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MB Sample ID: SA258352
| Local Sample ID: | ap1calAP1GR_cytokinins_3 |
| Subject ID: | SU002672 |
| Subject Type: | Plant |
| Subject Species: | Arabidopsis thaliana |
| Taxonomy ID: | 3702 |
| Genotype Strain: | ap1 cal AP1-GR and drmy1 ap1 cal AP1-GR |
| Age Or Age Range: | Bolting (40-50 days after germination) |
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Subject:
| Subject ID: | SU002672 |
| Subject Type: | Plant |
| Subject Species: | Arabidopsis thaliana |
| Taxonomy ID: | 3702 |
| Genotype Strain: | ap1 cal AP1-GR and drmy1 ap1 cal AP1-GR |
| Age Or Age Range: | Bolting (40-50 days after germination) |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| ap1calAP1GR_cytokinins_3 | SA258352 | FL033156 | ap1calAP1GR | Genotype |
Collection:
| Collection ID: | CO002665 |
| Collection Summary: | When sepals initiated from the floral meristems (on the fourth day after three daily inductions), inflorescence samples (including inflorescence meristems and buds under stage 6) were collected and immediately put into liquid nitrogen. Five samples were collected for ap1 cal 35S::AP1-GR and six for drmy1 ap1 cal 35S::AP1-GR. |
| Sample Type: | Plant |
| Collection Method: | Liquid Nitrogen |
| Collection Frequency: | Once |
| Volumeoramount Collected: | Around 250 mg per sample |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR002684 |
| Treatment Summary: | The ap1 cal 35S::AP1-GR and drmy1 ap1 cal 35S::AP1-GR plants were grown in soil under continuous light at 16°C to prevent premature floral induction. After bolting, plants were induced daily with an aqueous solution containing 10 µM dexamethasone (Sigma-Aldrich), 0.01% (v/v) ethanol, and 0.015% (v/v) Silwet L-77 (Rosecare.com). |
| Treatment Compound: | Dexamethasone |
| Treatment Dose: | 10 µM |
| Treatment Doseduration: | 1 minute daily for 3 days |
| Treatment Vehicle: | Solution |
| Plant Growth Location: | In a Percival walk-in growth chamber with fluorescent light bulbs |
| Plant Light Period: | Continuous light |
| Plant Humidity: | 60% |
| Plant Temp: | 16 °C |
| Plant Watering Regime: | Daily |
| Plant Growth Stage: | Bolting |
| Plant Storage: | -80 °C |
Sample Preparation:
| Sampleprep ID: | SP002678 |
| Sampleprep Summary: | Samples were ground in liquid nitrogen and twice extracted in methanol : water : formic acid (15:4:1). 200 pg of BAP per sample was added as an internal control. Extracts were centrifuged at 14,650 rpm in -4°C for 30 min, and supernatant was evaporated of methanol and reconstituted in 1% (v/v) acetic acid. Samples were passed through an Oasis MCX SPE column (Waters 186000252), washed with 1% acetic acid, washed with methanol, and eluted with 0.35 M ammonia in 70% methanol. Eluents were evaporated to complete dryness, reconstituted in 5% acetonitrile, and sent for LC-MS. |
| Processing Storage Conditions: | -80℃ |
| Extraction Method: | Modified Bieleski buffer, methanol : water : formic acid (15:4:1) |
| Extract Enrichment: | An Oasis MCX SPE column (Waters 186000252) was used to enrich for cytokinins |
| Extract Storage: | -80℃ |
| Sample Resuspension: | 5% acetonitrile |
| Sample Spiking: | 200 pg of BAP per sample was added as an internal control |
Combined analysis:
| Analysis ID | AN004236 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Waters Acquity |
| Column | Waters ACQUITY UPLC BEH C18 (100 x 2.1mm,1.7um) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive HF hybrid Orbitrap |
| Ion Mode | POSITIVE |
| Units | Peak area |
Chromatography:
| Chromatography ID: | CH003143 |
| Chromatography Summary: | 1 µl of each sample was injected into a Thermo Fisher Scientific Vanquish Horizon UHPLC System coupled with a Thermo Q Exactive HF hybrid quadropole-orbitrap high-resolution mass spectrometer equipped with a HESI ion source. Samples were separated on a C18 ODS column (AQUITY UPLC BEH C18, 1.7 μm, 2.1 × 100 mm, Waters), at a flow rate of 0.3 ml/min, with linear gradients of solvent A (0.1% formic acid) and solvent B (0.1% formic acid in methanol) according to the following profile: 0 min, 99.0% A + 1.0% B; 4.0 min, 55.0% A + 45.0% B; 7 min, 30.0% A + 70.0% B; and then with isocratic conditions: 8 min, 1.0% A + 99.0% B; 12 min, 99.0% A + 1.0% B. |
| Methods Filename: | Protocol_SK.PDF |
| Instrument Name: | Waters Acquity |
| Column Name: | Waters ACQUITY UPLC BEH C18 (100 x 2.1mm,1.7um) |
| Column Temperature: | 60 °C |
| Flow Gradient: | 0 min, 99.0% A + 1.0% B; 4.0 min, 55.0% A + 45.0% B; 7 min, 30.0% A + 70.0% B; and then with isocratic conditions: 8 min, 1.0% A + 99.0% B; 12 min, 99.0% A + 1.0% B |
| Flow Rate: | 0.3 ml/min |
| Internal Standard: | BAP |
| Solvent A: | 100% water; 0.1% formic acid |
| Solvent B: | 100% methanol; 0.1% formic acid |
| Preconditioning: | Mili-Q H2O |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS003983 |
| Analysis ID: | AN004236 |
| Instrument Name: | Thermo Q Exactive HF hybrid Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Cytokinins were detected using the positive ion mode. For tZ, tZR, iP, iPR, and the internal control BAP, peaks were identified from an external standard mix composed of 0.1 µg/ml each of BAP (Alfa Aesar A14678), tZ (Sigma Z0876), tZR (Sigma Z3541), iP (Cayman Chemical 17906), and iPR (Cayman chemical 20522) in 5% acetonitrile. For cZ and cZR, peaks were identified based on previously reported precursor m/z and retention time. Using Xcalibur (Thermo Scientific), peak area was quantified for each cytokinin in each sample, normalized against the peak area of BAP (internal control) and sample fresh weight, and then normalized against the average abundance of tZ in WT samples. |
| Ion Mode: | POSITIVE |
