Project description: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. Experiment Overall Design: Number of plants pooled:20 Experiment Overall Design: Biological replicates: 2

Project description: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. Keywords: strain_or_line_design

Project description: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. Experimenter name: Yinbo Gan Experimenter phone: 01524 592935 Experimenter fax: 01524 843854 Experimenter department: Lancaster University Experimenter address: Department of Biological Sciences Experimenter address: Lancaster University Experimenter address: Bailrigg Experimenter address: Lancaster Experimenter zip/postal_code: LA1 4YQ Experimenter country: UK Keywords: genetic_modification_design; growth_condition_design

Project description: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. Experimenter name: Yinbo Gan; Experimenter phone: 01524 592935; Experimenter fax: 01524 843854; Experimenter department: Lancaster University; Experimenter address: Department of Biological Sciences; Experimenter address: Lancaster University; Experimenter address: Bailrigg; Experimenter address: Lancaster; Experimenter zip/postal_code: LA1 4YQ; Experimenter country: UK Experiment Overall Design: 6 samples were used in this experiment

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: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. Experiment Overall Design: Number of plants pooled:100

Project description:Arable soils are extremely heterogeneous in spatial nutrient distribution. It is well documented that lateral roots (LRs) proliferate in nitrate-rich patch. Yet less information is available as to which genes are involved in this process, in particular in cereals. To understand the molecular mechanism for local nitrate induced LR growth in maize (Zea mays L.), we analyzed the gene expression profiling in maize root in early response (1 hour) to local nitrate stimulation by using Maize Oligonucleotide Array (http://www.maizearray.org) and a split-root system. Selected differentially expressed genes were further confirmed by semi-quantitative RT-PCR. The results showed that reception and/or transduction of local NO3- signal involve some important protein kinases and protein phosphatases (histidine kinases, serine/threonine kinases, protein phosphatase 2A etc.) and transcription factors (F-box, Zinc finger, Myb and bZIP transcription factors and response regulator). Increasing expression of genes encoding auxin response factor 7b, ethylene receptor, and cytokinin oxidase suggests strong interaction among hormonal pathways and local NO3- signaling pathways. Genes involving NO3- uptake and assimilation (NRT2.1, NR, etc.), sugar transport (a sugar transporter) and utilization (a sucrose synthase) were enhanced in the N-fed root. Furthermore, local NO3- induces rapid expression of genes related to cell division and expansion such as alpha-expansin, celluose synthase, kinesin, plasma membrane and tonoplast aquaporins. Based on the differentially expressed genes, a putative model which combines both the 'NO3- signal' and 'metabolic sink' theories is proposed to explain the molecular mechanism controlling local nitrate induced LR elongation in maize. plants were pre-cultured in solution containing 0.5 mmol L-1 NO3- for 6 days and then transferred to N-free solution to grow for another 2 days. Then these plants were transferred to a two-compartment split-root system with only half root supplied with 1.0 mmol L-1 NO3-. Supply of Ca2+ in the N-free half was compensated by adding 1.0 mmol L-1 CaCl2. One hour after the local nitrate treatment, root segments (15cm from root tip to lateral root elongation zone) were sampled for RNA extraction. Two biological replicates were performed for each treatment.

Project description: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.

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

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Transcription profiling of DEX treated Arabidopsis genotypes (ANGR4-12 and wild type) to analyse nutritional control of plant development: molecular analysis of the NO3- response pathway in Arabidopsis roots

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