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MB Sample ID: SA093778
| Local Sample ID: | 6HUKR2 |
| Subject ID: | SU001363 |
| Subject Type: | Plant |
| Subject Species: | Triticum durum |
| Taxonomy ID: | 4567 |
| Genotype Strain: | cv. Opera |
| Age Or Age Range: | 10 days |
| Gender: | Not applicable |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU001363 |
| Subject Type: | Plant |
| Subject Species: | Triticum durum |
| Taxonomy ID: | 4567 |
| Genotype Strain: | cv. Opera |
| Age Or Age Range: | 10 days |
| Gender: | Not applicable |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| 6HUKR2 | SA093778 | FL013422 | 6h | Time |
Collection:
| Collection ID: | CO001358 |
| Collection Summary: | Durum wheat seeds (Triticum durum L. cv. Opera) were germinated in Petri dishes (9 cm ) in a growth chamber at 25°C, 70% humidity and with a photoperiod of 16 : 8 (light : dark) and light intensity of 90 mol m-2 s-1 supplied by a cool white fluorescent lamp (Polylux XL FT8, 55W 8440). Immediately after germination uniform seedlings were transferred to a 4.5 l hydroponic system and grown in a modified Hoagland solution formulated as follows: KNO3 (1 mM); MgSO4 (100 µM); CaSO4 (400 µM); KCl (5 µM); K2SO4 (200 µM); KH2PO4 (175 µM); H3BO3 (2.5 µM); MnSO4 (0.2 µM); ZnSO4 (0.2 µM); NaMoO4 (0.05 µM); CuSO4 (0.05 µM); Fe-EDTA (200 µM). |
| Sample Type: | Plant |
| Storage Conditions: | -80℃ |
| Collection Vials: | 2ml Eppendorf Screwcapped Round Bottom tubes |
| Storage Vials: | 2ml Eppendorf Screwcapped Round Bottom tubes |
| Collection Tube Temp: | 4 C |
| Additives: | None |
Treatment:
| Treatment ID: | TR001378 |
| Treatment Summary: | Seedlings (10 day old) were treated for 0 h (T0), 6 h (T1), 12 h (T2), 24 h (T3) and 96 h (T4) with nitrate (1 mM) and/ or umbelliferone (100 uM) |
| Treatment Protocol Comments: | Durum weath seeds were directly sown in hydroponic systems the 11/March/2018 and allowed to establish for 10 days. Nutritional Regime:Plant were grown in hydroponic solution composed as follow: KNO3 (1 mM); MgSO4 (100 µM); CaSO4 (400 µM); KCl (5 µM); K2SO4 (200 µM); KH h2PO4 (175 µM); H h3BO3 (2.5 µM); MnSO4 (0.2 µM); ZnSO4 (0.2 µM); NaMoO4 (0.05 µM); CuSO4 (0.05 µM); Fe-EDTA (200 µM). Plant Watering Regime: plants were cropped in hydroponic solution daily changed |
| Treatment: | Umbelliferone (Inhibitor: 100 uM), Time (0, 6, 12, 24, 48, 96 h) |
| Treatment Compound: | Umbelliferone (Inhibitor: 100 uM) |
| Treatment Route: | Roots- Hydropony |
| Treatment Dose: | 100 uM |
| Treatment Dosevolume: | Umbelliferone (Inhibitor: 100 uM), Nitrate (1 mM) |
| Treatment Doseduration: | Time (0, 6, 12, 24, 48, 96 h) |
| Treatment Vehicle: | Hoagland Solution |
| Plant Growth Location: | Growth chambers belonging to the University Mediterranea of Reggio Calabria |
| Plant Light Period: | 18h/6h light/dark |
| Plant Humidity: | 60% Relative Humidity |
| Plant Temp: | 25°C |
| Plant Watering Regime: | See protocol comments |
| Plant Nutritional Regime: | See protocol comments |
| Plant Estab Date: | 2018-03-11 |
| Plant Harvest Date: | 2018-03-25 |
| Plant Growth Stage: | Seedlings collected at the formation of second true leaves |
| Plant Metab Quench Method: | Immediately after collection snap frozen in liquid Nitrogen |
| Plant Harvest Method: | Full plants harvested manually (5 plants for replicate) |
| Plant Storage: | Snap frozen and powdered plant material stored at -80°C |
