#METABOLOMICS WORKBENCH sykong15_20230420_081441 DATATRACK_ID:3872 STUDY_ID:ST002571 ANALYSIS_ID:AN004236 PROJECT_ID:PR001658 VERSION 1 CREATED_ON April 20, 2023, 11:41 am #PROJECT PR:PROJECT_TITLE DRMY1 promotes robust morphogenesis by sustaining translation of a hormone PR:PROJECT_TITLE signaling protein PR:PROJECT_TYPE MS quantitative analysis PR:PROJECT_SUMMARY Robustness is the invariant development of phenotype despite environmental PR:PROJECT_SUMMARY changes and genetic perturbations. In the Arabidopsis flower bud, four sepals PR:PROJECT_SUMMARY initiate at robust positions and times and grow to equal size to enclose and PR:PROJECT_SUMMARY protect the inner floral organs. We previously characterized the mutant PR:PROJECT_SUMMARY development related myb-like1 (drmy1), where 3-5 sepals initiate at irregular PR:PROJECT_SUMMARY positions and variable times and grow to different sizes, compromising their PR:PROJECT_SUMMARY protective function. The molecular mechanism underlying this loss of robustness PR:PROJECT_SUMMARY was unclear. Here, we show that drmy1 has reduced TARGET OF RAPAMYCIN (TOR) PR:PROJECT_SUMMARY activity, ribosomal content, and translation. Translation reduction decreases PR:PROJECT_SUMMARY the protein level of ARABIDOPSIS RESPONSE REGULATOR7 (ARR7), a rapidly PR:PROJECT_SUMMARY synthesized and degraded cytokinin signaling inhibitor. The resultant PR:PROJECT_SUMMARY upregulation of cytokinin signaling disrupts the robust positioning of auxin PR:PROJECT_SUMMARY signaling, causing variable sepal initiation. Our work shows that the PR:PROJECT_SUMMARY homeostasis of translation, a ubiquitous cellular process, is crucial for the PR:PROJECT_SUMMARY robust spatiotemporal patterning of organogenesis. PR:INSTITUTE Cornell University PR:DEPARTMENT Plant Biology Section PR:LABORATORY Roeder Lab PR:LAST_NAME Kong PR:FIRST_NAME Shuyao PR:ADDRESS 239 Weill Hall, 526 Campus Road, Ithaca, NY, 14850, USA PR:EMAIL sk3245@cornell.edu PR:PHONE 6072629684 PR:FUNDING_SOURCE Research reported in this publication was supported by the National Institute of PR:FUNDING_SOURCE General Medical Sciences of the National Institutes of Health (NIH) under award PR:FUNDING_SOURCE numbers R01GM134037 (A.H.K.R.), DP5OD023072 (J.O.B.), and R01GM145814 (J.O.B.); PR:FUNDING_SOURCE Cornell Graduate School new student fellowship (S.K.); and in part by a PR:FUNDING_SOURCE Schmittau-Novak Grant from the School of Integrative Plant Science, Cornell PR:FUNDING_SOURCE University (M.Z.). H.G.G. was supported by NIH Director’s New Innovator Award PR:FUNDING_SOURCE (DP2 OD024541-01) and NSF CAREER Award (1652236), NIH R01 Award (R01GM139913), PR:FUNDING_SOURCE and the Koret-UC Berkeley-Tel Aviv University Initiative in Computational PR:FUNDING_SOURCE Biology and Bioinformatics. H.G.G. is also a Chan Zuckerberg Biohub PR:FUNDING_SOURCE Investigator. PR:CONTRIBUTORS Shuyao Kong, Mingyuan Zhu, M. Regina Scarpin, David Pan, Longfei Jia, Ryan E. PR:CONTRIBUTORS Martinez, Simon Alamos, Batthula Vijaya Lakshmi Vadde, Hernan G. Garcia, PR:CONTRIBUTORS Shu-Bing Qian, Jacob O. Brunkard, Adrienne H. K. Roeder #STUDY ST:STUDY_TITLE Quantification of cytokinins in ap1 cal AP1-GR and drmy1 ap1 cal AP1-GR ST:STUDY_TITLE inflorescences using LC-MS ST:STUDY_TYPE Quantification using mass spectrometry ST:STUDY_SUMMARY Robustness is the invariant development of phenotype despite environmental