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Carbohydrate- and redox-regulation of gene expression in a TPT mutant

ABSTRACT: The triose-phosphate/phosphate translocator (TPT) of the chloroplast inner envelope membrane mediates the counter-exchange of stromal triose phosphates derived from CO2 fixation with cytosolic phosphate, thus providing the cytosol with precursors for sucrose synthesis. We have isolated an Arabidopsis mutant (tpt-1) in which the gene encoding TPT is disrupted by a T-DNA insertion. During growth in low light tpt-1 plants are phenotypically normal, but in high light photosynthesis is inhibited and growth is retarded relative to wildtype. This mutant compensates for the absence of TPT by diverting photosynthate into starch which is hydrolysed and exported from the chloroplast as glucose that is subsequently phosphorylated by hexokinase. In low light the capacity of the pathway of starch synthesis is sufficient to accommodate the normal rate of CO2 fixation, but in high light it is unable to match the potential rate of CO2 fixation. Consequently, in high light-grown plants there are measurable effects on the redoxstate of several components. Thus, the tpt-1 mutation influences the carbohydrate status within the cell, alters the form in which carbon is received by the cytosol, and changes the redox signals that are important in photosynthetic acclimation.Plants will be grown in a 8h light:16h dark regime at both 400 and 100 Âµmol PAR m-2 s-1. Total RNA will be extracted from pools of individual illuminated leaves from at least eight plants at growth stage 3.70. Leaves will be harvested 2 h into the photoperiod to maximises differences between plant lines in the expression of genes of photosynthesis and carbohydrate metabolism. We anticipate that expression of many genes will be affected by the absence of TPT and by changes in light intensity. However, by comparing differences in transcript levels between wildtype and TPT mutant grown in high light with the differences that occur in plants grown in low light we will discriminate between genes whose expression is affected by changes in the pattern of carbohydrate metabolism and those influenced by redox poise of the thylakoid photochemical components. These comparisons will also highlight genes affected by recently identified regulatory interactions between sugar-sensing and redox-sensing. A genome-wide expression study will establish the extent to which gene expression is altered by the absence of TPT in leaves, and will provide the basis for more detailed analysis of a selected range of transcripts whose levels of expression differ between plant lines. Experimenter name = Robin Walters Experimenter phone = 01865 275000 Experimenter fax = 01865 275074 Experimenter address = Dept of Plant Sciences Experimenter address = University of Oxford Experimenter address = South Parks Road Experimenter address = Oxford Experimenter zip/postal_code = OX1 3RB Experimenter country = UK Keywords: growth_condition_design; genetic_modification_design;

