11-11	The aim of this project is to identify endogenous targets of yet unidentified epigenetic mutants. Mutants were discovered in a forward genetic approach using T-DNA insertion mutagenesis.Pools of each 50 seedlings, grown in-vitro, will be used to examine changes in gene-expression.
NASCARRAY453 Natural variation of auxin response 2009-11-02	To assess natural variation of downstream auxin responses we subjected 7 different arabidopsis ecotypes to a time course of auxin treatments. 7d-old seedlings grown in liquid culture have been treated for 0, 30 min, 1h and 3h with 1 ÂµM IAA.
NASCARRAY454 Arabidopsis thaliana mutant leaves treated with Pseudomonas syringae ES4326 2008-04-02	In order to better understand the transcriptional networks triggered by pathogen inoculation, we monitored gene expression in leaves of mutant Arabidopsis plants, inoculated with Pseudomonas syringae ES4326 and wild type Col-0 plants grown in parallel. Individual leaves were injected in the morning using a needle-less syringe with 10E5 cfu cm-2 PsmES4326 (suspended in 5 mM MgSO4). For the wild type, leaves were also mock treated with 5 mM MgSO4. Leaves were harvested 24 hours later. Plants were grown in pots with BM-2 soil (Berger Peat Moss Ltd, Quebec, Canada) at a density of 9 plants per pot and kept at 22 degrees Celsius with 75% humidity and a 12 hour day length.
NASCARRAY455 pad4 vs. pad4 est1b 2009-11-11	This experiment was designed to identify transcripts which are changed in plants deficient for evolutionary conserved EST1B gene. A mutation in est1b leads to a constitutive up-regulation of a PAD4-dependent pathogen response pathway. To eliminate secondary effects of the constitutive pathogen response in est1b mutants we will analyze transcriptoms in plants in which lack PAD4.
NASCARRAY457 Transcriptome of Arabidopsis APS kinase null mutants 2009-01-13	The aim of the experiment is to look at transcriptional changes in null mutants of APS kinase. There are four APS kinase isoforms in Arabidopsis, we are interested in the double mutant APK1/APK2, created by crossing two single salk lines. The transcriptomes of WT (Col-0) plants and the double knockout (ab) will be compared. The plants were grown under controlled environment conditions of 10h day, 14h night, 22 degrees centigrade, 60% humidity for five weeks. 10 whole rosettes were pooled for each replicate, there are three replicates for each genotype.
NASCARRAY460 Analysis of RLI on RNA stability in A. thaliana 2009-11-10	A number of RNA encoding different functions have been detected in the plant vasculature. An RNase L inhibitor, similar to that present in humans have been identified in the phloem sap of cucurbits. We are interested in analyze the role of this putative inhibitor on the transcript levels that could be present in the vasculature and mainly in the phloem stream. For this purpose, we obtained an RLI T-DNA insertion mutant and generated A. thaliana overexpressing lines using the ORF of the two alleles found in the database.
NASCARRAY463 Priming for enhanced MeJA-responsive gene expression in Arabidopsis plants expressing P. fluorescens 2008-07-15	Upon appropriate stimulation, plants can develop an enhanced capacity to express infection-induced cellular defense responses, a phenomenon known as the primed state. Colonization of the roots of Arabidopsis thaliana by the beneficial rhizobacterial strain Pseudomonas fluorescens WCS417r primes the leaf tissue for enhanced pathogen- and insect-induced expression of jasmonate (JA)-responsive genes, resulting in an induced systemic resistance (ISR) that is effective against different types of pathogens and insect herbivores. Here we investigated the molecular mechanism of this rhizobacteria-induced priming response by following a whole-genome transcript profiling approach. To this end we profiled the transcriptome of the leaves of 5-week-old Arabidopsis Col-0 plants at 0, 1, 3, and 6 hours after exogenous application of 50 Ã‚ÂµM MeJA. This was done with Col-0 plants that were grown in soil with or without ISR-inducing Pseudmonas fluorescens WCS417r rhizobacteria. This microarray analysis is described in Pozo, M.J., Van der Ent, S., Van Loon, L.C. and Pieterse, C.M.J. (2008). The transcription factor MYC2 is involved in priming for enhanced defense during rhizobacteria-induced systemic resistance in Arabidopsis. New Phytologist, in press.
NASCARRAY466 Epigenetic mutant gene expression analysis_2 2009-11-18	The aim of this project is to identify endogenous targets of yet unidentified epigenetic mutants. Mutants were discovered in a forward genetic approach using T-DNA insertion mutagenesis. Pools of each 25 seedlings, grown in-vitro, will be used to examine changes in gene-expression.
NASCARRAY468 Arabidopsis thaliana/Phytophthora parasitica compatible interaction transcriptome 2009-11-18	Oomycetes from the genus Phytophthora are fungus-like plant pathogens that are devastating for agriculture and natural ecosystems. Due to particular physiological characteristics, no treatments against diseases caused by oomycetes are presently available. To develop such treatments, it appears essential to dissect the molecular mechanisms that determine the interaction between Phytophthora species and host plants. The present project is focused on the molecular mechanisms that underlie the compatible plant-oomycete interaction and plant disease.The laboratory developed a novel interaction system involving the model plant, Arabidopsis thaliana and Phytophthora parasitica, a soil-borne pathogen infecting a wide host range, thus representing the majority of Phytophthora species. A characteristic feature of the compatible Arabidopsis/P. parasitica interaction is an extended biotrophic phase, before infection becomes necrotrophic. Because the initial biotrophic phase is extremely short on natural (e.g. solanaceous) hosts, the Arabidopsis system provides the opportunity to analyze, for both interaction partners, the molecular events that determine the initiation of infection and the switch to necrotrophy.The present project aims at analyzing the compatible interaction between A. thaliana roots and P. parasitica. The Affymetrix A. thaliana full genome chip will be used to characterize modulations of the transcriptome occurring over a period of 24h from the onset of plant root infection to the beginning of necrotrophy. Parallel to this study, a custom designed P. parasitica biochip will enable analyzing of P. parasitica gene expression during the same stages.
NASCARRAY470 Molecular responses to external pH changes in roots of Arabidopsis thaliana 2009-11-03	Plants live in soils that vary considerably, both spatially and over time, in terms of nutrient composition and pH. Consistently, plants have to recognize and adapt to these changes by altering their structure and metabolism. The goal of this array analysis is to characterize the global transcriptional response to external pH changes in roots, which to date is almost unexplored. Arabidopsis thaliana (Columbia-0) were grown in hydroponic cultures in basic nutrient solution. Two days before treatment the media was shifted to nutrient solution containing 5mM MES, pH 6. At the time of the treatment start (4 hours after light on) the plants were shifted to nutrient solutions of pH 4.5 and 6.0 (control). Root RNA samples from time point 1 and 8 hour after treatment start is used for array analyzes.
NASCARRAY472 Effect of the explosive compound TNT on wild type sedlings 2008-08-06	The aim of this experiment was to identify the genes involved in the detoxification of the toxic pollutant and explosive compound 2,4,6-trinitrotolune (TNT).Fourteen-day-old, liquid culture grown, Arabidopsis seedlings, ecotype Col0 (NASC stock code N1093), were dosed with 60 uM TNT dissolved in 60 ul dimethyl formamide (DMF) solvent, or 60 ul DMF only. After six hours, RNA was extracted and used for the microarray analysis.Further details and characterisation of glucosyltransferases identified using this method are presented in: Gandia-Herrero F, Lorenz A, Larson T, Graham I, Bowles D, Rylott E, Bruce N. Detoxification of the explosive 2,4,6-trinitrotoluene in Arabidopsis- discovery of bifunctional O- and C-glucosyltransferases (2008). The Plant Journal Dec;56(6):963-74.
NASCARRAY474 Identification of circadian transcripts that are co-regulated with [Ca2+]cyt 2011-05-05	Our aim is to identify circadian transcripts that are co-regulated with [Ca 2+]cyt, with the eventual goal of identifying genetic regulators and targets for circadian oscillations of [Ca 2+]cyt. We have identified two conditions in which [Ca 2+]cyt behaves differently to other circadian outputs. 1. Treatment of plants with nicotinamide, a metabolic inhibitor of ADPR cyclase, abolishes the circadian oscillations of [Ca 2+]cyt however, leaf movement, CCA1, LHY, TOC1 and CAB transcript abundance and CAB promoter activity are all rhythmic albeit with a longer period (Dodd et al., 2007). 2. The toc1-1 mutant, which shortens the circadian period of all other rhythms tested, has no effect on the period of [Ca 2+]cyt oscillations (Xu et al., 2007). We will measure the circadian regulation of transcript abundance in wild type (C24), toc1-1 and nicotinamide (C24) treated plants.METHOD: Wild type (C24) and toc1-1 seeds were sown on 1/2MS 0.8% agar plates and imbibed at 4 C for 48 hours. Seedlings were grown in LD cycles of 12hL:12hD at 19 C for 11 days to entrain the oscillator. Following transfer to LL at dawn on the 12th day, 50% of the wild type seedlings were dosed with 50 mM nicotinamide every 2 hours over the entire course of the experiment. Wild type, toc1-1 and nicotinamide treated seedlings (app 100 for each sample, excluding roots) were harvested at 4 hour intervals from 49 to 93 hours in LL (12 time points covering the entire third and fourth circadian cycles.) Two independent replicates of the whole experiment will be hybridised.
NASCARRAY475 partial complementation of SALK_074069 2011-08-18	RNA extracted from total floral tissue (stage 1-13 buds and the floral meristem) and rosette leaf. Plant background: Col-0. Short day (10 h photoperiod).
NASCARRAY477 Expression comparison for white light grown Wt vs knockout arabidopsis 2009-11-18	The aim of this experiment is determine regulation of downstream factors through comparison of Wildtype vs gene knockout Arabidopsis lines. We have grown seedlings for 6 days under 16 hour white light and 8 hours dark. Seedlings were sampled 8 hours into the light cycle of the 6th day.
NASCARRAY478 Expression analysis in HDA6 mutants 2009-11-04	In addition to the canonical RNAi pathways that targets mRNAs in the cytoplasm, several RNA-dependent pathways operate in the nucleus to induce sequence-specific epigenetic modifications. One specialized type of RNAi-mediated pathway is RNA-directed DNA methylation (RdDM). During RdDM, nuclear DNA with sequence identity to the trigger dsRNA is de novo methylated at almost all cytosine residues, providing a mark for the formation of transcriptionally silent heterochromatin. Genetic forward screens identified the RPD3-like histone deacetylase HDA6 as the enzyme responsible for the histone deacetylation step of RdDM and suggest that HDA6 might have acquired specific functions for RNA-directed transcriptional silencing processes [Aufsatz et al., 2002a; Aufsatz et al., 2002b].Complete loss-of-function mutants for AtHDA6 (rts1-1; RNA-mediated transcriptional gene silencing) exhibit reactivation of RdDM-silenced promoters, despite the continuous presence of the silencing-inducing RNA signal. One of the found mutant alleles, rts1-5, has an amino acid substitution located at a conserved position within the HDAC domain (Naumann et al., manuscript in preparation). This mutant is characterized by strong reactivation of an RdDM-silenced target promoter, despite maintaining wild-type levels of cytosine methylation.An evaluation of the transcription pattern among the mutants with opposite methylation phenotype and wild-type plants should show which genes are differentially regulated between the mutants in comparison to wild-type plants.
NASCARRAY479 Treatment of Arabidopsis with low concn. of nitrate 2008-11-10	The genomic response to low levels of nitrate was studied in Arabidopsis using the Affymetrix ATH1 chip containing more than 22,500 probe sets. Arabidopsis plants were grown hydroponically in sterile liquid culture on ammonium as the sole source of nitrogen for 10 d, then treated with 250 ÃŽÂ¼m nitrate for 20 min. The response to nitrate was much stronger in roots (1,176 genes showing increased or decreased mRNA levels) than in shoots (183 responding genes). In addition to known nitrate-responsive genes (e.g. those encoding nitrate transporters, nitrate reductase, nitrite reductase, ferredoxin reductase, and enzymes in the pentose phosphate pathway), genes encoding novel metabolic and potential regulatory proteins were found. These genes encode enzymes in glycolysis (glucose-6-phosphate isomerase and phosphoglycerate mutase), in trehalose-6-P metabolism (trehalose-6-P synthase and trehalose-6-P phosphatase), in iron transport/metabolism (nicotianamine synthase), and in sulfate uptake/reduction. In many cases, only a few select genes out of several in small gene families were induced by nitrate. These results show that the effect of nitrate on gene expression is substantial (affecting almost 10% of the genes with detectable mRNA levels) yet selective and affects many genes involved in carbon and nutrient metabolism.