ST:STUDY_SUMMARY changes and genetic perturbations. In the Arabidopsis flower bud, four sepals ST:STUDY_SUMMARY initiate at robust positions and times and grow to equal size to enclose and ST:STUDY_SUMMARY protect the inner floral organs. We previously characterized the mutant ST:STUDY_SUMMARY development related myb-like1 (drmy1), where 3-5 sepals initiate at irregular ST:STUDY_SUMMARY positions and variable times and grow to different sizes, compromising their ST:STUDY_SUMMARY protective function. This loss of robustness was caused by a uniform increase in ST:STUDY_SUMMARY cytokinin signaling, as revealed by the TCS::GFP reporter, in the floral ST:STUDY_SUMMARY meristem before sepal initiation. We hypothesized that the increase in cytokinin ST:STUDY_SUMMARY signaling in drmy1 was due to an increase in the level of cytokinins. To test ST:STUDY_SUMMARY this idea, we extracted cytokinins from induced inflorescences of wild-type (5 ST:STUDY_SUMMARY bio-reps) and drmy1 (6 bio-reps) in ap1 cal AP1-GR background. We measured the ST:STUDY_SUMMARY level of three cytokinin bases, trans-Zeatin (tZ), cis-Zeatin (cZ), and ST:STUDY_SUMMARY isopentenyladenine (iP), and their corresponding nucleosides (tZR, cZR, and ST:STUDY_SUMMARY iPR), using liquid chromatography-mass spectrometry. We found that there was no ST:STUDY_SUMMARY statistically significant differences in cytokinin levels between these ST:STUDY_SUMMARY genotypes, indicating that the increase in cytokinin signaling in the drmy1 ST:STUDY_SUMMARY floral meristem is not due to increased cytokinin levels. ST:INSTITUTE Cornell University ST:DEPARTMENT Plant Biology Section ST:LABORATORY Roeder Lab ST:LAST_NAME Kong ST:FIRST_NAME Shuyao ST:ADDRESS 239 Weill Hall, 526 Campus Road, Ithaca, NY 14853 ST:EMAIL sk3245@cornell.edu ST:PHONE 6072629684 ST:NUM_GROUPS 2 ST:TOTAL_SUBJECTS 11 #SUBJECT SU:SUBJECT_TYPE Plant SU:SUBJECT_SPECIES Arabidopsis thaliana SU:TAXONOMY_ID 3702 SU:GENOTYPE_STRAIN ap1 cal AP1-GR and drmy1 ap1 cal AP1-GR SU:AGE_OR_AGE_RANGE Bolting (40-50 days after germination) #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - ap1calAP1GR_cytokinins_1 Genotype:ap1calAP1GR RAW_FILE_NAME=ap1calAP1GR_cytokinins_1.raw SUBJECT_SAMPLE_FACTORS - ap1calAP1GR_cytokinins_2 Genotype:ap1calAP1GR RAW_FILE_NAME=ap1calAP1GR_cytokinins_2.raw SUBJECT_SAMPLE_FACTORS - ap1calAP1GR_cytokinins_3 Genotype:ap1calAP1GR RAW_FILE_NAME=ap1calAP1GR_cytokinins_3.raw SUBJECT_SAMPLE_FACTORS - ap1calAP1GR_cytokinins_4 Genotype:ap1calAP1GR RAW_FILE_NAME=ap1calAP1GR_cytokinins_4.raw SUBJECT_SAMPLE_FACTORS - ap1calAP1GR_cytokinins_5 Genotype:ap1calAP1GR RAW_FILE_NAME=ap1calAP1GR_cytokinins_5.raw SUBJECT_SAMPLE_FACTORS - drmy1ap1calAP1GR_cytokinins_1 Genotype:drmy1ap1calAP1-GR RAW_FILE_NAME=drmy1ap1calAP1GR_cytokinins_1.raw SUBJECT_SAMPLE_FACTORS - drmy1ap1calAP1GR_cytokinins_2 Genotype:drmy1ap1calAP1-GR RAW_FILE_NAME=drmy1ap1calAP1GR_cytokinins_2.raw SUBJECT_SAMPLE_FACTORS - drmy1ap1calAP1GR_cytokinins_3 Genotype:drmy1ap1calAP1-GR RAW_FILE_NAME=drmy1ap1calAP1GR_cytokinins_3.raw SUBJECT_SAMPLE_FACTORS - drmy1ap1calAP1GR_cytokinins_4 Genotype:drmy1ap1calAP1-GR RAW_FILE_NAME=drmy1ap1calAP1GR_cytokinins_4.raw SUBJECT_SAMPLE_FACTORS - drmy1ap1calAP1GR_cytokinins_5 Genotype:drmy1ap1calAP1-GR RAW_FILE_NAME=drmy1ap1calAP1GR_cytokinins_5.raw SUBJECT_SAMPLE_FACTORS - drmy1ap1calAP1GR_cytokinins_6 Genotype:drmy1ap1calAP1-GR