#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