NASCARRAY480 WT vs NR null mutant high nitrate concn treatment 2008-11-10	A nitrate reductase (NR)-null mutant of Arabidopsis was constructed that had a deletion of the major NR gene NIA2 and an insertion in the NIA1 NR gene. This mutant had no detectable NR activity and could not use nitrate as the sole nitrogen source. Starch mobilization was not induced by nitrate in this mutant but was induced by ammonium, indicating that nitrate was not the signal for this process. Microarray analysis of gene expression revealed that 595 genes responded to nitrate (5 mM nitrate for 2 h) in both wild-type and mutant plants. This group of genes was overrepresented most significantly in the functional categories of energy, metabolism, and glycolysis and gluconeogenesis. Because the nitrate response of these genes was NR independent, nitrate and not a downstream metabolite served as the signal. The microarray analysis also revealed that shoots can be as responsive to nitrate as roots, yet there was substantial organ specificity to the nitrate response.
NASCARRAY481 Arabidopsis treated with nitrite and nitrate 2008-11-10	Nitrate serves as a potent signal to control gene expression in plants and algae, but little is known about the signaling role of nitrite, the direct product of nitrate reduction. Analysis of several nitrate-induced genes showed that nitrite increases mRNA levels as rapidly as nitrate in nitrogen-starved Arabidopsis (Arabidopsis thaliana) roots. Both nitrite and nitrate induction are apparent at concentrations as low as 100 nm. The response at low nitrite concentrations was not due to contaminating nitrate, which was present at <1% of the nitrite concentration. High levels of ammonium (20 mm) in the growth medium suppressed induction of several genes by nitrate, but had varied effects on the nitrite response. Transcriptome analysis using 250 or 5 microm nitrate or nitrite showed that over one-half of the nitrate-induced genes, which included genes involved in nitrate and ammonium assimilation, energy production, and carbon and nitrogen metabolism responded equivalently to nitrite; however, the nitrite response was more robust and there were many genes that responded specifically to nitrite. Thus, nitrite can serve as a signal as well as if not better than nitrate.
NASCARRAY482 Identification of early gibberellin (GA) responsive genes in Arabidopsis hypocotyls 2009-11-03	The aim of the experiment was to identify early gibberellin (GA) responsive genes in the roots of an Arabidopsis GA deficient mutant.The GA deficient mutant used in this study is a transgenic line overexpressing the PcGA2ox1 gene. This mutant has an identical phenotype to ga1-3, but it does not require exogenous GA treatment for germination.Seeds were germinated on 1xMS + 1% (w/v) sucrose plates containing 0.7% gelrite, and grown under continous light. The plates were orientated vertically.After six days growth the plates were treated with or without 5uM GA4 for 0, 30, 60 and 180 minutes.Approximately 400 hypocotyls were harvested per timepoint using a razorblade and after excision the hypocotyls were frozen in liquid nitrogen giving a total of 7 experimental samples (1: untreated, 2: 30 mins GA4, 3: 30 mins untreated, 4: 60 mins GA4, 5: 60 mins untreated, 6: 180 mins GA4, 7: 180 mins untreated. Three biological replicates were performed giving a total of 21 root samples. Total RNA was isolated from the roots using the QIAGEN RNeasy method.
NASCARRAY483 Biological function of Pip3 2009-11-25	Ralstonia solanacearum is the causal agent of bacterial wilt. Arabidopsis thaliana monogenic resistance to the strain GMI1000 is conferred by the RRS1-R gene, present in Nd-1 resistant ecotype and absent in the susceptible ecotype Col-0. The corresponding protein is recognized by the avirulence protein PopP2 of R. solanacearum. In order to identify the plant proteins involved in the PopP2/RRS1-R perception complex, the screening of a root cDNA library in yeast was performed using PopP2 as bait. One interactor found was called Pip3, and we would like understanding better its biological function. Thereby we choose to compare two RNAi lines with their wild type Nd-1 background, and one KO line with its wilt type Col-0 background. Also, two overexpressor lines will be compared with their wilt type Nd-1 background and two with their Col-0 background.
NASCARRAY484 Inhibitor of auxin response LI5 2009-11-18	LI5 is an inhibitor of auxin response identified in chemical genetics screen for lateral root emergence inhibitors. Chemicals identified in this screen blocked the auxin induction of LAX3 in the outer tissues (Swarup et al, Nat Cell Biol, 2008). We observed that LI5 affected many other auxin responsive genes (unpublished data, November 2008). Our aim is to identified the overall effects on auxin response using the Arabidopsis thaliana chip Ath1. 3 days old Col-0 Arabidopsis seedlings, germinated on NPA 10uM, were treated for 6 hours with [DMSO, LI5 (5uM), NAA (10uM) or LI5 (5uM) + NAA (10uM)], the shoot and root tip removed and RNA was extracted from the mature part of the root.
NASCARRAY485 Systems approach identifies an organic nitrogen-responsive gene network that is regulated by the mas 2008-11-20	To identify genome-wide responses to such organic N signals, Arabidopsis seedlings were transiently treated with ammonium nitrate in the presence or absence of MSX, an inhibitor of glutamine synthetase, resulting in a block of Glu/Gln synthesis. Genes that responded to organic N were identified as those whose response to ammonium nitrate treatment was blocked in the presence of MSX. We showed that some genes previously identified to be regulated by nitrate are under the control of an organic N-metabolite. Using an integrated network model of molecular interactions, we uncovered a subnetwork regulated by organic N that included CCA1 and target genes involved in N-assimilation. We validated some of the predicted interactions and showed that regulation of the master clock control gene CCA1 by Glu or a Glu-derived metabolite in turn regulates the expression of key N-assimilatory genes. This data is part of Gutierrez et al. PNAS. 2008 March; 105(12): 4939-4944.
NASCARRAY486 Transcriptom of the exo70A1 mutant 2009-11-02	We characterized two independent insertional mutants of the AtEXO70A1 gene, encoding a subunit of the exocyst complex. Homozygous mutants exhibit a suite of phenotypic defects: polar growth of root hairs and stigmatic papillae is disturbed; organs are generally smaller; plants show loss of apical dominance and an indeterminate growth where instead of floral meristems new lateral inflorescences are initiated in a reiterative manner; exo70A1 mutants have dramatically reduced fertility. These results suggest that EXO70A1 is involved in cell and organ morphogenesis.
NASCARRAY487 Response to gamma-irradiation in Arabidopsis thaliana mutant and wild-type seedlings 2010-06-22	Background: At5g24280 is an ATM dependent gamma-irradiation inducible gene (Culligan et al., The Plant Journal, 2006). To evaluate its possible role in the transcriptional response upon ionizing radiation, we collected total RNA from wildtype and one At5g24280 T-DNA insertion line upon gamma-irradiation. Experimental Setup: Seeds were sterilized, washed and imbibed in ddH2O for 3 days at 4Â°C, sown on 1/2 MS plates and grown under continuous light at 22Â°C for 5 days. Plants were irradiated using a Cs137 source (100Gy), returned back into the growth cabinet and harvested 1.5h post irraditaion. RNA was extracted from irradiated and control seedlings using Tri-Reagent (Sigma), then DNase-treated with TurboDNase (Ambion) following the manufacturers suggestions. Additional purification with PCI /CI extraction was followed by EtOH-precipitation. Strains: wildtype plants and the At5g24280 T-DNA insertion line (SALK_036931) are in a Columbia (Col-O) background.
NASCARRAY489 Investigating the molecular response of Arabidopsis to combined nematode and dehydration stress usin 2009-11-25	Plants respond to stress by activating differential signalling pathways leading to tolerance and resistance. Response to individual abiotic and biotic stresses has been well studied. There are key elements in common between the two pathways including the interplay of plant hormones, transcription factors and signal transduction cascades. It is thought that the two pathways may also act antagonistically in order to prioritise stress response, a process governed by the hormones abscisic acid and jasmonic acid (Anderson et al., 2004 Plant Cell 16: 3460-3479). Little is known about the molecular effect of simultaneous abiotic and biotic stress in plants. This study will characterise transcriptome changes in response to combined abiotic and biotic stress, namely dehydration and nematode infection. The response to these stresses will be analysed in roots and leaves. Arabidopsis Col-0 plants were grown on half MS media for 18 days (growth stage 1.08-1.12, Boyes et al., 2001 Plant Cell 13:1499-1510). Half the plants were challenged with rigorously sterilised, infective Heterodera schachtii nematodes as described before (Fuller et al., 2007 Molecular Plant Pathology 8: 595-609). Each plant was treated with a total of c. 175 juveniles in sterile distilled water at five separate infection points. Control plants were mock inoculated with sterile water. At 10 days post infection (growth stage 3.2-3.5, Boyes et al, 2001) half the plants from each group were subjected to dehydration stress. Plants were carefully lifted off the agar and desiccated for 15 minutes in plastic Petri dishes under a clean stream of air. During this time the plants lost 10-15% of their fresh weight. Plants were returned to the agar in the growth cabinet for a further 30 minutes before harvesting the tissue. Control plants were lifted off the agar and then immediately pla ced back in the growth cabinet for 45 minutes. Root and leaf tissue was harvested separately from each of the four treatment groups and RNA isolated using an RNeasy plant RNA preparation kit (Qiagen). The experiment will be carried out three times to provide biological replicates.
NASCARRAY490 Cell-specific nitrogen responses in the Arabidopsis root 2009-03-05	Overview: The organs of multicellular species are comprised of cell types that must function together to perform specific tasks. One critical organ function is responding to internal or external change but little is known about how responses are tailored to specific cell types or coordinated among them on a global level. Here we use cellular profiling of five Arabidopsis root cell types in response to a limiting resource, nitrogen, to uncover a vast and predominantly cell-specific response that was largely undetectable using traditional methods. These methods reveal a new class of cell-specific nitrogen responses. As a proof-of-principle, we dissected one cell-specific response circuit that mediates nitrogen-induced changes in root branching from pericycle cells. Thus, cellular response profiling links gene modules to discrete functions in specific cell types. Aim: We used microarrays to detail the global programme of gene expression involved in the plants response to nitrogen in the root and identified distinct classes of up- and down-regulated genes during this process. We further distinguished the nitrogen-responsive genes into (i) five cell types, and (ii) inorganic nitrogen (nitrate) or organic nitrogen (amino acid) responsive genes. Experimental setup: The whole experiment was carried out in at least triplicate. The nitrogen response in 5 FACS-sorted root cell types (LRC, lateral root cap; EpiC, epidermis and cortex; EndoP, endodermis and pericycle; Peri, pericycle; Stele, stele), protoplasts and Col-0 whole roots was assayed using microarrays. 5mM KNO3 was used as a nitrogen treatment for 2 hours. 5mM KCl was used as a control treatment for the same length of time. 1mM MSX in combination with 5mM KNO3 (or with 5mM KCl as a control) was used to block the assimilation of KNO3, and 5mM Gln added to restore it in order to separate KNO3 effects from assimilated-nitrogen effects. 5mM KNO3 was either maintained in the protoplast-generating solution (continuous treat) or not (transitory treat) to test how to best preserve the cellular nitrogen-response during the 1 hr protoplast generating procedure. As controls we used protoplasts, whole roots frozen directly after the 2hr treatment or 3.5hr treatment, or whole roots incubated in protoplast-generating solution (minus enzymes; transitory or continuous treated) and then frozen. After FACS-sorting or mock FACS-sorting cells were frozen. Reference: Gifford ML, Dean A, Gutierrez RA, Coruzzi GM and Birnbaum KD; PNAS 2009
NASCARRAY492 AtTRP1 encodes a novel TPR protein that interacts with the ethylene receptor ERS1 and modulates deve 2011-08-25	The aim is to analyse the transcriptional changes cuased by overexpression of AtTPR1 in plants.
NASCARRAY493 Analysis of circadian gene expression in mature shoots and roots 2009-03-23	Most previous experiments on circadian gene expression in Arabidopsis have used whole seedlings grown on agar plates, usually in the presence of sucrose, and with the whole seedling exposed to the prevailing light conditions. We reasoned that, since the circadian clock controls many aspects of metabolism and metabolism is inherently organ-specific, the behaviour of the clock and its outputs might be organ-specific. Our aim was to test for organ specificity in circadian behaviour. In this experiment we grew Arabidopsis thaliana Col-0 plants hydroponically, with their roots enclosed in light-tight black boxes, for 5 weeks in 12 h light (150 ÂµE/m2/s):12 h dark at 20Â°C. Plants were transferred to constant light and harvested every 3 h for 48 h. Rosette leaves (shoots) and roots were harvested separately; tissue was pooled from 9-13 plants and 6 independent RNA preparations were combined for each sample. The first timepoint (time 0) was taken 12 h after dawn, i.e. at the first subjective dusk. Note that roots are functionally in the dark throughout. The data show that both the central oscillator and some outputs are indeed organ-specific. Some of the data have been published in James et al., The circadian clock in Arabidopsis roots is a simplified slave version of the clock in shoots, Science 322, 1832-1835, 2008, see for example Figs. S4, S5 and S8.
