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MB Sample ID: SA301636
| Local Sample ID: | 583_04 |
| Subject ID: | SU002917 |
| Subject Type: | Plant |
| Subject Species: | Arabidopsis thaliana |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002917 |
| Subject Type: | Plant |
| Subject Species: | Arabidopsis thaliana |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| 583_04 | SA301636 | FL036610 | GAS2 | Treatment |
Collection:
| Collection ID: | CO002910 |
| Collection Summary: | Recombinant GAS2 and AtGA20ox1 were produced as previously described [5,6]. [5] Pimenta Lange, M. J. et al. Functional characterization of gibberellin oxidases from cucumber, Cucumis sativus L. Phytochemistry 90, 62-69 (2013). [6] Pimenta Lange, M. J. et al. Cucumber gibberellin 1-oxidase/desaturase initiates novel gibberellin catabolic pathways. J. Biol. Chem. 295, 8442-8448 (2020). |
| Sample Type: | Bacterial cells |
Treatment:
| Treatment ID: | TR002926 |
| Treatment Summary: | Recombinant GAS2 and AtGA20ox1 were produced as previously described [5,6]. [5] Pimenta Lange, M. J. et al. Functional characterization of gibberellin oxidases from cucumber, Cucumis sativus L. Phytochemistry 90, 62-69 (2013). [6] Pimenta Lange, M. J. et al. Cucumber gibberellin 1-oxidase/desaturase initiates novel gibberellin catabolic pathways. J. Biol. Chem. 295, 8442-8448 (2020). |
Sample Preparation:
| Sampleprep ID: | SP002923 |
| Sampleprep Summary: | 3’,5’,5’,7’,7’,7’-d6-labelled ABA and 17,17-d2-labelled GA12 were purchased from OlChemIm, Czech Republic. PA and 7’,7’,7’-d3-PA were gifts from Professor Eiji Nambara (University of Toronto, Canada). Preparations of E. coli cell lysates were incubated in a total volume of 100 µl containing 100 mM Tris-HCl, pH 7.0 at 30°C for 16 h with 2-oxoglutarate and ascorbate (100mM each, final concentrations), FeSO4 (0.5 mM), catalase (1mg/ml), and the substrates (5 µl in methanol for ABA, 3’,5’,5’,7’,7’,7’-d6-labeled ABA (500 ng), PA (500 ng), and 7’,7’,7’-d3-labeled PA (50 ng), and 2 µl in methanol per 5 ng 17,17-d2-labelled GA12. Variations to the standard incubation conditions are indicated in the individual experiments. Incubation products were extracted and analyzed by reverse-phase HPLC as described previously12 using gradients of increasing methanol in water, containing 1% acetic acid, at 1 ml.min-1 as follows: 50% methanol, followed by five 0.5-min steps to 57.4%, 60.6%, 61.8%, 62.3%, 62.5%, one 5-min step to 62.7%, one 1-min step to 63.2% and seven 2-min steps to 64.3%, 67.2%, 70.5%, 74.9%, 81%, 89% and 100% methanol. PA and compound A eluted between 3 and 6 min, and ABA between 6 and 9 min. The dried HPLC-fractions were redissolved in 100 µl methanol and methylated with 100 µl ethereal diazomethane. |
Combined analysis:
| Analysis ID | AN004570 |
|---|---|
| Analysis type | MS |
| Chromatography type | GC |
| Chromatography system | Thermo Scientific Trace 1300 |
| Column | Agilent DB5-MS (30m x 0.25mm, 0.25um) |
| MS Type | EI |
| MS instrument type | Single quadrupole |
| MS instrument name | Thermo Scientific ISQ 7000 |
| Ion Mode | POSITIVE |
| Units | Peak area |
Chromatography:
| Chromatography ID: | CH003435 |
| Instrument Name: | Thermo Scientific Trace 1300 |
| Column Name: | Agilent DB5-MS (30m x 0.25mm, 0.25um) |
| Column Temperature: | 60-280 |
| Flow Gradient: | 60-280 |
| Flow Rate: | 1.2 ml/min |
| Solvent A: | Helium |
| Solvent B: | Helium |
| Chromatography Type: | GC |
MS:
| MS ID: | MS004316 |
| Analysis ID: | AN004570 |
| Instrument Name: | Thermo Scientific ISQ 7000 |
| Instrument Type: | Single quadrupole |
| MS Type: | EI |
| MS Comments: | ThermoScientific ISQ7000 with Advanced Electron Ionization (AEI) source. Software used was Chromeleon 7.2.10 |
| Ion Mode: | POSITIVE |