RAW_FILE_NAME=drmy1ap1calAP1GR_cytokinins_6.raw #COLLECTION CO:COLLECTION_SUMMARY When sepals initiated from the floral meristems (on the fourth day after three CO:COLLECTION_SUMMARY daily inductions), inflorescence samples (including inflorescence meristems and CO:COLLECTION_SUMMARY buds under stage 6) were collected and immediately put into liquid nitrogen. CO:COLLECTION_SUMMARY Five samples were collected for ap1 cal 35S::AP1-GR and six for drmy1 ap1 cal CO:COLLECTION_SUMMARY 35S::AP1-GR. CO:SAMPLE_TYPE Plant CO:COLLECTION_METHOD Liquid Nitrogen CO:COLLECTION_FREQUENCY Once CO:VOLUMEORAMOUNT_COLLECTED Around 250 mg per sample CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY The ap1 cal 35S::AP1-GR and drmy1 ap1 cal 35S::AP1-GR plants were grown in soil TR:TREATMENT_SUMMARY under continuous light at 16°C to prevent premature floral induction. After TR:TREATMENT_SUMMARY bolting, plants were induced daily with an aqueous solution containing 10 µM TR:TREATMENT_SUMMARY dexamethasone (Sigma-Aldrich), 0.01% (v/v) ethanol, and 0.015% (v/v) Silwet L-77 TR:TREATMENT_SUMMARY (Rosecare.com). TR:TREATMENT_COMPOUND Dexamethasone TR:TREATMENT_DOSE 10 µM TR:TREATMENT_DOSEDURATION 1 minute daily for 3 days TR:TREATMENT_VEHICLE Solution TR:PLANT_GROWTH_LOCATION In a Percival walk-in growth chamber with fluorescent light bulbs TR:PLANT_LIGHT_PERIOD Continuous light TR:PLANT_HUMIDITY 60% TR:PLANT_TEMP 16 °C TR:PLANT_WATERING_REGIME Daily TR:PLANT_GROWTH_STAGE Bolting TR:PLANT_STORAGE -80 °C #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Samples were ground in liquid nitrogen and twice extracted in methanol : water : SP:SAMPLEPREP_SUMMARY formic acid (15:4:1). 200 pg of BAP per sample was added as an internal control. SP:SAMPLEPREP_SUMMARY Extracts were centrifuged at 14,650 rpm in -4°C for 30 min, and supernatant was SP:SAMPLEPREP_SUMMARY evaporated of methanol and reconstituted in 1% (v/v) acetic acid. Samples were SP:SAMPLEPREP_SUMMARY passed through an Oasis MCX SPE column (Waters 186000252), washed with 1% acetic SP:SAMPLEPREP_SUMMARY acid, washed with methanol, and eluted with 0.35 M ammonia in 70% methanol. SP:SAMPLEPREP_SUMMARY Eluents were evaporated to complete dryness, reconstituted in 5% acetonitrile, SP:SAMPLEPREP_SUMMARY and sent for LC-MS. SP:PROCESSING_STORAGE_CONDITIONS -80℃ SP:EXTRACTION_METHOD Modified Bieleski buffer, methanol : water : formic acid (15:4:1) SP:EXTRACT_ENRICHMENT An Oasis MCX SPE column (Waters 186000252) was used to enrich for cytokinins SP:EXTRACT_STORAGE -80℃ SP:SAMPLE_RESUSPENSION 5% acetonitrile SP:SAMPLE_SPIKING 200 pg of BAP per sample was added as an internal control #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY 1 µl of each sample was injected into a Thermo Fisher Scientific Vanquish CH:CHROMATOGRAPHY_SUMMARY Horizon UHPLC System coupled with a Thermo Q Exactive HF hybrid CH:CHROMATOGRAPHY_SUMMARY quadropole-orbitrap high-resolution mass spectrometer equipped with a HESI ion CH:CHROMATOGRAPHY_SUMMARY source. Samples were separated on a C18 ODS column (AQUITY UPLC BEH C18, CH:CHROMATOGRAPHY_SUMMARY 1.7 μm, 2.1 × 100 mm, Waters), at a flow rate of 0.3 ml/min, with CH:CHROMATOGRAPHY_SUMMARY linear gradients of solvent A (0.1% formic acid) and solvent B (0.1% formic acid CH:CHROMATOGRAPHY_SUMMARY in methanol) according to the following profile: 0 min, 99.0% A + 1.0% B; CH:CHROMATOGRAPHY_SUMMARY 4.0 min, 55.0% A + 45.0% B; 7 min, 30.0% A + 70.0% B; and then with CH:CHROMATOGRAPHY_SUMMARY isocratic conditions: 8 min, 1.0% A + 99.0% B; 12 min, 99.0% CH:CHROMATOGRAPHY_SUMMARY A + 1.0% B. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters Acquity CH:COLUMN_NAME Waters ACQUITY UPLC BEH C18 (100 x 2.1mm,1.7um) CH:SOLVENT_A 100% water; 0.1% formic acid CH:SOLVENT_B 100% methanol; 0.1% formic acid CH:FLOW_GRADIENT 0 min, 99.0% A + 1.0% B; 4.0 min, 55.0% A + 45.0% B; 7 min, 30.0% CH:FLOW_GRADIENT A + 70.0% B; and then with isocratic conditions: 8 min, 1.0% A + 99.0% CH:FLOW_GRADIENT B; 12 min, 99.0% A + 1.0% B CH:FLOW_RATE 0.3 ml/min CH:COLUMN_TEMPERATURE 60 °C CH:METHODS_FILENAME Protocol.PDF CH:INTERNAL_STANDARD BAP CH:PRECONDITIONING Mili-Q H2O #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME Schroeder Lab AN:OPERATOR_NAME Brian Curtis AN:ACQUISITION_DATE 11/29/2022 #MS MS:INSTRUMENT_NAME Thermo Q Exactive HF hybrid Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Cytokinins were detected using the positive ion mode. For tZ, tZR, iP, iPR, and MS:MS_COMMENTS the internal control BAP, peaks were identified from an external standard mix MS:MS_COMMENTS composed of 0.1 µg/ml each of BAP (Alfa Aesar A14678), tZ (Sigma Z0876), tZR MS:MS_COMMENTS (Sigma Z3541), iP (Cayman Chemical 17906), and iPR (Cayman chemical 20522) in 5% MS:MS_COMMENTS acetonitrile. For cZ and cZR, peaks were identified based on previously reported MS:MS_COMMENTS precursor m/z and retention time. Using Xcalibur (Thermo Scientific), peak area MS:MS_COMMENTS was quantified for each cytokinin in each sample, normalized against the peak MS:MS_COMMENTS area of BAP (internal control) and sample fresh weight, and then normalized MS:MS_COMMENTS against the average abundance of tZ in WT samples. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Peak area MS_METABOLITE_DATA_START Samples ap1calAP1GR_cytokinins_1 ap1calAP1GR_cytokinins_2 ap1calAP1GR_cytokinins_3 ap1calAP1GR_cytokinins_4 ap1calAP1GR_cytokinins_5 drmy1ap1calAP1GR_cytokinins_1 drmy1ap1calAP1GR_cytokinins_2 drmy1ap1calAP1GR_cytokinins_3 drmy1ap1calAP1GR_cytokinins_4 drmy1ap1calAP1GR_cytokinins_5 drmy1ap1calAP1GR_cytokinins_6 Factors Genotype:ap1calAP1GR Genotype:ap1calAP1GR Genotype:ap1calAP1GR Genotype:ap1calAP1GR Genotype:ap1calAP1GR Genotype:drmy1ap1calAP1-GR Genotype:drmy1ap1calAP1-GR Genotype:drmy1ap1calAP1-GR Genotype:drmy1ap1calAP1-GR Genotype:drmy1ap1calAP1-GR Genotype:drmy1ap1calAP1-GR 6-benzylaminopurine 385584658 83246054 39033337 132372832 177557397 401130047 115982026 160421998 183067099 98971472 88287065 trans-Zeatin 1716133 874739 275772 1226646 723662 1220409 797487 692453 793464 669812 765451 cis-Zeatin 62200 61568 9339 69469 56568 60070 62216 60122 60000 45810 76144 trans-Zeatin riboside 7064052 2910724 959418 4733385 2127527 16588038 8219521 4034722 5209907 2942599 4150934 cis-Zeatin riboside 2401320 1041915 366361 1443106 872840 1761909 2314636 1195987 1535159 1227279 1232073 isopentenyladenine 636100 348188 63285 381568 279091 433740 527505 420459 522856 463835 2191694 isopentenyladenosine 1306154 634831 144258 903474 512581 1116567 1084024 620301 709810 563960 584653 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Pubchem Id 6-benzylaminopurine 62389 trans-Zeatin 449093 cis-Zeatin 688597 trans-Zeatin riboside 6440982 cis-Zeatin riboside 13935024 isopentenyladenine 92180 isopentenyladenosine 24405 METABOLITES_END #END