#METABOLOMICS WORKBENCH lerland_20191220_161535 DATATRACK_ID:1881 STUDY_ID:ST001296 ANALYSIS_ID:AN002157
VERSION                          	1
CREATED_ON                       	03-03-2022
#PROJECT
PR:PROJECT_TITLE                 	Metabolomics and Hormonomics to Crack the Code of Filbert Growth
PR:PROJECT_TYPE                  	Plant Untargeted MS Metabolomics
PR:PROJECT_SUMMARY               	Introduction: Plants respond to changes in their environments through hormonal
PR:PROJECT_SUMMARY               	activation of a physiological cascade that redirects metabolic resources and
PR:PROJECT_SUMMARY               	growth. In filberts (Corylus sp.), chelated iron promotes the growth of new
PR:PROJECT_SUMMARY               	shoots but the mechanism(s) are not understood. Objectives: To use untargeted
PR:PROJECT_SUMMARY               	metabolomics and hormonomics approaches to generate novel hypotheses for the
PR:PROJECT_SUMMARY               	morphoregulatory role of ferric ethylenediamine-N,N'-di-(ortho-hydroxyphenyl)
PR:PROJECT_SUMMARY               	acetic acid (Fe-EDDHA) in filbert shoot organogenesis in vitro. Methods: Data
PR:PROJECT_SUMMARY               	were generated using previously optimized standardized untargeted metabolomics
PR:PROJECT_SUMMARY               	protocols with time of flight mass spectrometry. Multivariate statistical tools
PR:PROJECT_SUMMARY               	(principal component and partial least squares discriminant analysis) did not
PR:PROJECT_SUMMARY               	detect significant differences. Discovery tools Significance Analysis of
PR:PROJECT_SUMMARY               	Microarrays (SAM), multiple linear regression analysis, Bayesian analysis,
PR:PROJECT_SUMMARY               	logical algorithms, machine learning, synthetic biotransformations, targeted
PR:PROJECT_SUMMARY               	hormonomics, and online resources including MetaboAnalyst were used. Results:
PR:PROJECT_SUMMARY               	Starch/sucrose metabolism and shikimate pathway metabolites were increased. Dose
PR:PROJECT_SUMMARY               	dependent decreases were found in polyphenol metabolism, specifically ellagic
PR:PROJECT_SUMMARY               	acid and its methylated derivative 3,4,3'-tri-O-methylellagic acid. Hormonomics
PR:PROJECT_SUMMARY               	analysis revealed significant differences in phytohormones and their conjugates.
PR:PROJECT_SUMMARY               	FeEDDHA treatment reduced indole-3-acetic acid, abscisic acid, salicylic acid,
PR:PROJECT_SUMMARY               	jasmonic acid conjugates (JA-Trp, JA-Ile, OH-JA) and dihydrozeatinglucoside in
PR:PROJECT_SUMMARY               	regenerating explants. Serotonin (5HT) was decreased in FeEDDHA-treated
PR:PROJECT_SUMMARY               	regenerating tissues while the related metabolite melatonin was increased. Eight
PR:PROJECT_SUMMARY               	phenolic conjugates of 5HT and eight catabolites were affected by FeEDDHA
PR:PROJECT_SUMMARY               	indicating that metabolism to sequester, deactivate and metabolize 5HT was
PR:PROJECT_SUMMARY               	induced by Fe(III). Tryptophan was metabolized through kynurenine but not
PR:PROJECT_SUMMARY               	anthranilate. Conclusion: Seven novel hypotheses were generated to guide future
PR:PROJECT_SUMMARY               	studies to understand the regulatory control(s) of shoot organogenesis.