NASCARRAY494 Fenclorim safening of Arabidopsis 2011-05-05	Wildtype arabidopsis thaliana Col-0 root cultures, were treated with fenclorim or 4-chloro-6-methyl-2-phenylpyrimidine dissolved in acetone to achieve a final concentration of 100uM. The final acetone concentration of 0.1% was replicated in control root cultures. Samples were taken at four and twenty-four hours post addition in biological triplicate. Root cultures were routinely maintained at 25oC in the dark.
NASCARRAY497 Transcriptome analysis of cho1 mutant during and after seed germination 2009-04-16	Arabidopsis chotto1 (cho1) mutants show resistance to an ABA analog, (-)-R-ABA, in seed germination. CHO1 gene encodes a double AP2 domain transcription factors (At5g57390), and act in the ABI4-dependent genetic pathway. cho1 mutants are also resistant to high-concentrations of nitrate (>25 mM KNO3) during seed germination and post-germination growth. To reveal the role of CHO1 gene in seed germination and post-germination growth, we carried out gene expression analysis of cho1 and wild type (Col) seeds under 25-mM nitrate condition. Seeds were imbibed on the filter paper moistened with 25 mM KNO3 in 24 or 48 hours. Total RNA was prepared from dry seeds and imbibed seeds and was used for the microarray analysis. We carried out duplicate microarray analysis using independent seed batches.Ref):Nambara et al., Genetics 161: 1247-1255 (2002).Yamagishi et al., Plant Cell Physiol. 50: 330-340 (2009)
NASCARRAY498 Transcriptome analysis during axillary shoot outgrowth 2009-04-20	In plant axillary bud dormancy and outgrowth are regulated by phytohoromones, but it is still unknown about its molecular mechanism. We reveal that Arabidopsis axillary buds located at axil of rosette leaves show dormancy and that this is broken by the decapitation of main stem, resulting in the bud outgrowth. To investigate about the molecular mechanisms of dormancy and outgrowth, we carried out gene expression analysis during axillary shoot outgrowth in Arabidopsis wild type Columbia accession. Since axillary buds did not initiate outgrowth (dormancy) at 5 day after bolting of main stem, we used 5-day bolted plants as a control (before decapitation). Then, main stems were decapitated, and axillary shoots were collected at 24 hours after decapitation (named as growing shoot). Total RNA was prepared from either control or growing shoots and used for the microarray analysis. We carried out duplicate microarray analysis using independent plant materials.Ref):Tatematsu et al., Plant Physiol. 138: 757-766 (2005).
NASCARRAY499 Transcriptome analysis of Col and Cvi accession seeds immediately after imbibition 2009-06-03	Arabidopsis Cvi seeds show deep dormancy compared with Col seeds. To reveal the difference of gene expression profile between Col and Cvi within short time after imbibition, we carried out transcriptome analysis using imbibed Cvi and Col seeds. Seeds were imbibed on filter paper moistened with water for 15 min, 30 min, 1 hour and 3 hours. Dry seeds were used as 0 hour samples. Total RNA was prepared from these seed samples and was used for the microarray analysis. We carried out triplicate microarray analysis using independent three seed batches.
NASCARRAY502 Cell type specific analysis of root epidermis 2012-02-15	The experiment aims to analyse auxin signalling in root epidermis. We used two GFP marker lines (pCOBL9:GFP, pGL2:GFP) and fluorescent activated cell sorting to generate samples of trichoblast and atrichobast cells. Before protoplasting the roots were either mock treated or exposed to IAA (10ÂµM) for 2 hours. Eventually the study will establish the topology of the auxin response system in these cell types and together with data from other sources will enable us to develop a model of auxin signalling in root epidermis. These samples represent a scaled down experiment aimed at testing the efficiency of our experimental protocol from cell sorting to microarray analysis.
NASCARRAY503 Auxin induced in-vitro cambium initiation in Atrabidopsis thaliana 2009-03-17	An in-vitro system has been used to induce Cambium initiation in Arabidopsis thaliana stems via Auxin treatments. A time course experiment has been performed and LCM harvested samples from different tissue types have been hybridized. The aim of the project is to isolate and characterize important regulators for cambium initiation.
NASCARRAY504 Gene expression changes in Arabidopsis thaliana treated with Myzus persicae saliva 2009-06-11	Reference:De Vos and Jander, 2009 - Plant, Cell, and Environment Aim:Identification of genes responding to aphid saliva. Background:During feeding on phloem sap aphids repeatedly salivate into the sieve element. It is thought that compounds in aphid saliva play a role in sustainable feeding. These compounds may include proteins and small molecules, which can function as virulence factors. Growth conditions Plants: Seeds of wild-type Arabidopsis thaliana (Col-0) were obtained from the were kept in 0.1% Phytagar (Invitrogen, Carlsbad, CA) for 24 h at 4Â°C prior to planting on Cornell mix with Osmocoat fertilizer. Plants were grown in Conviron growth chambers in 20- x 40-cm nursery flats at a photosynthetic photon flux density of 200 mmol m-2 s-1 and a 16-h photoperiod. The temperature in the chambers was 23Â°C and the relative humidity was 50%. Plants were grown for 3 weeks and used in experiments before flowering. Aphids: All experiments were conducted with a tobacco-adapted red lineage of M. persicae. Aphids were raised on cabbage (Brassica oleracea) with a 16-h day (150 mmol m-2 s-1 at 24Â°C) and an 8-h night (19Â°C) at 50% relative humidity. Experimental set-up/treatment: Fifty aphids were allowed to feed from 50 ÂµL artificial diet, containing sucrose and amino acids (Kim and Jander, 2007) between two layers of Parafilm. After 24 h, artificial diet from 20 aphids and control (0 aphids) diet cups was collected and infiltrated into leaves of intact Arabidopsis plants using a 1-mL syringe without the needle. Plants for control diet and aphid saliva containing diet were grown in the same pot to allow for a paired comparison. Eighteen leaves (3 leaves from 6 plants) treated with control and aphid saliva containing diet were harvested and immediately frozen in liquid nitrogen. This experiment was repeated 3 times to function as independent biological replicates. RNA extraction + processing: RNA was extracted using the Qiagen Plant RNeasy kit. RNA quality and quantity was assessed with an Agilent BioAnalyser 2100. Samples were processed by the Cornell Microarray facility. Whole genome gene expression profiling was done using Affymetrix ATH1 GeneChips. Data analysis: Raw data from the microarrays was normalized at probe-level using gcRMA algorithm. The detection calls (present, marginal, absent) for each probe set was obtained using the GCOS system. Significance of gene expression was determined using the LIMMA (Smyth, 2004) program and raw p values of multiple tests were corrected using False Discovery Rate (FDR).
NASCARRAY505 GA-dependant SPT targets 2009-11-17	Aim: find GA-dependant targets of the transcription factor SPT. We used a Col nul allele of spt, and treated 4 days old red-light grown seedlings with 10uM of GA. Samples were harvested 30 minutes post treatment and 24 hours post-treatment.
NASCARRAY517 Interaction Arabidopsis thaliana vs. Ralstonia solanacearum 2008-11-20	Herein, whole genome expression analysis was conducted on rosettes of resistant and susceptible Arabidopsis thaliana accessions challenged with Ralstonia solanacearum. Two disease situations were considered: Col-5 plants inoculated with the virulent GMI1000 strain, as well as Nd-1 plants challenged with the same bacterial strain deleted of the avr PopP2 gene (DeltaPopP2), in both cases developing wilt disease symptoms. In contrast, Nd-1 plants challenged with GMI1000 are fully resistant to the pathogen and served as a control.Samples were harvested at the time of inoculation, as well as at 6, 12, 24 hours -at these times no symptoms were visible-, 5 days after inoculation when the first wilt symptoms became visible (25% of wilted leaves: disease index 1, D1), and finally 8 days after inoculation when 75 % of leaves were completely wilted (disease index 3, D3).
NASCARRAY529 Calcium and the clock 2011-02-15	We are aiming to analyze calcium effect on the circadian clock. Untrained plants lacking a calcium rhythm and artificially restored rhythm via ectopic calcium pulse. Plants were grown 10days in continuous light and then subjected to calcium/water treatment. Ramping from the subjective dawn: 0.5, 1, 5, 10, 20, 50, 60 mM CaCl2 for an hour. Every point (sampling every 4h) made in duplicate, with a water treated control. WT col used.
NASCARRAY533 Gene expression in arabidopsis lateral root development 2012-05-22	Gene expression in arabidopsis lateral root development
NASCARRAY534 Transcript profiling of PPDK and PCK mutant seedlings 2012-02-15	This experiment aims to assess effects on transcriptional profiles of knocking out genes encoding two enzymes of C4 photosynthesis, PPDK (At4g15530) and PCK (At4g37870), both individually and in combination in Arabidopsis seedlings. This is to be achieved using T-DNA insert lines N616158 and N532133 and a double mutant created by a cross between these two lines. Each will be compared to the wild type Col-0. Tissue has been collected from seedlings which have been grown in the dark at 24oC for two days after a 30 min light pulse. Three biological replicates have been used for both Col-0 and each line.
NASCARRAY536 RSL4 2009-12-17	Aims to look at the targets of the bHLH transcription factor in Arabiodpsis roots.
NASCARRAY538 Temperature stress on nrpd2A and rts1-1 mutants 2011-10-12	The role of NRPD2A and RTS1 on transcriptional regulation induced by heat stress.
NASCARRAY539 Transcriptional profiling in stem segments of Columbia wild type and the wox4 mutant 2011-05-05	Columbia wild type and wox4 mutant stem segments were analysed for transcriptional profiling
NASCARRAY541 SCR-LRP x Col0 comparison 2011-10-12	A digenic mutant in the Col-0 was isolated. The mutant has pleiotropic effects on plant growth and development. It will be compared with it's corresponding wild-type to identify gene expression differences that contribute to the phenotypic differences.
NASCARRAY542 abi4 x vtc2 2011-10-12	We crossed the Arabidopsis low vitamin C mutant, vtc2, which has about 25% of wild-type ascorbic acid, and the abscisic acid (ABA) insensitive mutant, abi4.The aim of this work is investigate the cross talk between the abcisic acid and ascorbic acid pathways. Plants were grown for 6 weeks at 22ÂºC under low (250 Âµmol m-2 s-1) light with a 10-h photoperiod and 70% humidity.
NASCARRAY544 Wt vs gonst1-2 seedling 2011-05-05	Aim of this experiment was to compare aerial tissue from 14d old seedlings of Wt (Col-0) vs gonst1-2 (SALK_043593) knockout plant. This will also be used to compare to a previous microarray experiment using callus with the same genotype.
NASCARRAY545 pCS19 2011-10-12	Identification of genes regulated by ATHB4. Seedlings grown for 7 days under continuous white light of the pCS19.5 line were treated for 4 h with or without a combination of DEX and/or CHX. Reference: Sorin et al. (2009). Plant J 59, 266, 277.
NASCARRAY546 The effect of gravitropism on the meristem and accelerating elongation zone of Col-0 and arf7arf19 r 2011-10-12	Gravitropism plays a role in the development of the root system. Upon gravistimulation, a complex response initiated in the root cap eventually triggers the gravitropic root curvature. Previous studies have demonstrated that gravity-induced lateral auxin transport can promote a differential growth response on opposite flanks of the elongation zone; cell elongation rate is reduced at the bottom side of the distal elongation zone, thus promoting the root to bend downwards. Auxin-response factors (ARFs), ARF7 and ARF19 are the major transcriptional regulators involved in root gravitropism (Okushima et al, 2005).The global transcriptomic analysis approach will allow us to construct and infer gene networks for the action of gravity. This will link the initial gravity signalling to downstream genes regulated by the ARF7/ARF19 pair.Two zones of Arabidopsis root will be considered in this experiment: zone 1 or meristem (from the root tip to the top of the lateral root cap-approx. 350 um) and zone 2 or accelerating elongation zone (from the top of the lateral root cap to the first visible root hair bulge-approx. 500 um). In order for statistical networks to be built, a time series of data is needed. This study has used 0, 15, 30, 60, 120, 240 and 480 minutes of gravity stimulation by 90Â° reorientation.