Sample Preparation:
| Sampleprep ID: | SP001371 |
| Sampleprep Summary: | Freshly homogenized (100 mg) plant material were obtained for each biological sample (plant) and replicates. These were transferred to a 2 ml microcentrifuge round bottom screw cap tubes (Eppendorf). Extraction was done by adding 1400 µl of methanol (at -20°C) and vortexing for 10 s after addition of 60 µl ribitol (0.2 mg/ml stock in dH2O) as an internal quantitative standard for the polar phase. Samples were transferred in a thermomixer at 70C and were shaken for 10 min (950 rpm) and were then further centrifuged for 10 min at 11000 g. The supernatants were collected and transferred to glass vials where 750 µl CHCl3 (-20°C) and 1500 µl dH2O (4°C) were sequentially added. All the samples were vortexed for 10 s and then centrifuged for another 15 min at 2200 g. Upper polar phase ((150 µl ) for each replicate were collected, transferred to a 1.5 ml tube and were dried in a vacuum concentrator without heating. Before freezing and storing at -80°C, the tubes were filled with argon and placed in a plastic bag with silica beads (for avoiding moisture and hydration during short-term storage). Before derivatization, stored samples, were placed in a vacuum concentrator for 30 minutes to eliminate any trace of humidity. Then, to the dried samples, 40 µl methoxyamine hydrochloride (20 mg/ml in pyridine) were added and incubated for 2 h in a Thermomixer (950 rpm) at 37 C. Methoxyaminated samples were then silylated by adding 70 µl of MSTFA to the aliquots. Samples were further shaken for 30 min at 37 C. |
| Sampleprep Protocol ID: | NA |
| Sampleprep Protocol Filename: | NA |
| Sampleprep Protocol Comments: | NA |
| Processing Method: | Freshly homogenized (100 mg) plant material were obtained for each biological sample (plant) and replicates. These were transferred to a 2 ml microcentrifuge round bottom screw cap tubes (Eppendorf). Extraction was done by adding 1400 µl of methanol (at -20°C) and vortexing for 10 s after addition of 60 µl ribitol (0.2 mg/ml stock in ddH2O) as an internal quantitative standard for the polar phase. Samples were transferred in a thermomixer at 70C and were shaken for 10 min (950 rpm) and were then further centrifuged for 10 min at 11000 g. The supernatants were collected and transferred to glass vials where 750 µl CHCl3 (-20°C) and 1500 µl ddH2O (4°C) were sequentially added. All the samples were vortexed for 10 s and then centrifuged for another 15 min at 2200 g. Upper polar phase (150 µl ) for each replicate were collected, transferred to a 1.5 ml tube and were dried in a vacuum concentrator without heating. Before freezing and storing at -80°C, the tubes were filled with argon and placed in a plastic bag with silica beads (for avoiding moisture and hydration during short-term storage). |
| Processing Storage Conditions: | On ice |
| Extraction Method: | Freshly homogenized (100 mg) plant material were obtained for each biological sample (plant) and replicates. These were transferred to a 2 ml microcentrifuge round bottom screw cap tubes (Eppendorf). Extraction was done by adding 1400 µl of methanol (at -20°C) and vortexing for 10 s after addition of 60 µl ribitol (0.2 mg/ml stock in ddH2O) as an internal quantitative standard for the polar phase. Samples were transferred in a thermomixer at 70C and were shaken for 10 min (950 rpm) and