PR:INSTITUTE                     	University of British Columbia
PR:DEPARTMENT                    	Chemistry
PR:LABORATORY                    	PlantSMART
PR:LAST_NAME                     	Murch
PR:FIRST_NAME                    	Susan
PR:ADDRESS                       	3247 University Way
PR:EMAIL                         	susan.murch@ubc.ca
PR:PHONE                         	250-807-9566
PR:DOI                           	http://dx.doi.org/10.21228/M8B382
PR:CONTRIBUTORS                  	Lauren A E Erland, Christina E Turi, Praveen K Saxena, Susan J Murch
#STUDY
ST:STUDY_TITLE                   	Metabolomics and Hormonomics to Crack the Code of Filbert Growth
ST:STUDY_SUMMARY                 	Introduction: Plants respond to changes in their environments through hormonal
ST:STUDY_SUMMARY                 	activation of a physiological cascade that redirects metabolic resources and
ST:STUDY_SUMMARY                 	growth. In filberts (Corylus sp.), chelated iron promotes the growth of new
ST:STUDY_SUMMARY                 	shoots but the mechanism(s) are not understood. Objectives: To use untargeted
ST:STUDY_SUMMARY                 	metabolomics and hormonomics approaches to generate novel hypotheses for the
ST:STUDY_SUMMARY                 	morphoregulatory role of ferric ethylenediamine-N,N'-di-(ortho-hydroxyphenyl)
ST:STUDY_SUMMARY                 	acetic acid (Fe-EDDHA) in filbert shoot organogenesis in vitro. Methods: Data
ST:STUDY_SUMMARY                 	were generated using previously optimized standardized untargeted metabolomics
ST:STUDY_SUMMARY                 	protocols with time of flight mass spectrometry. Multivariate statistical tools
ST:STUDY_SUMMARY                 	(principal component and partial least squares discriminant analysis) did not
ST:STUDY_SUMMARY                 	detect significant differences. Discovery tools Significance Analysis of
ST:STUDY_SUMMARY                 	Microarrays (SAM), multiple linear regression analysis, Bayesian analysis,
ST:STUDY_SUMMARY                 	logical algorithms, machine learning, synthetic biotransformations, targeted
ST:STUDY_SUMMARY                 	hormonomics, and online resources including MetaboAnalyst were used. Results:
ST:STUDY_SUMMARY                 	Starch/sucrose metabolism and shikimate pathway metabolites were increased. Dose
ST:STUDY_SUMMARY                 	dependent decreases were found in polyphenol metabolism, specifically ellagic
ST:STUDY_SUMMARY                 	acid and its methylated derivative 3,4,3'-tri-O-methylellagic acid. Hormonomics
ST:STUDY_SUMMARY                 	analysis revealed significant differences in phytohormones and their conjugates.
ST:STUDY_SUMMARY                 	FeEDDHA treatment reduced indole-3-acetic acid, abscisic acid, salicylic acid,
ST:STUDY_SUMMARY                 	jasmonic acid conjugates (JA-Trp, JA-Ile, OH-JA) and dihydrozeatinglucoside in
ST:STUDY_SUMMARY                 	regenerating explants. Serotonin (5HT) was decreased in FeEDDHA-treated
ST:STUDY_SUMMARY                 	regenerating tissues while the related metabolite melatonin was increased. Eight
ST:STUDY_SUMMARY                 	phenolic conjugates of 5HT and eight catabolites were affected by FeEDDHA
ST:STUDY_SUMMARY                 	indicating that metabolism to sequester, deactivate and metabolize 5HT was
ST:STUDY_SUMMARY                 	induced by Fe(III). Tryptophan was metabolized through kynurenine but not
ST:STUDY_SUMMARY                 	anthranilate. Conclusion: Seven novel hypotheses were generated to guide future
ST:STUDY_SUMMARY                 	studies to understand the regulatory control(s) of shoot organogenesis.
