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IdentificationOf genes responsive to non-metabolised glucose analogs: approach to hexokinase-independent glucose sensing

ABSTRACT: 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. Experimenter name = Dorthe Villadsen Experimenter phone = 0131 650 5318 Experimenter fax = 0131 650 5392 Experimenter address = Institute of Cell and Molecular Biology Experimenter address = University of Edinburgh Experimenter address = The King_s Buildings Experimenter address = Mayfield Road Experimenter address = Edinburgh Experimenter zip/postal_code = EH9 3JH Experimenter country = UK Keywords: growth_condition_design

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE5723 | GEO | 2007/01/08

SECONDARY ACCESSION(S): PRJNA97021

REPOSITORIES: GEO

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Project description: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. Experimenter name = Dorthe Villadsen; Experimenter phone = 0131 650 5318; Experimenter fax = 0131 650 5392; Experimenter address = Institute of Cell and Molecular Biology; Experimenter address = University of Edinburgh; Experimenter address = The King_s Buildings; Experimenter address = Mayfield Road; Experimenter address = Edinburgh; Experimenter zip/postal_code = EH9 3JH; Experimenter country = UK Experiment Overall Design: 6 samples were used in this experiment

Project description: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 4°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 22°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. Experimenter name = Martin Broadley Experimenter phone = 0115 951 6382 Experimenter fax = 0115 951 6334 Experimenter institute = University of Nottingham Experimenter address = Plant Sciences Division Experimenter address = School of Biosciences Experimenter address = University of Nottingham Experimenter address = Sutton Bonington Experimenter address = Loughborough Experimenter zip/postal_code = LE12 5RD Experimenter country = UK Keywords: growth_condition_design

Project description: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.Some questions on the form are omitted as we are not using mutant or transgenic lines. This is our first application. Experimenter name = Peter Edward Urwin Experimenter phone = 0113 343 3035/2909 Experimenter fax = 0113 343 3144 Experimenter address = Centre for Plant Science Experimenter address = University of Leeds Experimenter address = Leeds Experimenter zip/postal_code = LS2 9JT Experimenter country = UK Keywords: pathogenicity_design

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IdentificationOf genes responsive to non-metabolised glucose analogs: approach to hexokinase-independent glucose sensing

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