Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling by array of Arabidopsis mutant for abi1, aba1, max4, axr1, AtMYB50 or AtMYB61

ABSTRACT: 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). Experiment Overall Design: Number of plants pooled:200-400 per RNA sample

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: Nottingham Arabidopsis Stock Centre (NASC)

PROVIDER: E-GEOD-6151 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description: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. Experiment Overall Design: Number of plants pooled:over 100

Project description:This proposal is aimed at providing transcriptome data to underpin a long-term joint research programme of Steve Smith and Alison Smith. We are jointly studying starch synthesis and breakdown in Arabidopsis leaves, and individually studying other enzymes of carbohydrate metabolism (eg. sucrose synthases, invertases, sugar transporters). Collectively these enzymes are encoded by up to 100 known genes, but there are many others of relevance to our studies. For several years we have employed a defined set of growth conditions for this work (resulting in numerous publications). We have extensive data for changes in the amounts of starch, malto-oligosaccharides and sugars throughout the diurnal cycle in these plants, and we intend to quantitate numerous key enzymes. We now wish to profile changes in transcripts in these plants, so that this information can be correlated with changes in the amounts of key enzymes and metabolites. Analysis of the data sets will help us to relate the synthesis of certain enzymes to particular metabolic functions, and also to help identify genes encoding novel proteins involved in carbohydrate metabolism. Current research is aimed at discovering such novel proteins using proteomics approaches (BBSRC Grant: _Discovery and Functional Analysis of the Starch Proteome_). While the growth conditions (12h photoperiod, 150 umol/m2/s, 20C) and ecotype (Col-0) are specifically tailored to our experiments, they will be of value to the wider community, and could form the basis for collaborative studies with other groups. The experiment has involved sampling leaves at eleven different time points as follows: 0, 1, 2, 4, 8, 12, 13, 14, 16, 20, and 24 h (where time 0 is the onset of dark and 12 h is the onset of light). The 24 h time point is a repeat of 0 h. This sequence provides relatively more samples immediately after the light-dark transitions, when changes in metabolism are most pronounced. Plants were grown in a controlled environment chamber under defined conditions, and selected for harvest according to a random number generator. Three fully expanded leaves were harvested from each of 20 plants for each sample. Leaves were frozen at -80 C and a portion saved for metabolite and enzyme assays while RNA was isolated from the remainder. The RNA has been purified and is ready to send to Nottingham. Experiment Overall Design: Number of plants pooled:20 Experiment Overall Design: Biological replicates: 2

Project description:Arabidopsis genome sequencing has revealed the presence of at least three extensive gene families that may encode protein ligands. One of these, the SPH (S-protein homologue) family, was identified as a direct result of our studies on self-incompatibility in Papaver. The Arabidopsis SPH gene family consists of 81 members. We have initiated experimental work on a subset of these. RT-PCR studies indicate that many, if not all, SPH genes are expressed. Each SPH gene encodes an N-terminal signal peptide sequence and thus SPH proteins are likely to be secreted. Until recently none of the genes in this family had known function. However we have evidence that one member of the family, SPH1 is involved in leaf vascular development. In order to determine the function of SPH1, Arabidopsis plants were transformed with an SPH1 antisense construct. Analysis of the mutant phenotype shows that whilst plants appear as wt until principal growth stage 1.04, they subsequently show severe morphological defects. Plants are severely dwarfed with twisted rosette leaves at ~ 30% wt length and width as well as shortened inflorescence stem. Closer examination revealed aberrant leaf vasculature and severe reduction in expansion of parenchyma cells surrounding the primary leaf vein. We have conducted preliminary immunolocalisation studies with antibody raised to SPH1. These suggest that SPH1 protein is secreted by cells within the developing vasculature of the immature leaf (leaves<6mm in length). The data that we have so far obtained leads us to believe that SPH1 protein acts as a signalling molecule during early leaf development. As SPH1 is likely to be a signalling protein it is assumed that its interaction with a cognate receptor results in initiation of developmental processes within leaf tissue. The purpose of the experiment is to determine the network of genes within the normally developing rosette leaf whose expression is altered by SPH1. This will be accomplished by comparing transcriptional levels in rosette leaves of antisense-SPH1 plants with wt plants. We propose to make target RNA from rosette leaves taken from plants at principal stage 1.05 (immediately after initial appearance of developmental abnormality) and from plants at principal stage 1.14 (where gross changes are apparent). We propose to use replicate slides for each hybridisation, i.e. 2 hybridised to RNA from wt leaves at 1.05, 2 antisense at 1.05, 2 wt at 1.14 and 2 antisense at 1.14. Experiment Overall Design: Number of plants pooled:40