ST:INSTITUTE                     	University of British Columbia
ST:DEPARTMENT                    	Chemistry
ST:LABORATORY                    	PlantSMART
ST:LAST_NAME                     	Murch
ST:FIRST_NAME                    	Susan
ST:ADDRESS                       	3247 University Way
ST:EMAIL                         	susan.murch@ubc.ca
ST:PHONE                         	250-807-9566
ST:SUBMIT_DATE                   	2019-12-20
#SUBJECT
SU:SUBJECT_TYPE                  	Plant
SU:SUBJECT_SPECIES               	Corylus avellana;Corylus americana
SU:TAXONOMY_ID                   	13451;78632
SU:GENOTYPE_STRAIN               	Corylus avellana;Corylus americana(cv Geneva)
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data
SUBJECT_SAMPLE_FACTORS           	-	10_18_2011 M1A	FeEDDHA (µM):-	RAW_FILE_NAME=10_18_2011 M1A.raw
SUBJECT_SAMPLE_FACTORS           	-	10_18_2011 M1B	FeEDDHA (µM):-	RAW_FILE_NAME=10_18_2011 M1B.raw
SUBJECT_SAMPLE_FACTORS           	-	10_18_2011 M1C	FeEDDHA (µM):-	RAW_FILE_NAME=10_18_2011 M1C.raw
SUBJECT_SAMPLE_FACTORS           	-	10_18_2011 M2A	FeEDDHA (µM):230	RAW_FILE_NAME=10_18_2011 M2A.raw
SUBJECT_SAMPLE_FACTORS           	-	10_18_2011 M2B	FeEDDHA (µM):230	RAW_FILE_NAME=10_18_2011 M2B.raw
SUBJECT_SAMPLE_FACTORS           	-	10_18_2011 M2C	FeEDDHA (µM):230	RAW_FILE_NAME=10_18_2011 M2C.raw
SUBJECT_SAMPLE_FACTORS           	-	10_18_2011 M3A	FeEDDHA (µM):460	RAW_FILE_NAME=10_18_2011 M3A.raw
SUBJECT_SAMPLE_FACTORS           	-	10_18_2011 M3B	FeEDDHA (µM):460	RAW_FILE_NAME=10_18_2011 M3B.raw
SUBJECT_SAMPLE_FACTORS           	-	10_18_2011 M3C	FeEDDHA (µM):460	RAW_FILE_NAME=10_18_2011 M3C.raw
SUBJECT_SAMPLE_FACTORS           	-	10_18_2011 M4A	FeEDDHA (µM):690	RAW_FILE_NAME=10_18_2011 M4A.raw
SUBJECT_SAMPLE_FACTORS           	-	10_18_2011 M4B	FeEDDHA (µM):690	RAW_FILE_NAME=10_18_2011 M4B.raw
SUBJECT_SAMPLE_FACTORS           	-	10_18_2011 M4C	FeEDDHA (µM):690	RAW_FILE_NAME=10_18_2011 M4C.raw
#COLLECTION
CO:COLLECTION_SUMMARY            	Cultures were sampled in triplicate on day 35 of the culture period,
CO:SAMPLE_TYPE                   	Plant
#TREATMENT
TR:TREATMENT_SUMMARY             	Filbert (C. avellana L. × C. americana Marshall cv. Geneva; Grimo Nut Nursery,
TR:TREATMENT_SUMMARY             	Niagara-on-the-Lake, ON, Canada) shoot cultures were provided from germplasm
TR:TREATMENT_SUMMARY             	maintained at the Gosling Research Institute for Plant Preservation (GRIPP;
TR:TREATMENT_SUMMARY             	University of Guelph, Guelph, ON). Plantlets were grown in GA-7 vessels
TR:TREATMENT_SUMMARY             	according to methods previously described (Garrison et al. 2013; Latawa et al.
TR:TREATMENT_SUMMARY             	2016). In brief, cultures were grown on semi-solid modified NCGR-COR medium (Yu
TR:TREATMENT_SUMMARY             	and Reed 1995) supplemented with 10 g L-1 myo-inositol, 200 mg L-1 glycine, 100
TR:TREATMENT_SUMMARY             	mg L-1 nicotinic acid, 100 mg L-1 thiamine (PhytoTechnology Laboratories), 17.6
TR:TREATMENT_SUMMARY             	µM benzylaminopurine (BAP; Sigma-Aldrich), 0.014 µM indole-3-butyric acid
TR:TREATMENT_SUMMARY             	(IBA; Sigma-Aldrich), 0.29 µM gibberellic acid (GA3; PhytoTechnology
TR:TREATMENT_SUMMARY             	Laboratories), and 30 g L-1 glucose with 0, 230, 460 and 690 µM Fe-EDDHA
TR:TREATMENT_SUMMARY             	(Sigma, St. Louis, MO) and the pH of the medium was adjusted to 5.7 before
TR:TREATMENT_SUMMARY             	autoclaving at 121 ºC for 20 min. Cultures were maintained in a growth room at
TR:TREATMENT_SUMMARY             	23 ± 2oC with a 16 h photoperiod of 40 µmol m-2 s-1 provided by cool-white
TR:TREATMENT_SUMMARY             	fluorescent lamps (Osram Sylvania Ltd., Mississauga, ON, Canada).