were then further centrifuged for 10 min at 11000 g. The supernatants were collected and transferred to glass vials where 750 µl CHCl3 (-20°C) and 1500 µl ddH2O (4°C) were sequentially added. All the samples were vortexed for 10 s and then centrifuged for another 15 min at 2200 g. Upper polar phase (150 µl ) for each replicate were collected, transferred to a 1.5 ml tube and were dried in a vacuum concentrator without heating. Before freezing and storing at -80°C, the tubes were filled with argon and placed in a plastic bag with silica beads (for avoiding moisture and hydration during short-term storage). |
| Extract Enrichment: | NA |
| Extract Cleanup: | NA |
| Extract Storage: | 4℃ |
| Sample Resuspension: | Dried for Derivatization |
| Sample Derivatization: | Methoxyaminatin + trimethylsilylation (MSTFA + 1% TMCS) |
| Sample Spiking: | 60 µl ribitol (0.2 mg/ml stock in ddH2O) |
| Subcellular Location: | NA |
Combined analysis:
| Analysis ID | AN002145 |
|---|---|
| Analysis type | MS |
| Chromatography type | GC |
| Chromatography system | Trace GC 1310, Thermo Fisher Scientific |
| Column | TG-5MS |
| MS Type | EI |
| MS instrument type | GC-ITQ |
| MS instrument name | ISQ LT, Thermo Fisher Scientific |
| Ion Mode | POSITIVE |
| Units | Relative Abundance |
Chromatography:
| Chromatography ID: | CH001570 |
| Chromatography Summary: | The derivatized extracts were injected into a TG-5MS capillary column (30 m x 0.25 mm x 0.25 µm) (Thermo Fisher Scientific, Waltham, MA, USA) using a gas chromatograph apparatus (Trace GC 1310, Thermo Fisher Scientific, Waltham, MA, USA) equipped with a single quadrupole mass spectrometer (ISQ LT, Thermo Fisher Scientific, Waltham, Massachusetts, US). Injector and source were set at 250°C and 260°C temperature, respectively. One µl of sample was injected in splitless mode with a helium flow of 1 ml/min using the following programmed temperature: isothermal 5 min at 70 °C followed by a 5°C/ min ramp to 350 °C and a final 5 min heating at 330°C. |
| Methods ID: | NA |
| Instrument Name: | Trace GC 1310, Thermo Fisher Scientific |
| Column Name: | TG-5MS |
| Column Temperature: | programmed temperature: isothermal 5 min at 70 °C followed by a 5°C/ min ramp to 350 °C and a final 5 min heating at 330°C |
| Flow Rate: | 1 ml/min |
| Injection Temperature: | 250 |
| Internal Standard: | Adonitol (0.0002mg/ml of pure water) 60 μL per sample |
| Retention Index: | Alkanes mixture C10-C40 |
| Retention Time: | Alkanes mixture C10-C40 |
| Oven Temperature: | programmed temperature: isothermal 5 min at 70 °C followed by a 5°C/ min ramp to 350 °C and a final 5 min heating at 330°C |
| Transferline Temperature: | 250 |
| Sample Syringe Size: | 10 μL |
| Randomization Order: | YES |
| Chromatography Type: | GC |
MS:
| MS ID: | MS001997 |
| Analysis ID: | AN002145 |
| Instrument Name: | ISQ LT, Thermo Fisher Scientific |
| Instrument Type: | GC-ITQ |
| MS Type: | EI |
| MS Comments: | Mass spectra were recorded in electronic impact (EI) mode at 70 eV, scanning at 40-600 m/z range, scan time 0.2 sec. Mass spectrometric solvent delay was settled as 9 min. |
| Ion Mode: | POSITIVE |
| Fragmentation Method: | EI-MS |
| Helium Flow: | 1 ml/min |
| Ion Source Temperature: | 260 |
| Ionization Energy: | 70 eV |
| Mass Accuracy: | Unit Resolution |
| Precursor Type: | Full scan |
| Source Temperature: | 260 |
| Scan Range Moverz: | 40-600 |