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE5737 | GEO | 2007/01/08

SECONDARY ACCESSION(S): PRJNA97047

REPOSITORIES: GEO

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Project description:The triose-phosphate/phosphate translocator (TPT) of the chloroplast inner envelope membrane mediates the counter-exchange of stromal triose phosphates derived from CO2 fixation with cytosolic phosphate, thus providing the cytosol with precursors for sucrose synthesis. We have isolated an Arabidopsis mutant (tpt-1) in which the gene encoding TPT is disrupted by a T-DNA insertion. During growth in low light tpt-1 plants are phenotypically normal, but in high light photosynthesis is inhibited and growth is retarded relative to wildtype. This mutant compensates for the absence of TPT by diverting photosynthate into starch which is hydrolysed and exported from the chloroplast as glucose that is subsequently phosphorylated by hexokinase. In low light the capacity of the pathway of starch synthesis is sufficient to accommodate the normal rate of CO2 fixation, but in high light it is unable to match the potential rate of CO2 fixation. Consequently, in high light-grown plants there are measurable effects on the redoxstate of several components. Thus, the tpt-1 mutation influences the carbohydrate status within the cell, alters the form in which carbon is received by the cytosol, and changes the redox signals that are important in photosynthetic acclimation.Plants will be grown in a 8h light:16h dark regime at both 400 and 100 ÃÂµmol PAR m-2 s-1. Total RNA will be extracted from pools of individual illuminated leaves from at least eight plants at growth stage 3.70. Leaves will be harvested 2 h into the photoperiod to maximises differences between plant lines in the expression of genes of photosynthesis and carbohydrate metabolism. We anticipate that expression of many genes will be affected by the absence of TPT and by changes in light intensity. However, by comparing differences in transcript levels between wildtype and TPT mutant grown in high light with the differences that occur in plants grown in low light we will discriminate between genes whose expression is affected by changes in the pattern of carbohydrate metabolism and those influenced by redox poise of the thylakoid photochemical components. These comparisons will also highlight genes affected by recently identified regulatory interactions between sugar-sensing and redox-sensing. A genome-wide expression study will establish the extent to which gene expression is altered by the absence of TPT in leaves, and will provide the basis for more detailed analysis of a selected range of transcripts whose levels of expression differ between plant lines. Experimenter name = Robin Walters; Experimenter phone = 01865 275000; Experimenter fax = 01865 275074; Experimenter address = Dept of Plant Sciences; Experimenter address = University of Oxford; Experimenter address = South Parks Road; Experimenter address = Oxford; Experimenter zip/postal_code = OX1 3RB; Experimenter country = UK Experiment Overall Design: 12 samples were used in this experiment

Project description:The triose-phosphate/phosphate translocator (TPT) of the chloroplast inner envelope membrane mediates the counter-exchange of stromal triose phosphates derived from CO2 fixation with cytosolic phosphate, thus providing the cytosol with precursors for sucrose synthesis. We have isolated an Arabidopsis mutant (tpt-1) in which the gene encoding TPT is disrupted by a T-DNA insertion. During growth in low light tpt-1 plants are phenotypically normal, but in high light photosynthesis is inhibited and growth is retarded relative to wildtype. This mutant compensates for the absence of TPT by diverting photosynthate into starch which is hydrolysed and exported from the chloroplast as glucose that is subsequently phosphorylated by hexokinase. In low light the capacity of the pathway of starch synthesis is sufficient to accommodate the normal rate of CO2 fixation, but in high light it is unable to match the potential rate of CO2 fixation. Consequently, in high light-grown plants there are measurable effects on the redoxstate of several components. Thus, the tpt-1 mutation influences the carbohydrate status within the cell, alters the form in which carbon is received by the cytosol, and changes the redox signals that are important in photosynthetic acclimation. Method: Plants will be grown in a 8h light:16h dark regime at both 400 and 100 Âµmol PAR m-2 s-1. Total RNA will be extracted from pools of individual illuminated leaves from at least eight plants at growth stage 3.70. Leaves will be harvested 2 h into the photoperiod to maximise differences between plant lines in the expression of genes of photosynthesis and carbohydrate metabolism. We anticipate that expression of many genes will be affected by the absence of TPT and by changes in light intensity. However, by comparing differences in transcript levels between wildtype and TPT mutant grown in high light with the differences that occur in plants grown in low light we will discriminate between genes whose expression is affected by changes in the pattern of carbohydrate metabolism and those influenced by redox poise of the thylakoid photochemical components. These comparisons will also highlight genes affected by recently identified regulatory interactions between sugar-sensing and redox-sensing. A genome-wide expression study will establish the extent to which gene expression is altered by the absence of TPT in leaves, and will provide the basis for more detailed analysis of a selected range of transcripts whose levels of expression differ between plant lines.