NASCARRAY547 Microarray Analyses of Arabidopsis cgl1 (complex glycan 1) Mutants and Genetic Interaction with stt3 2011-05-05	We compare two Arabidopsis thaliana cgl1 alleles and report on genetic interaction with stt3a in double mutants. STT3a encodes a subunit of oligo-saccharyl-trans-ferase that affects efficiency of N-glycan transfer to nascent secretory proteins in the ER; cgl1 mutants lack N-acetyl-gluco-saminyl-trans-ferase-I activity and are unable to form complex N-glycans in the Golgi apparatus. In cgl1 C6, a point mutation affects the 3'-splice site of intron 14, resulting in frame shifts; and in cgl1-T, intron 11 fails to splice due to insertion of a T-DNA copy. Introgression of stt3a-2 did not restore complex glycan formation in cgl1 C6 or cgl1-T (Frank et.al.,2008). In this microarray experiment, Seeds were grown on 1/4 MS media supplemented with 0.5% sucrose and 0.8% agar in a growth chamber under long-day photoperiod (16-hr light, 8-hr darkness) at 24 Ã‚Â± 2Ã‚Â°C for 10 days. Then, seedlings were transferred into soil and grown three more weeks in a growth chamber at 25 Ã‚Â± 2Ã‚ÂºC under long-day photoperiod at a light intensity of 40 ÃŽÂ¼mol m-2s-1. Then, shoots from 5 plants were pooled and total RNAs were extracted according to Chomczynski (1993).
NASCARRAY549 Comparison of wt and biotin-deficient mutant 2011-02-15	In this experiment we have compared gene expression in wild-type Arabidopsis COL-ecotype and a T-DNA insertion mutant bio4-1 that has an altered biotin synthesis. The bio4-1 plants were either grown normally or watered with 200 ÂµM biotin solution.
NASCARRAY550 N9299 vs C24 2011-05-05	Six week old hypocotyls from the N9299 line were compared wild-type C24 controls.
NASCARRAY552 The effects of infection with wild-type or mutant cucumber mosaic virus on the Arabidopsis transcrip 2012-05-09	The cucumber mosaic virus (CMV) 2b counter-defense protein disrupts plant antiviral mechanisms mediated by RNA silencing and salicylic acid (SA). Using NASC ATH-1 microarrays to investigate defensive gene expression in 2b-transgenic Arabidopsis thaliana plants we found that, surprisingly, 2b inhibited expression of few SA-regulated genes and in some instances enhanced the effect of SA on certain genes. Strikingly, the 2b protein inhibited changes in the expression of 90% of genes regulated by jasmonic acid (NASCARRAYS-415: Lewsey et al. Molec. Plant-Microbe Interact. vol. 23, in press). We will extend this work to understand the effects of the 2b protein on plant gene expression during an authentic viral infection. By comparing the effects on the transcriptome of infection by wild-type CMV and the 2b gene deletion mutant, CMVÃ¢Ë†â€ 2b, we will reveal not only the influence of the 2b protein but also the effects of its interactions with other viral gene products (and the process of infection itself), on the host transcriptome.
NASCARRAY553 Changes in genome of selected lines in comparison to wildtype 2012-05-09	The aim of this microarray analysis is to analyse whole-genome changes in plants altered in the expression and/or activity of CESTA (CES), a transcription factor involved in the regulation of brassinosteroid responses.The lines analysed are the dominant mutant ces-D, and plants constitutively expressing a dominant negative version of CES (CES-SRDX) as compared to those of wild-type. The tissues used were ten-day-old whole seedlings grown in long-day conditions at 22Â°C.
NASCARRAY555 Identification of genes regulated by PAR1. 2012-05-25	Seedlings grown for 7 days under continuous white light of the pIR33.316 line were treated for 4 h with or without a combination of CHX and/or DEX.Reference, Roig-Villanova et al. (2007). EMBO J. 26,4756-4767
NASCARRAY561 Finlayson: Transcriptomics of Axillary Bud Outgrowth Regulation by Shade Signals in Arabidopsis. 2012-11-19	Aims: To determine the changes in the Arabidopsis axillary bud transcriptome in response to changes in the red light (R) to far red light (FR) ratio (R:FR).Background: The branching habit of plants is a key determinant of overall plant form and function with great relevance to modern agriculture. Shade signals transduced by phytochromes are major regulators of axillary bud outgrowth, and in turn control branching in both natural and agricultural environments. To continue our investigations into the regulation of branching by R:FR, we have developed a system using supplemental FR LEDs to tightly control the outgrowth of Arabidopsis axillary buds. Depending on the position of the bud in the rosette, outgrowth is either repressed (uppermost bud) or rapidly promoted (bud in the axil of the third leaf down) by the transition from low to high R:FR.Treatment: Two separate experiments were conducted to evaluate the effects of R:FR on transcriptome changes in the uppermost rosette bud (bud n) and the axillary bud in the axil of the third leaf from the top (bud n-2). WT Col-60000 was used as the experimental material. Plants were grown individually in 25 by 50 mm tubes and watered and fertilized optimally. Plants were grown in a split growth chamber (providing uniform temperature and PPFD but allowing for differential R:FR) with 18 h photoperiods (185 ÂMoles m-2 s-1 PPFD provided by T12 VHO CW fluorescent lamps) and 24oC/18oC day/night temperatures. One day after sowing the R:FR was reduced on both sides of the chamber from 3.5 to 0.08 using FR LEDs fixed in clear overhead arrays. Prior to anthesis the plants were matched and split into two treatment groups. In experiment 1 the FR source for one of the groups was switched off at 12:00 pm on the day of anthesis, causing the R:FR to increase to 3.5. Unelongated axillary buds in the axil of the uppermost leaves (approx. 2.5 mm long) were harvested for RNA preparation from both groups (low and transiently increased R:FR) 3 h after changing the R:FR. Each treatment was composed of three biological replicates, each containing buds from about 15-18 plants. Experiment 2 was conducted exactly the same as experiment 1, except the R:FR was altered 3 days after anthesis and the unelongated axillary buds in the axils of the third leaves down (approx. 1 mm long) were harvested for RNA preparation.
NASCARRAY562 Long recovery after heat shock 2012-05-09	Abiotic stress is a major factor for crop productivity, a problem likely to be exacerbated by climate change. Improving the tolerance to environmental stress is one of the most important goals of crop breeding programmes. While the early responses to abiotic stress in plants are well studied, plant adaptation to enduring or recurring stress conditions has received little attention. This project investigates the molecular mechanism of the maintenance of acquired thermotolerance as a model case of stress memory in Arabidopsis. Arabidopsis seedlings acquire thermotolerance through a heat treatment at sublethal temperatures. To investigate the underlying mechanisms, we are investigating changes in the transcriptome at two timepoints after a heat acclimation treatment using Arabidopsis thaliana seedlings.
NASCARRAY563 Transcriptomes of arabidopsis ttg1 mutant and overexpressor 2012-05-23	Transcriptomes of arabidopsis ttg1 mutant and overexpressor.
NASCARRAY565 The effect on cell wall remodelling enzymes gene expression when the gibberellin response is blocked 2012-05-09	Gibberellins (GAs) are plant hormones that act as essential endogenous signals regulating growth and developmental processes during the plant life cycle. At the cellular level, GAs have been shown to encourage both cell division and cell elongation. Both metabolic GA mutants, such as AtGA20ox1 (with reduced GA levels), and signalling GA mutants, such as gai (with reduced GA response) reveal a dwarf phenotype and short root. Ubeda-TomÃƒÂ¡s et al. (2008) showed that GA promotes root elongation in Arabidopsis thaliana by regulating cell elongation in the endodermis. The regulation of endodermal cell elongation seems to rely on the control of GA over the cell wall extensibility. In order to analyse the molecular mechanisms through which GAs control cell wall properties to regulate cell growth and therefore, root elongation in Arabidopsis thaliana, we are using a global transcriptomic analysis approach.The SCR::gai-GR system, an inducible system that blocks the GA response in the endodermis is being used in this experiment to analyse what the changes in cell wall remodelling enzymes gene expression are when the GA response is blocked in the endodermis of the Arabidopsis thaliana root. This experiment focuses on the meristem and elongation zone of Arabidopsis roots. A time series of data of induced GA blocked response after treating the roots for 0, 15, 30, 60, 120, and 240 minutes with 10uM of Dexamethasone has been used in this study.
NASCARRAY566 Comparison of silenced type II NAD(P)H dehydrogenase genes 2012-10-23	Type II NAD(P)H dehydrogenases bypass the energy-conserving steps in the plant mitochondrial electron transport chain. In this investigation we suppressed NDA1 and NDA2 with one RNAi construct, and separately, NDB1 with another construct. Both NDA1,2- and NDB1-suppressing plants displayed lowered level of transcript for the respective target genes. Also, the plants of both lines showed a small but significant decrease in biomass accumulation, as compared to the wild type. To investigate the putative transcriptomic effects of the gene silencing we grew plant in 10 h light period and harvested rosettes briefly before flowering for transcript analyses.
NASCARRAY567 AtMC9 knock-out root 2012-05-09	Analysis of two AtMC9 (At5g04200) knock-out lines SALK_075814 and GABI_540_H06. RNA was extracted from 3 day old roots to be analysed for transcritional changes in the mutant lines.
NASCARRAY569 Role of TGA factors in the salicylic acid/etylene crosstalk 2012-05-23	The aim of this study is to check which ethylene-induced genes are suppressed by salicylic acid. In addition we want to elucidate the role of TGA factors in the ethylene signaling network and how they connect this pathway with the salicylic acid pathway. To adress this question we treated Col-0 plants and tga256 mutant plants with water, ACC, SA and SA/ACC for 12h.Zander et al., 2010, Plant J.
NASCARRAY570 bob1-3 vs WT with and without a heat shock 2012-10-23	BOB1 is a non-canonical Arabidopsis small heat shock protein. This experiment contains four samples: WT (Col-O) and bob1-3 4 day old seedlings grown at 22 degrees Celsius with or without a 75 minute 37 degree Celsius heat shock.
NASCARRAY571 L.C EXP1/K.L EXP2 2012-05-23	Experiment 1Investigator: L. CivaleThe aim is to characterise and compare the gene expression profiles of Arabidopsis roots of mature plants treated to hypoxic stress (Sparged with 3% O2 Gas) grown in hydroponic culture, in wild-type (Col-0) and nia1 T-DNA insertion mutant line plants(SALK_148487C). NIA1 is the minor isoform of nitrate reductase in Arabidopsis.Experiment 2Investigator: K. LewandowskiThe aim is to characterise and compare the gene expression profiles of leaf and meristem tissue taken from wild-type (Col-0) Arabidopsis plants and a transgenic line overexpressing a potential novel miRNA, just before the transition to reproductive growth.
NASCARRAY572 ATHB1 mutant response to methyl jasmonate 2012-05-24	ATHB1 is a member of the HD-ZIP I family of transcription factors, peculiar to plants. To understand the functional role of ATHB-1 in Arabidopsis, we identified athb1-1 and athb1-2 loss-of-function insertional mutants. Both alleles do not show any phenotypic alteration respect to overall size and morphology under our growth conditions, likely due to a functional redundancy within the HD-ZIP I family. However, gene expression analysis suggests that ATHB1 could take part in plant responses to environmental stresses. In fact, ATHB1 expression is affected by flooding and wounding, and by ethylene and methyl jasmonate (MeJA). Moreover, although wild type and mutant plants were clearly impaired by MeJA in a dose dependent manner, both athb1 alleles were less affected and showed a reduced rate of seedling mortality.In order to investigate the role of ATHB-1 in the MeJA response at genomic level, a transcriptome analysis will be performed on wild type (Ler) and athb1-2 plants treated with 30 Î¼M MeJA. To this end, RNA samples have been prepared from 1 week-old plants treated with MeJA or ethanol (mock) for 1 hour and 4 hour.
NASCARRAY576 Hydrogen peroxide and light treatment 2012-05-18	Plant7days arabidopsis seedlingscontrol was grown in the darkHP was grown in the dark and treated with H2O2light was grown in the light.each sample has 2 biological replicates.
NASCARRAY577 Comparative gene expression analysis between Arabidopsis wt and wrky33 mutant plants upon Botrytis c 2011-12-13	Wrky33 mutant plants are highly susceptible to the fungus Botrytis cinerea (Bc) while wt is not. To identify differentially expressed genes 5 weeks old wt and wrky33 (GABI_324B11) plants were mock treated and spray infected with Bc (isolate 2100 Spanish Type Culture Collection) at 5x105 spores/ml, and kept for 14 hours at high humidity and constant light. To resuspend the spores and for mock treatment Vogel buffer was used (in 1l: 15g sucrose, 3g Na-citrate, 5g K2HPO4, 0.2g MgSO4 7H2O, 0.1g CaCl2 2H2O, 2 g NH4NO3). 100 mg of leaf tissue was collected and total RNA isolated using TRI reagent following the suppliers instructions (Ambion). Leaves from three plants were pooled to make one biological replicate, and samples from three independent biological replicates for each genotype and treatment were submitted to hybridisation against a total number of 12 Arabidopsis ATH1 GeneChip arrays (Affymetrix).
