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Growth of suspension-cultured cells

ABSTRACT: The growth of Arabidopsis cell cultures following their sub-culture into fresh media follows standard growth kinetics of a period of exponential increase associated with high rate of cell division, followed by a slowing of the rate of increase as cells approach stationary phase. For the analysis described here, MM2d cells were subcultured into fresh MSS-medium and samples were taken at day 1, day3, day 5 and day7. We have carried out transcriptional profiling analysis with the aim to follow growth stage specific gene expression during unperturbed growth using the near full genome ATH1 arrays (Menges et al., 2003). Journal Absract: (Plant Molecular Biology: 53, 2003) Plant cell suspension cultures are invaluable models for the study of cellular processes. Here we develop the recently described Arabidopsis suspension culture MM2d as a transcript profiling platform by means of Affymetrix ATH1 microarrays. Analysis of gene expression profiles during normal culture growth, during synchronous cell cycle re-entry and during synchronous cell cycle progression provides a unique integrated view of gene expression responses in a higher-plant system. Particularly striking is that expression of over 14 000 genes belonging to all defined categories can be reliably detected, suggesting that integrated and comparative analysis of data sets derived from transcript profiling of cultures is a powerful approach to identify candidate components involved in a wide range of biological processes. Combinatorial analysis of independent cell cycle synchrony methods allows the identification of genes that are apparently cell-cycle-regulated but are most likely responding to the induction of synchrony. We thus present an integrated genome-wide view of the transcriptional profile of a plant suspension culture and identify a refined set of 1082 cell cycle regulated genes largely independent of synchrony method. Experimenter name = Jim Murray Experimenter phone = 44 1223 334166 Experimenter fax = 44 1223 334162 Experimenter department = J Murray Laboratory Experimenter address = University of Cambridge Experimenter address = Institute of Biotechnology Experimenter address = Tennis Court Road Experimenter address = Cambridge Experimenter zip/postal_code = CB2 1QT Experimenter country = United Kingdom Keywords: time_series_design

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE5750 | GEO | 2007/01/08

SECONDARY ACCESSION(S): PRJNA97073

REPOSITORIES: GEO

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Project description:The growth of Arabidopsis cell cultures following their sub-culture into fresh media follows standard growth kinetics of a period of exponential increase associated with high rate of cell division, followed by a slowing of the rate of increase as cells approach stationary phase. For the analysis described here, MM2d cells were subcultured into fresh MSS-medium and samples were taken at day 1, day3, day 5 and day7. We have carried out transcriptional profiling analysis with the aim to follow growth stage specific gene expression during unperturbed growth using the near full genome ATH1 arrays (Menges et al., 2003). Journal Absract:; (Plant Molecular Biology: 53, 2003); Plant cell suspension cultures are invaluable models for the study of cellular processes. Here we develop the recently described Arabidopsis suspension culture MM2d as a transcript profiling platform by means of Affymetrix ATH1 microarrays. Analysis of gene expression profiles during normal culture growth, during synchronous cell cycle re-entry and during synchronous cell cycle progression provides a unique integrated view of gene expression responses in a higher-plant system. Particularly striking is that expression of over 14 000 genes belonging to all defined categories can be reliably detected, suggesting that integrated and comparative analysis of data sets derived from transcript profiling of cultures is a powerful approach to identify candidate components involved in a wide range of biological processes. Combinatorial analysis of independent cell cycle synchrony methods allows the identification of genes that are apparently cell-cycle-regulated but are most likely responding to the induction of synchrony. We thus present an integrated genome-wide view of the transcriptional profile of a plant suspension culture and identify a refined set of 1082 cell cycle regulated genes largely independent of synchrony method. Experimenter name = Jim Murray; Experimenter phone = 44 1223 334166; Experimenter fax = 44 1223 334162; Experimenter department = J Murray Laboratory; Experimenter address = University of Cambridge; Experimenter address = Institute of Biotechnology; Experimenter address = Tennis Court Road; Experimenter address = Cambridge; Experimenter zip/postal_code = CB2 1QT; Experimenter country = United Kingdom Experiment Overall Design: 4 samples were used in this experiment

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 Keywords: growth_condition_design

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

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Growth of suspension-cultured cells

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