Project description:Atmospheric CO2 concentrations can determine the number of stomata that form on plant leaves (Woodward & Kelly 1995 New Phyt 131: 311-327). The majority of species exhibit reduced stomatal densities at elevated CO2. However, not all plant species react in the same way to elevated CO2 levels and there is a spectrum of effects: Some species increase stomatal densities, some decrease stomatal densities, and some are unaffected. In addition to which, other environmental factors influence the number of stomata that a plant form. Light intensity has also been shown to affect stomatal numbers in various Arabidopsis ecotypes (Schluter et al. 2003 J Exp Bot 54 (383): 867-874; Lake et al. 2002 J Exp Bot 53 (367): 183-193), by increasing stomatal numbers with increasing light levels. There are many changes in gene expression under elevated CO2 conditions, so pinpointing specific genes involved in the stomatal response to CO2 is difficult. In addition, if there is crosstalk between the various signalling pathways affecting ultimate stomatal numbers this complicates further the task of finding genes specifically involved the stomatal response to CO2. Therefore we propose to look at the interaction of two known influences on stomatal numbers, light and CO2, on one specific ecotype, Col-0. We aim to test the hypothesis that light signals interact the CO2 signals that affect stomatal development. Arabidopsis thaliana Columbia-0 ecotype has previously been shown to decrease stomatal numbers in response to a doubling of ambient CO2 concentrations. Col-0 has also been shown to increase stomatal numbers in response to high light intensities. Therefore we propose to grow A. thaliana Col-0 at three light intensities (50 mmol m-2 s-1, 150 mmol m-2 s-1 and 250 mmol m-2 s-1), in both ambient and elevated (double ambient) atmospheric CO2 concentrations. By looking in more detail at how gene expression differs between plants grown at ambient and elevated CO2 at the same light intensities, and also how gene expression differs between plants grown at the same CO2 concentration but different light intensities, we aim to identify those genes involved in the stomatal developmental response to CO2 and whether genes involved in the light response can also be isolated. Experimenter name = Susannah Bird Experimenter phone = (0114) 222 4649 Experimenter address = Animal and Plant Science Department Experimenter address = Alfred Denny Building Experimenter address = Western Bank Experimenter address = Sheffield Experimenter zip/postal_code = S10 2TN Experimenter country = UK Keywords: growth_condition_design

Project description:Atmospheric CO2 concentrations can determine the number of stomata that form on plant leaves (Woodward & Kelly 1995 New Phyt 131: 311-327). The majority of species exhibit reduced stomatal densities at elevated CO2. However, not all plant species react in the same way to elevated CO2 levels and there is a spectrum of effects: Some species increase stomatal densities, some decrease stomatal densities, and some are unaffected. In addition to which, other environmental factors influence the number of stomata that a plant form. Light intensity has also been shown to affect stomatal numbers in various Arabidopsis ecotypes (Schluter et al. 2003 J Exp Bot 54 (383): 867-874; Lake et al. 2002 J Exp Bot 53 (367): 183-193), by increasing stomatal numbers with increasing light levels. There are many changes in gene expression under elevated CO2 conditions, so pinpointing specific genes involved in the stomatal response to CO2 is difficult. In addition, if there is crosstalk between the various signalling pathways affecting ultimate stomatal numbers this complicates further the task of finding genes specifically involved the stomatal response to CO2. Therefore we propose to look at the interaction of two known influences on stomatal numbers, light and CO2, on one specific ecotype, Col-0. We aim to test the hypothesis that light signals interact the CO2 signals that affect stomatal development. Arabidopsis thaliana Columbia-0 ecotype has previously been shown to decrease stomatal numbers in response to a doubling of ambient CO2 concentrations. Col-0 has also been shown to increase stomatal numbers in response to high light intensities. Therefore we propose to grow A. thaliana Col-0 at three light intensities (50 mmol m-2 s-1, 150 mmol m-2 s-1 and 250 mmol m-2 s-1), in both ambient and elevated (double ambient) atmospheric CO2 concentrations. By looking in more detail at how gene expression differs between plants grown at ambient and elevated CO2 at the same light intensities, and also how gene expression differs between plants grown at the same CO2 concentration but different light intensities, we aim to identify those genes involved in the stomatal developmental response to CO2 and whether genes involved in the light response can also be isolated. Experimenter name = Susannah Bird; Experimenter phone = (0114) 222 4649; Experimenter address = Animal and Plant Science Department; Experimenter address = Alfred Denny Building; Experimenter address = Western Bank; Experimenter address = Sheffield; Experimenter zip/postal_code = S10 2TN; Experimenter country = UK Experiment Overall Design: 6 samples were used in this experiment