NASCARRAY578 K.Oracz 2012-05-21	Aim: The Xspecies-type cross-species hybridization with RNA samples extracted from cress seedsBackground and species/tissues: Lepidium sativum is a close relative of Arabidopsis. RNA is extracted from radicles and micropylar endosperm isolated from imbibed control and proteasome inhibitor (MG132) treated cress seeds.Literature: Linkies et al. PLANT CELL 21: 3803-3822 (2009)Other information: These arrays are part of the Alexander von Humboldt grant of Dr Krystyna Oracz and are sent to you by her from the lab of Gerhard Leubner (University of Freiburg, Germany, www.seedbiology.de). Neil Graham and/or Martin Broadley agreed to do the XSpecies post-hybridization masking as a collaborator of us. The given project code is for K.Oracz.krystyna.oracz@biologie.uni-freiburg.degerhard.leubner@biologie.uni-freiburg.de
NASCARRAY579 auxin response in thaliana and lyrata 2012-10-23	a. thaliana and a. lyrata seedlings have been induced with iaa for 0h, 1h and 3h
NASCARRAY580 Exogenous glucose treatment for Arabidopsis thaliana seedlings (Col-0) 2012-05-21	Glucose regulation of auxin (IAA) synthesis has recently been suggested in Maize (LeClere et al. 2010). The experiment in hand involves long glucose treatment of wild-type Arabidopsis thaliana seedlings. The purpose of the gene chip analysis is to pinpoint carbohydrate-regulated genes which are involved in IAA homeostasis and to monitor the metabolic status of the seedlings during the incubation with/without glucose.Wild type Arabidopsis thaliana (Col-0) seedlings were grown on 1% agarose plates (pH 5.7) under long day light period for 10 days. On day 10, one hour after the onset of light, the seedlings were transferred into liquid MS (1x, pH 5.7). After four hours of incubation in the dark, a treatment meant to drain the soluable carbohydrate pools of the plants, the material was transferred into liquid media (1xMS, ph 5.7) with varying amounts of glucose (0%, 1%, 5%). Pooled samples of approximately 30-50 whole seedlings were harvested at the point of glucose application (t0) and after 15 hours of incubation (t15) with/without exogenous glucose.LeClere et al. Plant Physiology (2010) 153, 306-318.
NASCARRAY581 Glabal expressional analysis of cold related mutants 2012-05-21	the aim of this study to look for the level of the expression of cor genes and other related genes in these mutantsLer-1 is the genetic background for all muatants used in this studywhole plants were used to extract total RNA using Qiagen mini Plant RNA easy kit
NASCARRAY582 Xylella fastidiosa infection of Arabidopsis thaliana Tsu-1 2011-02-14	The experiment compares Xylella fastidiosa (GFP labeled Temecula) inoculated Arabidopsis with mock inoculated tissue. Bacterial were mechanically inoculated into midrib of leaves. At 9dpi, midribs above the inoculation point were dissected out and frozen. Total RNA was isolated using Trizol, and hybridized to Affy ATH-1 arrays. Three biological replicate pairs of Xf and mock inoculated RNA were run.
NASCARRAY583 Comparison of floral stems of two clv1 KO mutants 2012-05-22	In this experiment, 2 knock out mutants in the CLV1 gene (clv1.12 and clv1.13) and WS-2 were grown under short day conditions (8h/16h) with 70% humidity and a temperature of 22Â°C in the night and 25Â°C during the day, until their floral stem reach 20cm. Then, the 5 first centimeters (from the lowest part) were harvested and flash frozen for further RNA extraction.
NASCARRAY584 Chemical compound induced somatic embryogenesis in Arabidopsis 2012-05-14	Dry seed of wild-type columbia were germinated in containers with liquid 0.5ms including 1ÂµM 2.4-D.After two days the seedlings were induced with 1ÂµM of 2,4-D incombination with 25ÂµM of the chemical compound. As a control we used seedlings incubated with DMSO.After 0.5hr induction the seedlings were harvested.RNA was extracted using the RNA isolation kit of invitek with an on-column DNAse treatment during the RNA isolation.
NASCARRAY585 Compatibility to Downy Mildew (Hyaloperonospora arabidopsidis) infection 2012-05-14	The analysis of physiological and molecular determinants accounting for successful infection by pathogenic oomycetes has become a topic of prime scientific interest during the last years. The hunt is now on for pathogen effectors subverting the host cell, and for the plant compatibility functions manipulated by these effectors. An understanding of the molecular mechanisms underlying successful infection should make it possible to develop new crop protection strategies based on interference with compatibility to prevent disease. We identified several Arabidopsis genes that account for full susceptibility to the Downy Mildew pathogen, Hyaloperonospora arabidopsidis. A common denominator is that mutants, in which these genes were knocked-out, are more resistant (= less susceptible) to infection. Here, we aim at analyzing the metabolic pathways that are responsible for the lowered susceptibility phenotype of three mutant lines, which are deficient for a microtubule-associated protein (MAP65-3), a leucine-rich repeat receptor-like kinase (PSKR1), and a glycosyltransferase.The Wassilewskija (Ws-4) wild-typ and the mutant lines dyc283, eyw110, and egy19 were from the T-DNA insertion collection at INRA, Versailles, France. Seeds were sown to high density on a soil/sand mixture in 6 cm x 6 cm pots, stratified for 3 days at 4 Ã‚Â°C, and then grown under a 16 h photoperiod in a growth chamber at 20 Ã‚Â°C. For control treatment and infection, 8-day-old plants were spray-inoculated to saturation with either water, or a H. arabidopsidis isolate Emwa1 spore suspension at 40,000 spores/ml, respectively. Plants were kept in a growth cabinet at 16Ã‚Â°C for 3 d with a 16 h photoperiod, before the aerial parts including hypocotyls, fully expanded cotyledons, and leaf primordia were pooled from ~500 plantlets for RNA extraction.
NASCARRAY587 DELLA-mediated regulation of seedling trascriptome 2010-12-15	Seedling growth in short days shows a rhythmic pattern occurring mostly at the end of the night. The increase in growth rate is related with changes in the expression of PIF4, PIF5 and several phytohormone genes as well as with an increment in auxin signaling in the seedling (Nozue, Covington et al. 2006; Covington and Harmer 2007; Michael, Breton et al. 2008). Gibberellins (GA) are main promoters of hypocotyl elongation, and the way in which they contribute to rhythmic growth is still unknown.We found that in short day grown seedlings, DELLA proteins accumulate during the day and show a minimum at the middle of the night, shortly before the period when hypocotyl growth increases. The circadian clock regulates the transcript level of two GA receptor genes; GID1A and GID1B. Changes in DELLA accumulation are associated with GID1s expression, indicating that DELLA proteins oscillation in the seedlings is regulated, at least in part, by the clock-mediated control of GID1s. The decrease in DELLA proteins at the end of the night is relevant for proper hypocotyl growth. In order to study the way in which DELLA oscillations contribute to rhythmic patterns of growth we performed transcriptome analysis of wild type and quintuple della mutant seedlings grown under SD. Samples were taken in two periods of the night: shortly after the dark period starts (ZT9), characterized by a strong DELLA accumulation and low growth rate, and near the end of the night (ZT21) where the opposite pattern is observed. Genes exhibiting a diurnal oscillation in circadian gene expression databases (e.g. Diurnal: http://diurnal.cgrb.oregonstate.edu/) and show a different pattern of regulation in della mutants compared with the wild type early in the night will be chosen for diurnal expression studies and functional analysis. References Covington, M. and S. Harmer (2007). "The Circadian Clock Regulates Auxin Signaling and Responses in Arabidopsis. "PLOS Biology 8: 1773-1784.Michael, T., G. Breton, et al. (2008). "A morning-specific phytohormone gene expression program underlying rhythmic plant growth. "PLOS Biology 6(9): e225. doi:10.1371/journal.pbio.0060225. Nozue, K., M. Covington, et al. (2006). "Rhythmic growth explained by coincidence between internal and external cues. "Nature(448): 358-363.
NASCARRAY588 ERFVII and hypoxia 2012-05-14	The aim of this analysis is to compare plants with increased expression of At1g53910 and decreased expression of At1g53910 and At3g14230 in their response to hypoxia (1% O2). HA-tagged overexpressors of At1g53910 and knock-out lines obtained by overexpression of an artificial microRNA against At1g53910 and At3g14230 were used. Plants were treated for 90 mins under hypoxic or normoxic conditions and then the above-ground tissues were collected and rapidly frozen in liquid nitrogen. RNA was extracted using the RNeasy (Qiagen) extraction kit.Control wt are also used to test differentially regulated genes in plants overexpressing an HA tagged version of At5g21960.
NASCARRAY590 P G62 study 2012-05-16	Analysis of root transcriptome of wild type (Col-0) and mutant (pen1-3) Arabidopsis plants after seed inoculation with the growth promoting rhizobacterium Pseudomonas sp. G62.
NASCARRAY591 Slow growth 2 2012-05-17	The purpose for this experiment is to find the gene expression differences between wildtype and our mutants
NASCARRAY592 Identification of putative directly-regulated STM target genes 2012-05-16	The KNOX gene SHOOT MERISTEMLESS (STM) encodes a homeodomain trascriptional regulator that is critical for shoot apical meristem (SAM)function in Arabidopsis. To identify target genes, STM function was induced using an STM-GR fusion protein activated by dexamethasone for 3 hours, after which total RNA was isolated and analysed using Affymetrix ATH1 arrays. This was compared to a mock-induced sample. Additionally, STM function was induced with dex in the presence of cycloheximide to identify genes that show differetial regulation in the absence of protein synthesis, which may therefore be directly-regulated STM target genes.
NASCARRAY593 Sugar responses in Col and pft1-2 2012-05-17	aims: compare gene expression in response to sugar treatment between Col and the pft1-2 mutant. mutant: pft1-2 (Salk_129555), tissues: seedlings, treatment: 3% Glucose for 6 hours, reference for the protocol: ISSN 1088-9051/06, experimental set up: seedlings were grown in vitro for 7 days on MS medium complemented with 0.5% Glucose. After 7 days, the seedlings were transfered in liquid culture in MS without glucose. After 24 hours, the medium was replaced by MS complemented with 3% Glucose for 6 hours.
NASCARRAY594 Understanding the affect of maternal environment on seed dormancy 2011-05-05	Maternal environment is an important regulator of seed dormancy, but the mechanisms underlying the process are poorly understood. We have found that genes in the circadian clock control dormancy, in part through their regulation of the canonical photoperiod pathway known from research into flowering time control. In this experiment we compare the effects of altering seed maturation temperature or maternal photoperiod on dry seed transcriptomes, and the photoperiod-insenstive ft-1 mutant to wt type Ler. In this way we are identifying gene expression programmes which result from the seed's response to maternal environmental experience.
NASCARRAY595 Identify direct targets of PGY genes in Arabidopsis 2012-05-17	Animal/human work may default to this text IF REQUIRED
NASCARRAY596 Effect of compound 3 on somatic embryogenesis 2012-06-15	The goal of this microarray experiment is to find the targets of compound 3 in Arabidopsis.on day 2,wildtype columbia seedlings were induced for 8 hours with compound 3.After 8 hours total RNA was isolated from whole seedlings including seedcoat.
NASCARRAY598 whole genome analysis 2012-10-23	The aim of this microarray analysis is to analyse whole-genome changes in plants in which the expression of a transcription factor involved in the regulation of brassinosteroid homeostasis is induced.The lines analysed expressed a TF under inducible promoter as compared to those of wild-type. The tissues used were ten-day-old whole seedlings grown in the light at 22Ã‚Â°C.
NASCARRAY599 Adaptation to phosphate starvation in roots 2012-05-23	Gene expression analysis in response to phosphate starvation in wt (Col-0) roots compared to roots of a phosphate deficiency response mutant (pdr3).
NASCARRAY602 Miguel Angel 2012-06-13	Arabidpsis thaliana subjected to a boron toxicity teratment.
NASCARRAY603 Compound induction 2012-05-23	wildtype Arabidopsis thaliana (ecotype:columbia) seedlings were induced with compound 5 and B at day 2 to form Somatic embryos. 2.4-D was present from the beginning. After 8 hour induction total seedlings were harvest and total RNA was isolated. The aim of this experiment is to see which genes are upregulated compared to a previous experiment were seedlings were induced with DMSO (control) and C3.
