Summary of Study ST002571
This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001658. The data can be accessed directly via it's Project DOI: 10.21228/M8CX2C This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.
| Study ID | ST002571 |
| Study Title | Quantification of cytokinins in ap1 cal AP1-GR and drmy1 ap1 cal AP1-GR inflorescences using LC-MS |
| Study Type | Quantification using mass spectrometry |
| Study Summary | Robustness is the invariant development of phenotype despite environmental changes and genetic perturbations. In the Arabidopsis flower bud, four sepals initiate at robust positions and times and grow to equal size to enclose and protect the inner floral organs. We previously characterized the mutant development related myb-like1 (drmy1), where 3-5 sepals initiate at irregular positions and variable times and grow to different sizes, compromising their protective function. This loss of robustness was caused by a uniform increase in cytokinin signaling, as revealed by the TCS::GFP reporter, in the floral meristem before sepal initiation. We hypothesized that the increase in cytokinin signaling in drmy1 was due to an increase in the level of cytokinins. To test this idea, we extracted cytokinins from induced inflorescences of wild-type (5 bio-reps) and drmy1 (6 bio-reps) in ap1 cal AP1-GR background. We measured the level of three cytokinin bases, trans-Zeatin (tZ), cis-Zeatin (cZ), and isopentenyladenine (iP), and their corresponding nucleosides (tZR, cZR, and iPR), using liquid chromatography-mass spectrometry. We found that there was no statistically significant differences in cytokinin levels between these genotypes, indicating that the increase in cytokinin signaling in the drmy1 floral meristem is not due to increased cytokinin levels. |
| Institute | Cornell University |
| Department | Plant Biology Section |
| Laboratory | Roeder Lab |
| Last Name | Kong |
| First Name | Shuyao |
| Address | 239 Weill Hall, 526 Campus Road, Ithaca, NY 14853 |
| sk3245@cornell.edu | |
| Phone | 6072629684 |
| Submit Date | 2023-04-20 |
| Num Groups | 2 |
| Total Subjects | 11 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | raw(Thermo) |
| Analysis Type Detail | LC-MS |
| Release Date | 2023-05-11 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR001658 |
| Project DOI: | doi: 10.21228/M8CX2C |
| Project Title: | DRMY1 promotes robust morphogenesis by sustaining translation of a hormone signaling protein |
| Project Type: | MS quantitative analysis |
| Project Summary: | Robustness is the invariant development of phenotype despite environmental changes and genetic perturbations. In the Arabidopsis flower bud, four sepals initiate at robust positions and times and grow to equal size to enclose and protect the inner floral organs. We previously characterized the mutant development related myb-like1 (drmy1), where 3-5 sepals initiate at irregular positions and variable times and grow to different sizes, compromising their protective function. The molecular mechanism underlying this loss of robustness was unclear. Here, we show that drmy1 has reduced TARGET OF RAPAMYCIN (TOR) activity, ribosomal content, and translation. Translation reduction decreases the protein level of ARABIDOPSIS RESPONSE REGULATOR7 (ARR7), a rapidly synthesized and degraded cytokinin signaling inhibitor. The resultant upregulation of cytokinin signaling disrupts the robust positioning of auxin signaling, causing variable sepal initiation. Our work shows that the homeostasis of translation, a ubiquitous cellular process, is crucial for the robust spatiotemporal patterning of organogenesis. |
| Institute: | Cornell University |
| Department: | Plant Biology Section |
| Laboratory: | Roeder Lab |
| Last Name: | Kong |
| First Name: | Shuyao |
| Address: | 239 Weill Hall, 526 Campus Road, Ithaca, NY, 14850, USA |
| Email: | sk3245@cornell.edu |
| Phone: | 6072629684 |
| Funding Source: | Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health (NIH) under award numbers R01GM134037 (A.H.K.R.), DP5OD023072 (J.O.B.), and R01GM145814 (J.O.B.); Cornell Graduate School new student fellowship (S.K.); and in part by a Schmittau-Novak Grant from the School of Integrative Plant Science, Cornell University (M.Z.). H.G.G. was supported by NIH Director’s New Innovator Award (DP2 OD024541-01) and NSF CAREER Award (1652236), NIH R01 Award (R01GM139913), and the Koret-UC Berkeley-Tel Aviv University Initiative in Computational Biology and Bioinformatics. H.G.G. is also a Chan Zuckerberg Biohub Investigator. |
| Contributors: | Shuyao Kong, Mingyuan Zhu, M. Regina Scarpin, David Pan, Longfei Jia, Ryan E. Martinez, Simon Alamos, Batthula Vijaya Lakshmi Vadde, Hernan G. Garcia, Shu-Bing Qian, Jacob O. Brunkard, Adrienne H. K. Roeder |
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:
Subject type: Plant; Subject species: Arabidopsis thaliana (Factor headings shown in green)
| mb_sample_id | local_sample_id | Genotype |
|---|---|---|
| SA258348 | ap1calAP1GR_cytokinins_1 | ap1calAP1GR |
| SA258349 | ap1calAP1GR_cytokinins_5 | ap1calAP1GR |
| SA258350 | ap1calAP1GR_cytokinins_4 | ap1calAP1GR |
| SA258351 | ap1calAP1GR_cytokinins_2 | ap1calAP1GR |
| SA258352 | ap1calAP1GR_cytokinins_3 | ap1calAP1GR |
| SA258353 | drmy1ap1calAP1GR_cytokinins_6 | drmy1ap1calAP1-GR |
| SA258354 | drmy1ap1calAP1GR_cytokinins_5 | drmy1ap1calAP1-GR |
| SA258355 | drmy1ap1calAP1GR_cytokinins_1 | drmy1ap1calAP1-GR |
| SA258356 | drmy1ap1calAP1GR_cytokinins_2 | drmy1ap1calAP1-GR |
| SA258357 | drmy1ap1calAP1GR_cytokinins_3 | drmy1ap1calAP1-GR |
| SA258358 | drmy1ap1calAP1GR_cytokinins_4 | drmy1ap1calAP1-GR |
| Showing results 1 to 11 of 11 |
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 |
