Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of Arabidopsis dormant versus active axillary shoot apical mersitems

ABSTRACT: 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. Experimenter name: Sally Ward; Experimenter phone: 01904 328683; Experimenter fax: 01904 328682; Experimenter institute: University of York; Experimenter address: Dept of Biology (Area 11),; University of York,; Heslington,; York; Experimenter zip/postal_code: YO10 5DD; Experimenter country: UK Experiment Overall Design: 2 samples were used in this experiment

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: Nottingham Arabidopsis Stock Centre (NASC)

PROVIDER: E-GEOD-10324 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description: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. Experimenter name: Hsiu-Ling Yap; Experimenter phone: 01904 434 302/304; Experimenter fax: 01904 434 312; Experimenter institute: University of York; Experimenter address: Department of Biology; University of York; P.O.Box 373; York!Series_summary = Experimenter zip/postal_code: YO10 5YW; Experimenter country: UK Experiment Overall Design: 4 samples were used in this experiment

Project description: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. Experimenter name: Raffaella Greco; Experimenter department: Pereira Lab; Experimenter institute: Plant Research International; Experimenter address: Business Unit Genomics; Plant Research International; Postbus 16; Wageningen; Experimenter zip/postal_code: 6700 AA; Experimenter country: The Netherlands Experiment Overall Design: 2 samples were used in this experiment

Project description: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 Experiment Overall Design: 2 samples

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: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

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Transcription profiling of Arabidopsis dormant versus active axillary shoot apical mersitems

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