NASCARRAY605 Direct downstream targets of the transcription factor JAGGED in floral tissue 2012-06-11	The aim of the experiment was to identify direct downstream targets of the transcription factor JAGGED that has been reported to be a key regulator in floral organ outgrowth (Dinneny et al. 2004, Ohno et. al. 2004) downstream of floral organ identity genes such as AGAMOUS (Gomez et al. 2005). For the experiment, plant line 22.11.1.27 that is homozygous for the DEX inducible 35S::JAG:GR construct in Ler background and jag-1 mutant in Ler background were used. Plants were grown on soil under long day conditions with 16 h light, with 20Ã‚Â° C day temperature and 18Ã‚Â° C night temperature. At flowering, siliques and open flowers were removed from the main inflorescence. Plants were recovered from dissection for 18 h. For induction, main inflorescence was dipped into solutions containing 0.015 % SILWET supplemented with 0.1% EtOH for control treatment of 22.11.3.27 (treatment label E) and jag-1 mutant (treatment label JEG), 10Ã‚ÂµM Dexamethasone (treatment label DEX), 10 Ã‚ÂµM Dexamethasone and 10 Ã‚ÂµM Cycloheximide (treatment label D+C), and Cycloheximide (treatment label C). Plants were incubated for 5 hours for induction. Experiment was performed in 3 replications.
NASCARRAY606 Analysis of gene expression in developing siliques of a mutant in bZIP67 2012-06-12	The aim of this experiment is to analyse gene expression in developing seeds of a mutant in bZIP67 (At3g44460). Homozygous T-DNA insertion line SALK_085497 (N666597) and wild type plants were grown on soil to maturity in a growth chamber set to: 16 h light (21oC, 150 Ã‚Âµmol m-2 s-1) / 8 h dark (18oC). Total RNA was prepared from whole siliques containing developing seeds at stage 8 to 9.
NASCARRAY607 The role of ribosomes in plant development 2012-10-23	Ribosome pull downs are employed to study ribosome-associated transcripts in wild type and a ribosomal mutant (pgy1)
NASCARRAY608 Microarray analysis wild type versus mutant 2012-06-20	Phospholipid biosynthesis is essential for the construction of most eukaryotic cell membranes, but how this process isregulated in plants remains poorly understood. Here, we show that in Arabidopsis thaliana, two Mg2+-dependentphosphatidic acid phosphohydrolases called PAH1 and PAH2 act redundantly to repress phospholipid biosynthesis atthe endoplasmic reticulum (ER). Leaves from pah1 pah2 double mutants contain ;1.8-fold more phospholipid than the wildtype and exhibit gross changes in ER morphology, which are consistent with massive membrane overexpansion. The netrate of incorporation of [methyl-14C]choline into phosphatidylcholine (PC) is ;1.8-fold greater in the double mutant, and thetranscript abundance of several key genes that encode enzymes involved in phospholipid synthesis is increased. Inparticular, we show that PHOSPHORYLETHANOLAMINE N-METHYLTRANSFERASE1 (PEAMT1) is upregulated at the level oftranscription in pah1 pah2 leaves. PEAMT catalyzes the first committed step of choline synthesis in Arabidopsis and definesa variant pathway for PC synthesis not found in yeasts or mammals. Our data suggest that PAH1/2 play a regulatory role inphospholipid synthesis that is analogous to that described in Saccharomyces cerevisiae. However, the target enzymesdiffer, and key components of the signal transduction pathway do not appear to be conserved.
NASCARRAY610 Nomad mutant roots 2012-06-15	The aim is to define changes in global gene expression in roots of the nomad mutant.
NASCARRAY611 Identification of Genes Differentially Expressed in fry 2-1 Mutants 2011-08-18	Low temperature, drought, and high salinity induce the expression of many plant genes. Recessive mutations in FIERY2 (FRY2) result in super-induction of the DRE/CRT class of stress-responsive genes, suggesting that FRY2 negatively regulates stress and ABA activation of these genes. The aim of this experiment is to identify genes differentially expressed in fry 2-1 mutant and their comparison with C24 wild type Arabidopsis plants. For this purpose, both C24 and fry2-1 mutant seeds were surface sterilized by 70% ethanol and washed with distilled water for several times. Seeds were grown on 1/4 strength Murashigee and Skoog basal media containing 0.5 % sucrose, solidified with 0.8 % plant cell tissue grade agar. Approximately 200 seeds were sown on each petri dish and stratified for 4 days at 4Â°C. Then, they were transferred to a growth chamber providing 16 h of light and 8 h of darkness at 25Â°C.After 10 days of growth at these conditions, total RNA was extracted from 100mg of seedlings by using TRIzol (Invitrogen, Cat No. 15596-018) method according to the manufacturer's procedure. Then, total RNA samples were treated with DNase I (Qiagen, Cat. No. 79254) at 37Â°C for 1 hours and cleaned using Qiagen minicolumns.
NASCARRAY612 N6-adenosine methylation deficient plant 2011-08-18	The embryo leathal adenosine methylase (AT4G10760) tDNA knockout line SALK_074069 was partially complemented with its cDNA driven by the embryo specific ABI3 promoter. The partially complemented plants have reduced adenosine methylation and show pleiotropic phenotypes.Rosette leaves were harvested from 3-month-old plants maintained in growth camber under a 20 h photoperiod (23 degree C).
NASCARRAY613 elo3/WT 2011-08-23	We want to study the influnce of an elo3 deletion on the transcriptome of Arabidopsis thaliana. The samples are whole plant samples, RNA was extracted using the Trizol-Methode followed by RNA cleanup using RNeasy columes.
NASCARRAY614 Transcriptome analysis of DELAY OF GERMINATION1 (DOG1) mutant during seed maturation 2011-08-25	Seed dormancy controls the start of a plant's life cycle by preventing germination of a viable seed in an unfavorable season. The molecular mechanisms controlling seed dormancy are not well understood. DELAY OF GERMINATION 1 (DOG1) has been identified as a major Quantitative Trait Locus (QTL) for seed dormancy in a Recombinant Inbred Line (RIL) population between the lowly dormant accession Landsberg erecta (Ler) and the high dormant accession Cape Verde Islands (Cvi) (Alonso Blanco et al., 2003). DOG1 is a key regulator of seed dormancy because dog1 mutants are completely non-dormant and do not show any obvious pleiotropic phenotypes, apart from a reduced seed longevity (Bentsink et al., 2006). We compared the transcriptome of dog1-1 mutant with that of its wild-type background NIL DOG1_Cvi on siliques at mid-maturation phase when DOG1 is highly expressed. This analysis shows the influence of DOG1 on gene expression related to dormancy induction during seed maturation.
NASCARRAY615 Expression changes during polyploidisation 2011-08-25	Polyploidisation is frequent in higher plants and contributed significantly to their evolution and adaptation. The sucess of polyploid plants seems to be due to diversification that is least in part generated by major changes in genome organisation and epigenetic regulation. The aim of this experiment is to analyse changes in global gene-expression occurring after autopolyploidisation of an Arabidopsis thaliana line carrying a HPT transgene.Mittelsten Scheid O. et al., PNAS, 1996Mittelsten Scheid O. et al., Nature Genetics, 2003
NASCARRAY616 Expression changes during polyploidisation (2) 2011-08-25	Polyploidisation is frequent in higher plants and contributed significantly to their evolution and adaptation. The sucess of polyploid plants seems to be due to diversification that is least in part generated by major changes in genome organisation and epigenetic regulation. The aim of this experiment is to analyse changes in global gene-expression occurring after autopolyploidisation of an Arabidopsis thaliana line carrying a HPT transgene. Mittelsten Scheid O. et al., PNAS, 1996 Mittelsten Scheid O. et al., Nature Genetics, 2003
NASCARRAY617 Gene expression in root tips of curly leaf mutants 2011-08-30	Our group would like to compare gene expression between wildtype and curly leaf (clf) mutant roots. Wildtype and mutant plants were grown vertically in MS agar and the root tips harvested after 5 days. RNA was extracted using RNEasy Plant mini kit.
NASCARRAY618 Identification of brg11 targets 2011-08-30	The aim is to identify genes that are transcriptionally upregulated by Brg11 and related proteins. We use transgenic plants in which Brg11 and related proteins are induced by Estradiol. Thus we will have to compare a) Columbia without Estradiol application b) Columbia with Estradiol and c) Brg11 and related proteins under the control of the estradiol-inducible promoter.
NASCARRAY619 Heat stress-30h 2011-08-30	We applied 30 hours long heat stress to Arabidopsis Col plants with an aim to study the effects of heat stress on chromatin, epigenome stability and maintenance of transcriptional gene silencing.
NASCARRAY620 MSKJ521 microarray analysis 2011 2012-10-23	Analysis of the kluh (cyp78a5) mutant and two enhancers mutants of kluh: cyp78a5 cyp78a7 and cyp78a5 tik1 compared to the respective wild-type.
NASCARRAY621 Transcriptome comparison of a lignin-deficient mutant and the wildtype 2012-10-24	Gb is a novel lignin deficient mutant with reduced lignification in inflorescence stem. The objective of this transcript profiling analysis is to study the potential transcriptome difference between this Gb mutant and the wildtype.
NASCARRAY622 Transcriptomic analysis of inducible RNAi transgenic plants 2012-10-24	The objective of this experiment is to proof the concept of tetrapyrrole-mediated signaling by analysis of the primary response on nuclear transcription by using an inducible transgene expression system. Transgenic plants with chemical induced silencing (RNAi) of genes encoding enzymes of the Mg branch (chlH, chlM, chl27) will allow us to explore time-resolved early variations of nuclear gene expression upon induced changes of activities of these specific steps in the chlorophyll-synthesizing branch of tetrapyrrole biosynthesis.For transcriptome analysis we used green tissue of three weeks old arabidopsis seedlings treated with dexamethasone for 24 h. The seedlings were grown in little pots on soil under the same conditions.
NASCARRAY623 ABCG37 expression profiling 2012-10-29	Root-specific expression profiling of gain- and loss-of-function mutants in response to indole-3-propionic acid (7d-old, 1h 5 Ã‚ÂµM IPA)
NASCARRAY624 Characterization of FBK mutant and OX lines 2012-10-29	Characterization of transcriptional profiles of four F-box gene mutants/overexpressors (AT1G15670, AT1G80440, AT2G44130, AT3G59940; see also Schumann et al., Plant Physiol 155:835). Samples have been prepared from 9d-old seedlings and open flowers (stage 15, 45d-old).
NASCARRAY632 Role of FVE in plant development 2012-10-23	15-d-old seedlings grown in vitro on horizontal plates containing 1/2 MS, 0,5% sucrose, 0,4% phytoagar under LD (16h light / 8h dark) conditions were collected for analysis. Samples include Col-0 wt control, fve, and mutants of potential FVE-interactors. Three biological replicates are included for each genotype.RNA was prepared using the Qiagen Plant RNEasy kit with additional on-column DNaseI treatment.
NASCARRAY634 Effect of jasmonate of gene expression in the mutants jin1 and jut 2011-09-27	The jasmonate signal pathway regulates plant growth, plant growth in response to the environment, and responses to pests and pathogens. Its activation is associated with transcriptional re-profiling of as much as 10% of the genome. One transcription factor that regulates JA responses in JIN1/MYC2. We discovered another potentially important regulator of JA responses which we have named JUT. Here we examine gene expression induced by jasmonte in wild type, jin1-1, jut, and in the double mutant jin1-1/jut.
NASCARRAY635 Analysis MYB26 targets by using Dex inducible line 2012-12-14	MALE STERILITY 35 (MS35/ MYB26) is a R2R3-type MYB transcription factor (Steiner-Large et al., 2003). MYB26 is suggested to be a key regulator in the process of lignified secondary thickening in the anther endothecium which provides mechanical force for anther dehiscence (Dawson et al., 1999). This microarray experiment aims to identify genes controlled by MYB26 and thus establish the precise role MYB26 plays during anther development. Comparisons of gene expression will be carried out between functional complemented ms35 mutant line-MYB26promoter::MYB26gDNA-GRYFP and ms35 mutant at the time points 0h, 2h, 4h and 6h after Dex treatment to identify genes with altered expression due to the functional MYB26 released by the Dex induction. The Dex treated ms35 mutant was chosen to ensure that the changes of gene expression are not due to the Dex effect. Plants were grown in growth room at 22h of daylight (167 umol.m2.sec-1) at 24 C and 2h of night at 24 C. Flowering plants with 3-5 sterile siliques were sprayed with 25uM Dex; closed buds were collected from both lines at the time points 0h, 2h, 4h and 6h. RNA was extracted using RNeasy Mini Kit (Qiagen, USA) and digested with DNase to eliminate genomic DNA contamination.