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Cultures were sampled in triplicate on day 35 of the culture period, accurately
SP:SAMPLEPREP_SUMMARY            	weighed (50 mg), and homogenized in 1 mL of 70% ethanol for 30 s (Kontes Pellet
SP:SAMPLEPREP_SUMMARY            	Pestle, Fisher Scientific). Samples were centrifuged (16,000 x g) for 3 min to
SP:SAMPLEPREP_SUMMARY            	settle particulate matter and the supernatant was filtered (0.1 µm,
SP:SAMPLEPREP_SUMMARY            	Ultrafree-MC filtered centrifuge tubes; Millipore, MS, USA) prior to
SP:SAMPLEPREP_SUMMARY            	chromatography.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	Extracts and 70% ethanol blanks (n=3 for each treatment) were separated using a
CH:CHROMATOGRAPHY_SUMMARY        	Waters BEH Acquity C18 (2.1 X 150 mm, 1.7 µm) column with the following
CH:CHROMATOGRAPHY_SUMMARY        	gradient: 0.1% aqueous formic acid:acetonitrile (0.0-25 min, 95:5-5:95 v/v,
CH:CHROMATOGRAPHY_SUMMARY        	25.01-30.0 min, 95:5 v/v). The flow rate was set to 0.25 mL min-1 for 30 mins at
CH:CHROMATOGRAPHY_SUMMARY        	30 ◦C (Waters Acquity UPLC).
CH:INSTRUMENT_NAME               	Waters Acquity
CH:COLUMN_NAME                   	Waters BEH Acquity C18 (2.1 X 150 mm, 1.7 um)
CH:COLUMN_TEMPERATURE            	30
CH:FLOW_GRADIENT                 	0.0 - 10.0 min, : 95:5-5:95 v/v, 10.0-15.0 min, 5:95 v/v, 15.0-20.0min,
CH:FLOW_GRADIENT                 	5:95-95:5 v/v, 20.0-25.0min, 95:5 v/v)
CH:FLOW_RATE                     	0.25 ml/min
CH:SOLVENT_A                     	1% aqueous formic acid
CH:SOLVENT_B                     	acetonitrile
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Waters Micromass QTOF Premier
MS:INSTRUMENT_TYPE               	QTOF
MS:MS_TYPE                       	ESI
MS:MS_COMMENTS                   	A steady flow of leucine enkephalin (Waters 1525 HPLC binary solvent manager, 2
MS:MS_COMMENTS                   	ng mL-1) was used as the internal standard for calibration of the Micromass LCT
MS:MS_COMMENTS                   	Premier series ToF-MS (Waters Inc.). Time of flight mass spectrometry was used
MS:MS_COMMENTS                   	with previously published optimized conditions (Brown, Murch, et al. 2012; Brown
MS:MS_COMMENTS                   	et al. 2012) including: electrospray ionization and positive ion detection in W
MS:MS_COMMENTS                   	mode, mass range of 100-1000 amu and a scan time of 0.1s. Data were collected
MS:MS_COMMENTS                   	with MassLynx V4.1 and exported via MarkerLynx. Data were processed in Excel™
MS:MS_COMMENTS                   	to align retention times and remove multiply charged ions as described
MS:MS_COMMENTS                   	previously (Brown et al. 2012; Brown et al. 2012; Turi and Murch 2013; Turi et
MS:MS_COMMENTS                   	al. 2014).
MS:ION_MODE                      	POSITIVE
MS:MS_RESULTS_FILE               	ST001296_AN002157_Results.txt	UNITS:Peak Intensity	Has m/z:Yes	Has RT:Yes	RT units:Minutes
#END