Project description:We were awarded a BBSRC grant about a year ago to undertake some affymetrix gene chip profiling of light and CO2 systemic signalling in Arabidopsis. The design of the proposed experiment is given below and the appropriate funding has been provided by the BBSRC. The aim of the project is to identify the temporal profile of those genes that respond to light and CO2 systemic signals in developing leaves. Moreover, as thes two signals have opposing effects on leaf development to ascertain whether they involve similar or parallel signalling pathways. The experiment is to examine the effect of exposing mature leaves to high CO2 or low light or both on the gene expression profile of developing leaves. We already have data for maize that changes in gene expression profile occur within 4h and that there are a variety of temporal responses that differ between individual gene transcripts. We have also demonstrated that Arabidopsis leaf development is altered by these systemmic signals and that lesions in the jasmonate and ethylene signalling pathways block these responses. Our experimental design is shown below: We have 4 treatments and 7 timepoints. (0, 2, 4, 12, 24, 48, 96 h) We would sample from 5 individual plants that would be pooled for each RNA preparation. This would require 28 chips and this would include extra replication of the 0 time-point control (deemed by many as nessary). Experimental details: All plants were germinated for 7 days under the following conditions: Humax multi-purpose compost, ambient carbon dioxide (370 ppm) and ambient light (250 µmol/m/s), constant temperature of 20°C and a 10 h photoperiod (8 am until 6 pm). After a week the the seedlings were potted up into 104-cell plug trays for a further 2 weeks and then potted up into 10 cm pots and the bottom part of the signalling cuvette system attached (see Lake et al., Nature 10th May 2001 Vol. 411, pp 154). Twenty four, 4 week old plants, then had the top part of the signalling system attached, trapping leaf insertions 5-13. Humidified, ambient air was passed through them at 500 mls/min via an oil-free air compressor. The three target leaves (19-21) were then marked with non-toxic, acrylic paint. After a 24 h period (the plants were sealed into the cuvettes from 10 am until 10am) of adjustment, the experiment was started by harvesting the target leaves from 4 plants and immediately freezing the tissue in liquid nitrogen to give the 0 h sample before RNA extraction. The remaining 20 plants were divided into 4 groups of five and given one of the following treatments: Ambient carbon dioxide/ambient light (Control) (A) Elevated carbon dioxide (750 ppm)/ambient light (E) Ambient carbon dioxide/low light (50 µmol/m/s) (AS) Elevated carbon dioxide/low light (ES) - For the Elevated CO2, elevated CO2 was pumped in using a CT room next door set to same temperature but with a CO2 cylinder inside and the same pump as used in the ambient room to supply the elevated CO2 laden, humidified air into the signalling room using rubber tubing. - Shade treatment consisted of neutral density filter (Cat. 210 0.6ND, Lee Filters) that had a hole cut in the middle to allow the middle developing leaves to grow through. A timecourse of 2, 4, 12, 24, 48 and 96 h were carried out each using a batch of 24 plants. This whole process was repeated with another batch of 24 plants at the same developmental stage to give a 2, 4, 12, 24, 48 and 96 hour sample from each of the four treatments. The whole timecourse was then repeated 4 times. For the mature leaves: We had 8 chips left over so we devised this little experiment to assess the gene changes that were occurring in the enclosed, treated, mature leaves that were signalling the environment to the young developing leaves. Experimenter name = Simon Coupe Experimenter phone = 0114 222 4115 Experimenter fax = 0114 222 0002 Experimenter institute = University of Sheffield Experimenter address = Animal and Plant Sciences Experimenter address = University of Sheffield Experimenter address = Western Bank Experimenter address = Sheffield Experimenter zip/postal_code = S10 2TN Experimenter country = UK Keywords: development_or_differentiation_design; growth_condition_design

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Carbohydrate- and redox-regulation of gene expression in a TPT mutant

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