NASCARRAY636 Gene expression in arabidopsis lateral root development 2011-10-05
NASCARRAY637 Using transcriptome analysis to compare gene expression of prt6 and Col-0 seed during germination an 2011-10-05	Absisic Acid (ABA) signaling for seed germination and seedling establishment is controlled by the N-end rule pathway of targeted proteolysis (Holman et al., 2009). In Arabidopsis thaliana, two components of this pathway, PROTEOLYSIS6 (PRT6) and arginyl-tRNA: protein arginyltransferase (ATE) have been shown to regulate seedling lipid breakdown and ABA sensitivity to germination (Holman et al., 2009).In these experiments, the transcriptome profile of after-ripened prt6 seed during germination on water agarose will be compared with Col-0 seed at the same stage. Also, the transcriptome profile of prt6 seed in response to ABA will be compared to that of WT seed. Seed will be imbibed on water agarose with an ABA concentration that will prevent germination- 1 Ã‚ÂµM for prt6 and 10 Ã‚ÂµM for Col-0. This will aid our understanding of the N-end rule pathway in regulating ABA signaling controlling germination and dormancy.Reference List1. Holman TJ, Jones PD, Russell L, Medhurst A, Ubeda-Thomas S, Talloji P, Marquez J, Schmuths H, Swee-Ang T, Taylor I, Footitt S, Bachmair A, Theodoulou FL, Holdsworth MJ (2009). The N-end rule pathway promotes seed germination and establishment through removal of ABA sensitivity in Arabidopsis. PNAS. 106:4549-4554.
NASCARRAY638 DELLA-mediated regulation of seedling trascriptome 2012-06-15	Seedling growth in short days shows a rhythmic pattern occurring mostly at the end of the night. The increase in growth rate is related with changes in the expression of PIF4, PIF5 and several phytohormone genes as well as with an increment in auxin signaling in the seedling (Nozue, Covington et al. 2006; Covington and Harmer 2007; Michael, Breton et al. 2008). Gibberellins (GA) are main promoters of hypocotyl elongation, and the way in which they contribute to rhythmic growth is still unknown.We found that in short day grown seedlings, DELLA proteins accumulate during the day and show a minimum at the middle of the night, shortly before the period when hypocotyl growth increases. The circadian clock regulates the transcript level of two GA receptor genes; GID1A and GID1B. Changes in DELLA accumulation are associated with GID1s expression, indicating that DELLA proteins oscillation in the seedlings is regulated, at least in part, by the clock-mediated control of GID1s. The decrease in DELLA proteins at the end of the night is relevant for proper hypocotyl growth.In order to study the way in which DELLA oscillations contribute to rhythmic patterns of growth we performed transcriptome analysis of wild type and quintuple della mutant seedlings grown under SD. Samples were taken in two periods of the night: shortly after the dark period starts (ZT9), characterized by a strong DELLA accumulation and low growth rate, and near the end of the night (ZT21) where the opposite pattern is observed. Genes exhibiting a diurnal oscillation in circadian gene expression databases (e.g. Diurnal: http://diurnal.cgrb.oregonstate.edu/) and show a different pattern of regulation in della mutants compared with the wild type early in the night will be chosen for diurnal expression studies and functional analysis.ReferencesCovington, M. and S. Harmer (2007)."The Circadian Clock Regulates Auxin Signaling and Responses in Arabidopsis."PLOS Biology 8: 1773-1784.Michael, T., G. Breton, et al. (2008)."A morning-specific phytohormone gene expression program underlying rhythmic plant growth."PLOS Biology 6(9): e225. doi:10.1371/journal.pbio.0060225.Nozue, K., M. Covington, et al. (2006)."Rhythmic growth explained by coincidence between internal and external cues."Nature(448): 358-363.
NASCARRAY641 Identification of genes regulated by ATHB4 in plants grown under simulated shade. 2014-04-30	Seedlings of the pCS19.5 line, grown for 2 days under continuous white light and 5 additional days under simulated shade (W+FR), were treated for 4 h with a combination of CHX and/or DEX.Reference, Sorin et al. (2009). Plant J 59, 266-277.
NASCARRAY642 Expression changes in CK2mut (a dominant negative mutant of protein kinase CK2) 2011-11-10	Our aim is to identify transcriptional changes occurs in Arabidopsis plants depleted of CK2 kinase activity. CK2 is a pleiotropic enzyme, essential for maintaining life, and with multiple isoforms. These facts have made important to use a dominant-negative mutant approach, by overexpressing a CK2 alfa kinase inactive subunit (CKA3mut), cloned downstream of the GVG inducible promoter (Moreno-Romero et al. 2008). This stably transformed CK2mut plants showed that longtime inductions of the transgene were lethal. However, short-time inductions were not lethal and revealed broad phenotypic changes in CK2 activity depleted plants, some of them related to defects in auxin transport (Moreno-Romero et al. 2008; Marques-Bueno et al. 2011). For the transcriptome analysis, the use of the inducible promoter made important to include in the experimental design control plants transformed with empty vector (only GVG receptor and with similar levels of GVG expression to CK2mut) for discarding side effects of the inducible system. METHOD: Arabidopsis seeds were surface sterilized, cold-treated at 4 C for 48 hours, and then sown on MS plates and grown for five days. Afterwards, plantlets were transferred to MS plates supplemented with 1uM Dex and grown for two days. Total RNA was isolated from frozen plantlets with Trizol (Invitrogen) and later puried using Qiagen RNA easy Plant Mini Kit columns (Qiagen, Hilden, Germany). RNA quality was checked using Experion (Bio-Rad laboratories). Three independent RNA preparations were made from CK2mut pooled samples and two from Empty vector samples (Moreno-Romero et al. 2011). Moreno-Romero et al. 2008. A role for protein kinase CK2 in plant development: evidence obtained using a dominant-negative mutant. The Plant Journal 55, 118-130 [2008].Moreno-Romero et al. 2011. About the role of CK2 in plant signal transduction. Molecular and Cellular Biochemistry 356(1-2):233-40.Marques-Bueno et al. 2011. a dominant negative mutant of protein kinase CK2 exhibits altered auxin responses in Arabidopsis. The Plant Journal 67, 169-180.
NASCARRAY643 Identification of genes regulated by DRA2 under simulated shade. 2014-04-30	Seedlings of dra2-1 and its wild-type (Ws-2, PBL line) grown for 7 days under continuous white light, were treated for 0 and 1 h with simulated shade (White light suplemented with Far Red light, W+FR).Reference. The mutant dra2-1 was identified in the same screening described in Wang et al. (2011). Journal of Experimental Botany, doi:10.1093/jxb/err062. Still unpublished.
NASCARRAY647 Identification of genes that are directly regulated by NMD in Arabidopsis 2012-12-07	To understand the way NMD mediates responses it is important to distinguish between direct and indirect targets. We will accomplish this using a global analysis of mRNA stability in wild type and NMD mutant backgrounds. Direct NMD targets show enhanced stability exclusively in NMD mutants. We will use cordycepin to interrupt transcription in wild type and NMD mutant three week old seedlings and analyse samples at time intervals following treatment. We will take timepoints at 0hrs, 1hr, 3hrs and 6hrs. We will analyse global mRNA stability in seedlings of four of our NMD mutant lines (upf1-5, UPF2 (6-11E5), upf3-1 and smg7-1) and wild type. This experiment will generate a list of mRNAs that are directly destabilised by NMD, which can be compared and analysed for features that make them targets of NMD. Three crucial questions will be addressed by this experiment. Firstly, the mRNA decay data will allow us to identify the genes that are directly targeted by each subset of the NMD pathway. For example, targets that are independent of UPF3 will appear to have altered stability only in the upf1 and smg7 mutant backgrounds. Secondly, it will allow us to test the association of specific NMD trigger signals with subsets of NMD. Finally, the list of direct targets will facilitate identification and dissection of the downstream regulated processes.
NASCARRAY648 Expression analysis on low phosphate conditions 2012-12-07	Low phosphate conditions induce massive changes in the root system architecture (RSA). Our aim was to find differences in the expression pattern of Pi starvation related genes under high and low phosphate conditions in the wild type versus starvation hypersensitive and insensitive (double-) mutants pdr2 and lpr1lpr2, respectively. For this, we used RNA from root tissue of 5 days old seedlings which were grown for 4 days on high phosphate medium and subsequently transferred to low phosphate medium for 20h.
NASCARRAY654 Expression analysis of nuclear pore mutants in Arabidopsis 2013-06-06	The Nuclear Pore Complex (NPC) is an essential conduit for movement of protein and RNA between nucleus and cytoplasm. The NPC is a large macromolecular complex that is comprised of over 20 nucleoporins that are broadly conserved throughout eukaryote phyla. We have shown that Arabidopsis mutants with defects in the function of nucleoporin function exhibit pleiotrophic phenotypes including reduced root and hypocotyl elongation, early flowering and reduced stature of adult plants (Parry et al (2006), Plant Cell 18:1590-1603. We have identified mutants in different nucleoporins (nup62, nup54) that exhibit similar pleiotropic phenotypes.We have little knowledge regarding the gene expression changes that occur in these mutants. Therefore we have extracted RNA from 7do seedlings to assess the gene expression changes in nup62 (At2g45000), nup54 (At1g24310) and nup160/sar1 (At1g33410) mutants. We hope to identify suites of genes that are altered in these mutants and go some way to explain the phenotypic changes we observe. These novel insights will drive further experimentation into the critical role of the NPC in plant development.
NASCARRAY656 Effect of the ribosomal protein mutation pgy1-1 on total mRNAs and on ribosome-bound mRNAs in Arabid 2012-05-11	Mutations in ribosomal proteins are known to cause specific developmental defects. For example, six different ribosomal protein mutations have been isolated as modifiers of the leaf patterning gene ASYMMETRIC LEAVES 1 (AS1). These modifiers, called piggyback (pgy), all condition ectopic outgrowths on the upper side of as1 mutant leaves (Pinon et al. 2008, Development 135, 1315).The specific developmental phenotypes of as1 pgy mutants suggested that ribosomes serve as a control point in expression of development genes. It is possible that different subpopulations of ribosomes, containing different ribosomal protein isoforms, are preferentially associated with translation or the translational control of subsets of mRNAs with specific roles in development.To test this hypothesis, we compared mRNAs pulled down with immunoprecipitated ribosomes in the pgy1-1 mutant and in the PGY1 control. To tag ribosomes for immunoprecipitation, we used pgy3-1 mutant plants complemented with a functional PGY3-FLAG fusion. As controls for changes in steady-state mRNA levels, we also compared RNA from total homogenates from pgy1-1 pgy3-1 PGY3-FLAG and from pgy3-1 PGY3-FLAG plants. Two independent experiments were performed, each using three biological replicates per treatment, comparing RNA extracted from total homogenates and from immunoprecipitated ribosomes (see detailed methods).
NASCARRAY658 cpl4 microarray analysis 2012-11-30	An Arabidopsis thaliana gene called RNA polymerase II CTD phosphatase-like 4 (CPL4) is orthologous to fungal and metazoan FCP1, and is necessary for normal plant growth and development. RNAi suppression of CPL4 expression results in growth defects that range from transient retardation of seedling development to lethality prior to maturation. The aim of this experiment is to identify genes differentially expressed in CPL4 RNAi plants. For this purpose, seeds were surface sterilized by 70% ethanol and washed with distilled water for several times. Seeds were grown on Ã‚Â¼ strength MS basal media containing 0.5% sucrose, solidified with 0.8% plant cell tissue grade agar. Approximately 200 seeds were sown on each petri dish and stratified for 4 days at 4Ã‚Â°C. Then, they were transferred to a growth chamber providing 16h of light and 8h of darkness at 25Ã‚Â°C. After 10 days of growth at these conditions, total RNA was extracted from 100mg of seedlings by using TRIzol (Invitrogen, Cat. No. 15596-018) method according to the manufacturerÃ¢â‚¬â„¢s procedure. Then, total RNA samples were treated with DNase I (Qiagen, Cat No. 79254) at 37Ã‚Â°C for 1 hours and cleaned using Qiagen minicolumns.
NASCARRAY659 Ethylene signaling enhances salt tolerance in Arabidopsis thaliana 2012-08-13	Ethylene has been regarded as a stress hormone to regulate myriad stress responses. But how ethylene signaling is involved in the salt stress is poorly understood. To dissect the underlying molecular mechanisms, we performed transcriptome profiling of transgenic plants overexpressing EIN3 (EIN3ox), ein3eil1 double mutant and wild type Col-0. EIN3 and EIL1 are two key transcription factors in ethylene signaling pathway. For this analysis, 5-d-old seedlings treated with or without 200 mM NaCl for 6 h were used for RNA preparation. This design enabled us to examine the overlap in expression pattern among these plants, as well as to identify salt-induced and EIN3/EIL1-dependent genes.
NASCARRAY665 Study the affect of fhy3-4 and far1-2 mutation on the global gene expression of Arabidopsis thaliana 2012-07-25	Recently we showed that in Arabidopsis thaliana, FHY3, FAR1 and HY5, directly bind to the promoter of ELF4 and activate its expression during the day (Li et al., 2011). Through this microarray experiment we are interested to identify more target genes which are directly regulated by these transcription factors.
