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MB Sample ID: SA217798

Local Sample ID:2110-C-R2
Subject ID:SU002358
Subject Type:Plant
Subject Species:Hordeum vulgare
Taxonomy ID:4513

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Subject:

Subject ID:SU002358
Subject Type:Plant
Subject Species:Hordeum vulgare
Taxonomy ID:4513

Factors:

Local Sample IDMB Sample IDFactor Level IDLevel ValueFactor Name
2110-C-R2SA217798FL026438HOR 2110 - B1Genotype
2110-C-R2SA217798FL026438control temp_18/22°CCulture_condition

Collection:

Collection ID:CO002351
Collection Summary:Isolated embryos from barley seeds 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:Seeds
Storage Conditions:-80℃

Treatment:

Treatment ID:TR002370
Treatment Summary:Two barley genotypes (Hordeum vulgare L.), the Austrian landrace HOR 2110 (termed “B1”) and the breeding line HOR 4710 (termed “B2”) were used. For each genotype, seedlings were grown in a greenhouse in pots (30 x 30 x 15 cm, 4 plants per pot) at a 23/15 °C day/night cycle until anthesis. After half of the spikes had flowered, plants were kept for another seven days under the same conditions, and then randomly selected and subjected either to control conditions (C, 22/18 °C and regular watering) or elevated temperature (ET, 28/25 °C and regular watering,) or drought (D, 22/18 °C and 15 % field capacity). After harvest, seeds were cleaned, dried at 20 °C and 20 % relative humidity (RH) for eight weeks (for after-ripening), and then stored at 18 °C.

Sample Preparation:

Sampleprep ID:SP002364
Sampleprep Summary:For metabolite profiling, three biological replicates of 12 mg of barley embryos manually dissected from dry mature seeds were ground with mortar and pestle in liquid nitrogen and stored at -80 °C. All steps were performed in 2 ml Safelock Eppendorf tubes. The ground frozen samples (12 mg) were resuspended in 1 ml of frozen (-20°C) Water:Acetonitrile:Isopropanol (2:3:3 v/v/v/) containing Ribitol at 4 mg.L-1 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. 25 µL were collected and dried for 150 min in a SpeedVac. Fiehn et al (the Plant Journal (2008) 53, 691-704).
Processing Storage Conditions:-20℃
Extract Storage:-80℃

Combined analysis:

Analysis ID AN003714
Analysis type MS
Chromatography type GC
Chromatography system Agilent 7890A
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 5975C
Ion Mode POSITIVE
Units nmoles/mg DW and arbitrary/mg DW

Chromatography:

Chromatography ID:CH002751
Chromatography Summary:The instrument was an Agilent 7890A gas chromatograph coupled to an Agilent 5975C mass spectrometer. The column was an Rxi-5SilMS from Restek (30 m with 10 m integraguard column). The liner (Restek # 20994) was changed before each derivatization series. 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 7890A
Column Name:Restek Rxi-5Sil (30m x 0.25mm,0.25m) with 10m precolumn
Flow Rate:0.7 ml/min
Injection Temperature:250°C
Chromatography Type:GC

MS:

MS ID:MS003463
Analysis ID:AN003714
Instrument Name:Agilent 5975C
Instrument Type:Single quadrupole
MS Type:EI
MS Comments: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 as well as TargetSearch. AMDIS, Target Lynx and TargetSearch 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 and principal component analysis) were then made on them. 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
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