Project description:Nitric oxide (NO.) is a well-established signal in mammalian cells regulating e.g. smooth muscle tone, neurotransmission and apoptosis. NO interacts with superoxide (O2-) to derive the highly reactive peroxynitrite (ONOO-) radical leading to the generation of lipid hydroxy-peroxides (ROO.) and cell death. Thus the recent reports implicating of .NO in the Hypersensitive Response (HR) a form of programmed cell death elicited by pathogens in plantsis suggestive of parallel roles in animals and plants. As part of BBSRC funded programmes which are investigating oxidative signaling and cell death in Arabidopsis we have collaborated with the Trace Gas Detection facility at the University of Nijmegen to be the first to measure NO in planta from a developing HR (Mur et al.paper in prep). The challenge remains to understand the spatio-temporal role(s) of .NO during the HR and disease development. In line with other groups e.g. Klessig et al.(2000) PNAS 978849-55 we have observed that .NO mediated event are both SA- dependent and independent. We propose a two-stage programme to investigate NO events which will lead to subsequent BBSRC grant bids. Firstly to exploit the GARNET Affymetrix transcriptomic service to identify genes which are upregulated by .NO (see programme below). These will be compared with similar data generated by other members of the consortium; Dr. Steve Neill for oxidative stress (Desikan et al. (2001). Plant Physiol 127(1): 159-72; Clarke et al.2000 Plant J.; 24:667-77); Dr. Paul Kenton who is mainly interested in signaling by the oxylipid Jasmonic acid (Kentonet al. (1999). MPMI 12(1): 74-78) as well as from other sources. Though these data in themselves will suggest modes of NO action. However as a second approach we will clone the promoter of a strongly NO-responsive gene which will be fused to positive and negative selectable markers as well as reporter genes e.g. LUC. to identify Arabidopsis EMS mutants which are perturbed in NO-mediated events. Programme. Our in planta measurement show that from a baseline production of 1nl.g fwt-1h-1 NO levels rise to 6nl. g fwt-h-1 (_plus/- 1.1 SE) prior to cell death and to 25nl g fwt-h-1 (_plus/- 4.2 SE) at cell death following which the concentration falls. At this juncture we will intend to gas Arabidopsis to ~75ppb (the head-space concentration when NO production was at 6nl. g fwt-h-1). Prior to using the DNA RNA will establish that this does not elicit cell death over the proposed treatment period and that both PR1 (SA-dependent) and PAL1 (SA-independent) genes are induced. We will compare this plant with sealed but non-treated Arabidopsis. Future targets for DNA RNAs analysis induce plant treated with both NO (Sodium Nitro Prusside) and O2- (Xanthine oxidase) generators and depending on their effectiveness (to be assessed by NO measurement) HR lesions treated with Nitric Oxide Synthase inhibitors. Experiment Overall Design: Number of plants pooled:8

Project description:This application is the second part of a BBSRC-funded grant to compare and contrast the plastid-signalling pathways disrupted by Norflurazon and far-red light treatment of Arabidopsis seedlings. The first application of this laboratory to GARNet's Affymetrix service (2002-08-25-17.41.49_McCormac) addressed the Norflurazon pathway; this application addresses the far-red pathway. The assembly of photosynthetic complexes in developing chloroplasts is critical to the establishment of the autotrophic plant. This requires light-mediated upregulation of both nuclear- and chloroplast-encoded genes. The expression of such photosynthetically-associated nuclear genes is also often dependant on a retrograde plastid signal which emanates from chloroplasts to modulate nuclear transcription. Extensive studies using the herbicide Norflurazon to knock-out the plastid signal (including this lab's previous Affymetrix application to GARNet) are identifying the affected gene sets. However, genetic studies have indicated the existence of more than one plastid signalling pathway. We have recently investigated a phytochrome A-mediated, far-red (FR) input pathway which blocks subsequent chloroplast development under white light (WL). This has also been found to inhibit the transcription of a small group of known nuclear-encoded plastidic proteins. Here we wish to establish how wide-reaching this FR-effect is on nuclear transcription, and will directly compare the affected gene groups with those identified from our earlier (and other's) studies with a Norflurazon treatment. In this array experiment we will compare RNA from seedlings grown in complete darkness (D) before transfer to WL, with that of seedlings preconditioned under a restricted wavelength FR source before exposure to WL. This comparison of FR- and Norflurazon-affected gene groupings will indicate whether the plastid signalling pathways are likely to be the same, overlapping or highly divergent. As well as wild-type seedlings, the gun1,gun5 mutant line is to be used as both these alleles are well established as alleviating the Norflurazon-inhibited pathway, but their affect on the FR-pathway is less clear. A single replicate of a phyA-null mutant line will also be included in order to differentiate between specific phytochromeA-mediated responses and other FR effects. It is envisaged that, in general, D- and FR-treated samples of the phyA mutant line will respond in the same way as each other and as the wild-type D-treated samples and, thus, the lack of a biological repeat in this case is not a major short-fall. The proposed experiment will consist of: wild-type: D-pretreated (x2 biological replicates) wild-type: FR-preconditioned (x2) gun1,gun5: D-pretreated (x2) gun1,gun5: FR-preconditioned (x2) phyA: D-pretreated (x1) phyA: FR-preconditioned (x1) Experiment Overall Design: Number of plants pooled:300 seedlings

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Transcription profiling by array of Arabidopsis mutant for abi1, aba1, max4, axr1, AtMYB50 or AtMYB61

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