NASCARRAY670 Measurement of pre-existing and newly transcribed mRNA at two ambient temperatures 2012-10-22	In Arabidopsis thaliana a number of genes show altered transcript abundances at different ambient temperatures. As with other biochemical reactions, mRNA transcription and decay rates might be expected to increase passively with increasing temperature but it is not clear to what degree active temperature responses play a role in temperature-regulated genome-wide gene expression changes.Using a non-perturbing base analogue (4-thiouridine) labelling and separation method we have measured the relative abundances of both labelled, newly transcribed transcripts and unlabelled, pre-existing transcripts. Together with unseparated samples and samples taken at the start of the labelling time, these arrays have been used to calculate transcription and decay rates of the Arabidopsis thaliana transcriptome at two ambient temperatures.
NASCARRAY673 Ecotype specific nitrogen responses in the Arabidopsis root Ecotype specific nitrogen responses in 2013-09-06	OVERVIEWRoot branching in response to changes in nitrogen status in the soil is a dramatic example of the plant's remarkable developmental plasticity. To characterise natural variation in root architectural plasticity at the molecular level we compared root nitrogen responses across a panel of seven ecotypes.AIMIn recent work we investigated the genetic architecture of developmental plasticity in the root at the phenotypic, genetic, and transcriptional levels by combining phenoclustering and genome-wide association studies in 96 Arabidopsis thaliana ecotypes with expression profiling in seven ecotypes. This series contains the microarray expression data for seven ecotypes that represent a range of root branching strategies. The microarrays detailed the global programme of gene and identified distinct classes of up- and down-regulated genes in the seven different Arabidopsis ecotypes during this process.EXPERIMENTAL SETUPThe nitrogen response in seven Arabidopsis thaliana ecotype (Col0, Kas2, NFA8, SQ8, TAMM27, Ts5, Var2-1) whole roots was assayed using microarrays. Seedlings were grown hydroponically in basal MS containing no nitrogen except 0.5mM ammonium succinate and 3mM sucrose for 12 days in long day conditions in a Percival. At dawn on day twelve, 5mM KNO3 (nitrogen treatment) or 5mM (control) was added to the hydroponic medium for 2 hours. At the end of the treatment time roots were harvested and flash-frozen in liquid nitrogen for subsequent RNA extraction. Total RNA was extracted using Trizol, then standard Affymetrix protocols were used to label RNA and hybridise to the Affymetrix ATH1 Array which was stained using the 'EukGE-WS2v4_450' protocol and scanned using the Affymetrix GeneChip 3000 Scanner. The whole experiment was carried out in triplicate with 42 chips in total (14 experiments).
NASCARRAY674 Escherichia coli O157:H7 Induces Stronger Plant Immunity than Salmonella enterica Typhimurium SL1344 2012-11-26	There have been increased concerns of leafy vegetable contamination by human pathogens and food safety due to outbreaks associated with consumption of fresh produce. In this study, we assessed defense responses induced by the fully pathogenic bacteria Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium SL1344 in both Arabidopsis thaliana. Unlike SL1344, O157:H7 induces strong plant immunity at both pre-invasion and post-invasion steps of infection. For instance, O157:H7 triggers stomatal closure even under high relative humidity (RH); an environmental condition that generally weakens plant defenses against bacteria in the field and laboratory conditions. SL1344 instead induces a transient stomatal immunity. We also observed that O157:H7 modulates more components of innate immunity, i.e. genes and signaling pathways, as compared to SL1344. These results suggest that plants may recognize and respond to some human pathogens more effectively than others. Furthermore, stomatal immunity can diminish the penetration of human pathogens through the leaf epidermis resulting in low bacterial titers in the plant apoplast, which suggest that additional control measures can be employed to prevent food contamination. The understanding of how plant responses can diminish bacterial contamination is paramount in preventing outbreaks and improving the safety of food's supplies.
NASCARRAY675 Salt adaptation 2012-11-21	Comparison between a control cell line of tobacco BY-2 cells and a salt adapted cell line to 250 mM of NaCl.The cell cultures have been been grown in LS medium and in darkness at 26C.
NASCARRAY684 Crosstalk between the defense hormones salicylic acid (SA) and jasmonic acid (JA). 2013-01-30	Aims: Elucidating genes affected by hormonal crosstalk between SA and JA in Arabidopsis thaliana. Background: 5 week old Col-0 plants grown 8h day / 16h night cycle at 70% RH and 20 degrees Celsius. Tissues: All aboveground parts (whole rosette) of at least 5 plants per treatment were harvested for RNA extraction. Treatment: To all treatments 0.015% v/v silwet was added.Mock (Water), 1 mM SA, 0.1 mM MeJA or 1 mM SA + 0.1 mM MeJA Each treatment was performed on 3 independent biological replicates. Paper: Van der Does et al. (2013) The Plant Cell
NASCARRAY698 Global expression changes in CK2mut plants (a dominant negative mutant of protein kinase CK2) after 2013-04-30	CK2 is an essential protein kinase involved in a wide variety of cellular processes. The aim of these experiments was to decipher the role of CK2 kinase in Arabidopsis DNA damage response. It is well reported that a rapid transcriptional burst occurs when plants are exposed to gamma radiation. For this reason, we studied the global changes of gene expression in 7-day-old Arabidopsis seedlings expressing a dominant-negative mutant of CK2 (CK2mut plants). Those plants contain a CK2-alfa inactive subunit cloned downstream of a GVG inducible promoter, which is inducible by Dexametahone (Dex)(Moreno-Romero et al. 2008). The gene expression changes were analyzed in gamma-irradiated, Dex-treated mutant seedlings, and were compared to those in irradiated, Dex-untreated seedlings.METHOD: Arabidopsis seeds were surface sterilized, cold-treated at 4Ã‚ÂºC for 48 hours, and then sown on MS plates and grown for five days. Afterwards, plantlets were transferred to MS plates supplemented with 1uM Dex and after two days were treated with 100 Gy of gamma rays. Total RNA was isolated from frozen plantlets with Trizol (Invitrogen) and later purified using Qiagen RNA easy Plant Mini Kit columns (Qiagen, Hilden, Germany). RNA quality was checked using Experion (Bio-Rad laboratories). Three independent RNA preparations were made from CK2mut pooled samples in two different conditions: Dex-treated and irradiated, or mock-treated and irradiated. The data relative to controls necessary for this experiment (plants transformed with the empty vector, and non-irradiated plants) are deposited in NASC (ref. NASCARRAYS-642).Moreno-Romero et al. 2008. A role for protein kinase CK2 in plant development: evidence obtained using a dominant-negative mutant. The Plant Journal 55, 118-130 [2008].
NASCARRAY699 Modification of transcript profile of tobacco due to the transformation 2013-05-09	The aim of the experiment is to find out the contribution of the modification of the host-plant transcript profiling to the generation of a phenotype as a result of the transformation.The whole roots were used for the isolation of RNA.Transgenic and wild type plants were cultivated in hydroponics medium for 6 weeks. Three independent experiments were performed.In each experiment, roots from eight plants grown in different containers were pooled and frozen in liquid nitrogen.RNA was isolated with RNeasy Plant Mini Kit, Quiagen. RNA was treated with the RNAse-free DNAse (Promega).There were prepared three samples from the control material (wild-type tobacco), and three samples from transformed tobacco - grown in three independent experiments. Samples were sent on dry ice for analysis.
NASCARRAY702 Comparison between different Mediator subunit mutants in Arabidopsis 2013-06-17	To study Mediator function in development, we performed Affymetrix microarrays in different late flowering time mediator mutants; med8, med18, med25, and dreb2a mutant (Background Columbia). We included dreb2a because of its interaction with Med25 and it displays an early flowering time (Elvfing et al., 2011; Blomberg et al., 2012).Plants were grown in long days conditions with a day/night (16hr at 23Ã‚Â°C/8hr at 20Ã‚Â°C) period with a light intensity of ~110 ÃŽÂ¼mol m-2 s-1. Leaves of med18, med25, dreb2a, and Col-0 were collected at the bolting time of Col-0. Total RNA was isolated from the powder of leaves samples using RNeasy Plant Mini Kit (Qiagen). The experiments were repeated at least 3 times.N. Elfving et al., The Arabidopsis thaliana Med25 mediator subunit integrates environmental cues to control plant development. Proc Natl Acad Sci USA 108, 8245-8250 (2011).J. Blomberg et al., Interactions between DNA, transcriptional regulator Dreb2a and the Med25 mediator subunit from Arabidopsis thaliana involve conformational changes. Nucleic Acids Res 40, 5938-5950 (2012).
NASCARRAY703 Reprogramming of plants during systemic acquired resistance 2013-06-24	Aim: Characterization of biologically-induced systemic acquired resistance (SAR) at the whole genome level in Arabidopsis thaliana Col-0 wild-type and fmo1 mutant plants. The fmo1 mutant is unable to establish systemic acquired resistance (Mishina TE and Zeier J (2006). The Arabidopsis flavin-dependent monooxygenase FMO1 is an essential component of biologically induced systemic acquired resistance. Plant Physiol. 141, 1666-1675). Experimental setup: For the induction of SAR, plants were infiltrated between 10 AM and 11 AM into three lower (1°) rosette leaves (typically leaf 7 to 9) with a suspension of Pseudomonas syringae pv. maculicola ES4326 (Psm) in 10 mM MgCl2 (OD600 = 0.005). Infiltration with 10 mM MgCl2 served as the mock-control treatment. Upper (2°) leaves (typically leaf 10 to 12) were harvested and shock-frozen in liquid nitrogen two days after the treatment of 1° leaves for RNA extraction. Together, three biologically independent SAR experiments were performed for ATH1 chip microarray analyses. Reference: Gruner K, Griebel T, Návarová H, Attaran E and Zeier J (2013) Reprogramming of plants during systemic acquired resistance. Sample Information: 001 = SAR1 Col mock; 002 = SAR2 Col mock; 003 =SAR3 Col mock; 004= SAR1 Col Psm; 005= SAR2 Col Psm; 006 = SAR3 Col Psm; 007 = SAR1 fmo1 mock; 008 = SAR2 fmo1 mock; 009 = SAR3 fmo1 mock; 010 = SAR1 fmo1 Psm; 011 = SAR2 fmo1 Psm; 012 = SAR3 fmo1 Psm.
NASCARRAY704 Comparison of gonst1 mutant callus with WT 2013-06-26	The aim of this experiment is to compare two independent knockout mutations in the gonst1 gene (At2g13650) with WT. As the plants do not grow on soil, and poorly on agar, we have made callus cell culture lines, from which this RNA is prepared. GONST1 is thought to be a Golgi-localsied GDP-sugar transporter (Baldwin et al., 2001; Handford et al., 2004), although it's precise role in planta is unknown.Baldwin, TC, Handford, MG, Yuseff, I, Orellana, O and P Dupree. (2001) Identification and Characterization of GONST1, a Golgi-Localized GDP- Mannose Transporter in Arabidopsis. The Plant Cell, 13, 2283Ãƒâ€šÃ‚Â-2295Handford MG, Sicilia F, Brandizzi F, Chung JH and P Dupree. (2004) Arabidopsis thaliana expresses multiple Golgi-localised nucleotide-sugar transporters related to GONST1. Molecular and General Genomics, 272, 397-410
NASCARRAY705 Systemic response of Arabidopsis to Pseudomonas syringae pv maculicola ES4326 cfa6 2013-09-19	The aim of this experiment was to compare the systemic response of Arabidopsis thaliana accession Col-0 to Pseudomonas syringae pv maculicola (Psm) cfa6 with and without the effector avrRpt2, which elicits effector-triggered immunity, over a four-day time-course. The cfa6 mutant of Psm was chosen to remove the influence of coronatine. Systemic leaves (true leaves 6 and 7) were harvested 3, 48 and 96 hours after infiltration of true leaves 3, 4 and 5 with Psm cfa6 at an OD600 of 0.2. Expression values were calculated relative to mock-inoculated control plants, which were infiltrated with sterile 10 mM MgSO4.
NASCARRAY706 Expression differences of fc1-1 and fc2-1 2013-10-02	We want to see the expression pattern of fc1-1 anf fc2-1 in roots and leaves.
NASCARRAY708 Time series of translational dependent retrograde signaling Time series of translational dependent 2014-01-27	We took an ethanol inducible RNAi approach to dissect early transcriptome changes in response to a translation dependent retrograde stimulus.

Please write to all@arabidopsis.info if you experience problems with this form....

NASC's International Affymetrix Service

Ran from 2002 - 2013

Quick Index