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MB Sample ID: SA211291
| Local Sample ID: | Tsu-0_W+N+_P3ID34_a |
| Subject ID: | SU002287 |
| Subject Type: | Plant |
| Subject Species: | Arabidopsis thaliana |
| Taxonomy ID: | 3702 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002287 |
| Subject Type: | Plant |
| Subject Species: | Arabidopsis thaliana |
| Taxonomy ID: | 3702 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| Tsu-0_W+N+_P3ID34_a | SA211291 | FL025762 | Tsu-0 | Genotype |
| Tsu-0_W+N+_P3ID34_a | SA211291 | FL025762 | W+ | Drought |
| Tsu-0_W+N+_P3ID34_a | SA211291 | FL025762 | N+ | Nitrogen |
Collection:
| Collection ID: | CO002280 |
| Collection Summary: | Arabidopsis shoots were harvested and inserted in a previously weighed eppendorf tubes, frozen in liquid nitrogen and weighed fastly again. They were grinded by shaking with a metal ball. |
| Sample Type: | Plant |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR002299 |
| Treatment Summary: | We applied a combination of soil water content (SWC; adjusted with respect to complete soil saturation) and nutrient solution (adjusted to provide a specific amount of nitrate over the course of the experiment : 160.45 ± 7.96 ml of 5mM [Nitrate] nutrient solution in total for the control condition W+N+; 50.85 ± 4.67 ml of 5mM [Nitrate] in total for the single drought stress condition W-N+; 161.93 ± 11.07 ml of 0.5mM [Nitrate] in total for the single N-deficiency condition W+N-; 50.70 ± 4.10 ml of 1.5mM [Nitrate] in total for the combined stress condition W-N-) to establish the different conditions |
Sample Preparation:
| Sampleprep ID: | SP002293 |
| Sampleprep Summary: | All steps were performed in 2 ml Safelock Eppendorf tubes. The ground frozen samples (25 mg) were resuspended in 1 ml of frozen (-20°C) Water:Acetonitrile:Isopropanol (2:3:3) containing Ribitol at 4 µg/ml and extracted for 10 min at 4°C with shaking at 1400 rpm in an Eppendorf Thermomixer. Insoluble material was removed by centrifugation at 20000g for 5 min. 66 µl were collected and dried overnight at 35 °C in a Speed-Vac . Fiehn et al (the Plant Journal (2008) 53, 691-704). |
| Processing Storage Conditions: | -20℃ |
| Extract Storage: | -80℃ |
Combined analysis:
| Analysis ID | AN003604 |
|---|---|
| Analysis type | MS |
| Chromatography type | GC |
| Chromatography system | Agilent 7890B |
| Column | Restek Rxi-5Sil (30m x 0.25mm,0.25m) with 10m precolumn |
| MS Type | EI |
| MS instrument type | Single quadrupole |
| MS instrument name | Agilent 5977B |
| Ion Mode | POSITIVE |
| Units | µg/mg FW and arbitrary/mg FW |
Chromatography:
| Chromatography ID: | CH002664 |
| Chromatography Summary: | The instrument was an Agilent 7890B gas chromatograph coupled to an Agilent 5977B mass spectrometer. The column was an Rxi-5SilMS from Restek (30 m with 10 m integraguard column). The liner (Restek # 20994) was changed before the analysis. Oven temperature ramp was 70 °C for 7 min then 10 °C/min to 330 °C for 5 min (run length 38 min). Helium constant flow was 0.7 mL/min. Temperatures were the following: injector: 250°C, transfer line: 290°C, source: 250 °C and quadripole 150 °C. 5 scans per second were acquired spanning a 50 to 600 Da range. Instrument was tuned with PFTBA with the 69 m/z and 219 m/z of equal intensities. 5 scans per second were acquired. The split mode conditions were: 70°C for 2 min then 30°C per min to 330 °C for 5 min. Helium constant flow 1 mL/min. |
| Instrument Name: | Agilent 7890B |
| Column Name: | Restek Rxi-5Sil (30m x 0.25mm,0.25m) with 10m precolumn |
| Chromatography Type: | GC |
MS:
| MS ID: | MS003359 |
| Analysis ID: | AN003604 |
| Instrument Name: | Agilent 5977B |
| Instrument Type: | Single quadrupole |
| MS Type: | EI |
| MS Comments: | The instrument was an Agilent 7890B gas chromatograph coupled to an Agilent 5977B mass spectrometer. The column was an Rxi-5SilMS from Restek (30 m with 10 m integraguard column). The liner (Restek # 20994) was changed before the analysis. Oven temperature ramp was 70 °C for 7 min then 10 °C/min to 330 °C for 5 min (run length 38 min). Helium constant flow was 0.7 mL/min. Temperatures were the following: injector: 250°C, transfer line: 290°C, source: 250 °C and quadripole 150 °C. 5 scans per second were acquired spanning a 50 to 600 Da range. Instrument was tuned with PFTBA with the 69 m/z and 219 m/z of equal intensities. 5 scans per second were acquired. The split mode conditions were: 70°C for 2 min then 30°C per min to 330 °C for 5 min. Helium constant flow 1 mL/min.Data processing: Raw Agilent datafiles were converted in NetCDF format and analyzed with AMDIS http://chemdata.nist.gov/mass-spc/amdis/. An home retention indices/ mass spectra library built from the NIST, Golm , http://gmd.mpimp-golm.mpg.de/ and Fiehn databases and standard compounds was used for metabolites identification. Peak areas were also determined with the Targetlynx software (Waters) after conversion of the NetCDF file in masslynx format. AMDIS, Target Lynx in splitless and split 30 modes data were compiled in one single Excel File for comparison. After blank mean substraction peak areas were normalized to Ribitol and Fresh Weight. Statistical analysis was made with TMEV http://www.tm4.org/mev.html : univariate analysis by permutation (1way-anova and 2-way anova) were firstly used to select the significant metabolites (P-value < 0.01). Multivariate analysis (hierarchical clustering an principal component analysis) were then made on them. Mapman http://www.gabipd.org/projects/MapMan/ was used for graphical representation of the metabolic changes after Log2 transformation of the mean of the 3 replicates. Absolute quantification: A response coefficient was determined for 4 ng each of a set of 103 métabolites, respectively to the same amount of ribitol. This factor was used to give an estimation of the absolute concentration of the metabolite in what we may call a “one point calibration”. Metabolites rich in nitrogen (basic aminoacids and polyamines) gave several analytes (up to 5 for glutamine and asparagine). The peak area as TIC equivalent of these analytes were summed to express the contents of these metabolites. They are referred to “sum” in the tables. |
| Ion Mode: | POSITIVE |
| Helium Flow: | 0.7 ml/min |
| Ion Source Temperature: | 250 |
| Ionization: | EI |
| Source Temperature: | 250 |
