Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of Arabidopsis bountiful gain of function mutant

ABSTRACT: 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

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: Nottingham Arabidopsis Stock Centre (NASC)

PROVIDER: E-GEOD-10322 | 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: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.

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treatment of a new conditional root expansion mutant partially restores the expansion phenotype (JE100; Werner et al., unpublished).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

Project description: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.

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.

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Transcription profiling of Arabidopsis bountiful gain of function mutant

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