Project description:The transcript responses of both growing, trifoliate 6 and fully expanded, trifoliate 4 soybean leaves to elevated CO2 was investigated. We also compared the transcriptome of fully expanded vs. developing leaves in both ambient and elevated CO2. Keywords = soybean Keywords = elevated carbon dioxide Keywords = global change Keywords = leaf growth Keywords = plant Keywords: soybean leaf comparisons
Project description:The microalga Coccomyxa subellipsoidea C-169 possesses some features that may be valuable for lipid production, and, as demonstrated in this study, can be greatly induced to produce a high amount of fatty acid by CO2 supplementation. Here we have compared the transcriptome of air group (AG, cells cultured under 0.04% CO2) and CO2-supplemented group (CG, cells cultured under 2% CO2), and found that dramatic and collaborative regulation in central metabolic pathways as well as biochemical processes occured in response to CO2 supplementation. This study gains a broad understanding of how CO2 stress regulates gene expression and eventually reveals a fine-tuned strategy adopted by C-169 to sustain rapid cell growth and lipid production, which will be helpful for the implementation of biofuels production from oleaginous microalgae. Transcriptomic profiles of Coccomyxa subellipsoidea C-169 cultured for 4 days under two CO2 levels (0.04% and 2%, v/v) were generated by digital gene expression (DGE) analysis, in triplicate, using Illumina Hiseq2000.
Project description:The microalga Coccomyxa subellipsoidea C-169 possesses some features that may be valuable for lipid production, and, as demonstrated in this study, can be greatly induced to produce a high amount of fatty acid by CO2 supplementation. Here we have compared the transcriptome of air group (AG, cells cultured under 0.04% CO2) and CO2-supplemented group (CG, cells cultured under 2% CO2), and found that dramatic and collaborative regulation in central metabolic pathways as well as biochemical processes occured in response to CO2 supplementation. This study gains a broad understanding of how CO2 stress regulates gene expression and eventually reveals a fine-tuned strategy adopted by C-169 to sustain rapid cell growth and lipid production, which will be helpful for the implementation of biofuels production from oleaginous microalgae.
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
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
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
Project description:Introduction : By mid-century, global atmospheric carbon dioxide concentration ([CO2]) is predicted to reach 600 umol mol-1 with global temperatures rising by 2ºC. Rising [CO2] and temperature will alter the growth and productivity of major food and forage crops across the globe. Although the impact is expected to be greatest in tropical regions, the impact of climate-change has been poorly studied in those regions. Objectives : This experiment aimed to understand the effects of elevated [CO2] (600 umol mol-1) and warming (+ 2°C), singly and in combination, on Panicum maximum Jacq. (Guinea grass) metabolite and transcript profiles. Methods: We created a de novo assembly of the Panicum maximum transcriptome. Leaf samples were taken at two time points in the Guinea grass growing season to analyze transcriptional and metabolite profiles in plants grown at ambient and elevated [CO2] and temperature, and statistical analyses were used to integrate the data. Results: The MiSeq library was quantified by qPCR and sequenced on one MiSeq flowcell for 301 cycles using paired-end sequencing. HiSeq paired-end sequencing was done with four quantified libraries per treatment which were pooled in equimolar concentration, and sequenced on two lanes for 161 cycles. The final read lengths for MiSeq and HiSeq were 300 nt and 160 nt in length. A total of 635,649,277 reads were assembled from the MiSeq/HiSeq pools. Quality control for reads generated from sequencing was performed using FastQC. Quality reads were used to perform de novo transcriptome assembly using Trinity. The initial assembly consisted of 187,216 genes. A filter was applied to keep only those genes that had at least 10 reads (across the 4 replicates) for an individual treatment. The resulting transcriptome contained 45,073 genes and reads. Functional annotation of the genes was done by using BLAST against Arabidopsis thaliana, Zea mays, and Setaria italica.
Project description:Background: Microalgae are promising feedstocks for production of renewable biofuels and value-added bioproducts. Temperature and nitrogen supply are important environmental and nutritional factors affecting the growth and metabolism of microalgae, respectively. In this study, the growth and lipid accumulation of filamentous microalga Xanthonema hormidioides under different temperatures (5, 7, 10, 15, 20, 25, 27 and 30℃) and initial nitrogen concentrations (3, 9, 18 mM) were investigated, and its adaptive mechanisms of tolerance to low temperature and nitrogen stress were analysis by proteomics. Results: The optimum temperature range for the growth of X. hormidioides was between 15℃ and 20℃, and the algal cells had slow growth rate at 5℃ and could not survive at 30℃. The maximum biomass concentration was 11.73 g L-1 under the temperature of 20℃, and the highest total lipid content was 56.63% of dry weight. Low temperature did not change the fatty acids profiles but promoted the accumulation of unsaturated fatty acids of X. hormidioides. The maximum contents of palmitoleic acid, eicosapentaenoic acid and total fatty acid were 23.64%, 2.49% and 41.14% of dry weight, respectively. Proteomics was performed under three temperature (7、15、25℃), two nitrogen concentrations (3 and 18 mM) and two cultivation times (day3 and 12). A total of 6503 proteins were identified. In the low temperature, photosynthesis related proteins were down-regulation to protect the photosynthetic apparatus. The up-regulation of key enzymes DGAT and PDAT demonstrated the accumulation of TAGs under low nitrogen treatment. The proteins related to ribosome, phosphatidylinositol signaling system, antioxidant system and cold shock proteins (CSPs) in X. hormidioides were co-up-regulate under the treatment of low temperature, which can alleviate the damages induced by temperature stress and maintain the normal growth and metabolism of algal cells. Conclusions: X. hormidioides is a psychrotolerant microalga. It is an oleaginous filamentous microalga containing hyper palmitoleic acid and a certain amount of eicosapentaenoic acid with great potential for biofuel development, as well as for applications in nutritional health products and other industries.
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 Experiment Overall Design: 28 samples were used in this experiment