VERSION             	1
CREATED_ON             	December 22, 2020, 10:10 am
PR:PROJECT_TITLE                 	A combinatorial action of GmMYB176 and bZIP controls isoflavonoid biosynthesis
PR:PROJECT_TITLE                 	in soybean
PR:PROJECT_TYPE                  	Non-targeted high resolution MS of soybean GmMYB176-bZIP overexpressed hairy
PR:PROJECT_TYPE                  	roots.
PR:PROJECT_SUMMARY               	GmMYB176 is an R1 MYB transcription factor that regulates multiple genes in the
PR:PROJECT_SUMMARY               	isoflavonoid biosynthetic pathway thereby affecting their levels in soybean
PR:PROJECT_SUMMARY               	roots. While GmMYB176 is important for isoflavonoid synthesis, it is not
PR:PROJECT_SUMMARY               	sufficient for the function and requires additional cofactor(s). The aim of this
PR:PROJECT_SUMMARY               	study was to identify how the GmMYB176 protein complex affects the metabolome of
PR:PROJECT_SUMMARY               	soybean hairy roots using non-targeted high resolution mass spectrometry.
PR:INSTITUTE                     	Agriculture and Agri-Food Canada
PR:LAST_NAME                     	Renaud
PR:FIRST_NAME                    	Justin
PR:ADDRESS                       	1391 Sandford Street, London, Ontario, N5V 4T3, Canada
PR:EMAIL                         	justin.renaud@canada.ca
PR:PHONE                         	519-953-6698
ST:STUDY_TITLE                   	A combinatorial action of GmMYB176 and bZIP controls isoflavonoid biosynthesis
ST:STUDY_TITLE                   	in soybean.
ST:STUDY_SUMMARY                 	This study identified how the GmMYB176 protein complex affects the metabolome of
ST:STUDY_SUMMARY                 	soybean hairy roots using non-targeted high resolution mass spectrometry.
ST:INSTITUTE                     	Agriculture and Agri-Food Canada
ST:LAST_NAME                     	Renaud
ST:FIRST_NAME                    	Justin
ST:ADDRESS                       	1391 Sandford Street, London, Ontario, N5V 4T3, Canada
ST:EMAIL                         	justin.renaud@canada.ca
ST:PHONE                         	519-953-6698
ST:NUM_GROUPS                    	2
ST:TOTAL_SUBJECTS                	10
SU:SUBJECT_TYPE                  	Plant
SU:SUBJECT_SPECIES               	Glycine max
SU:TAXONOMY_ID                   	3847
SU:AGE_OR_AGE_RANGE              	6 days
#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           	-	Sa_001	Genotype:Wild-type	RAW_FILE_NAME=Sa_001_pos;Sa_001_neg
SUBJECT_SAMPLE_FACTORS           	-	Sa_002	Genotype:Wild-type	RAW_FILE_NAME=Sa_002_pos;Sa_002_neg
SUBJECT_SAMPLE_FACTORS           	-	Sa_003	Genotype:Wild-type	RAW_FILE_NAME=Sa_003_pos;Sa_003_neg
SUBJECT_SAMPLE_FACTORS           	-	Sa_004	Genotype:Wild-type	RAW_FILE_NAME=Sa_004_pos;Sa_004_neg
SUBJECT_SAMPLE_FACTORS           	-	Sa_005	Genotype:Wild-type	RAW_FILE_NAME=Sa_005_pos;Sa_005_neg
SUBJECT_SAMPLE_FACTORS           	-	Sa_006	Genotype:GmMYB176-GmbZIP5-overexpressed	RAW_FILE_NAME=Sa_006_pos;Sa_006_neg
SUBJECT_SAMPLE_FACTORS           	-	Sa_007	Genotype:GmMYB176-GmbZIP5-overexpressed	RAW_FILE_NAME=Sa_007_pos;Sa_007_neg
SUBJECT_SAMPLE_FACTORS           	-	Sa_008	Genotype:GmMYB176-GmbZIP5-overexpressed	RAW_FILE_NAME=Sa_008_pos;Sa_008_neg
SUBJECT_SAMPLE_FACTORS           	-	Sa_009	Genotype:GmMYB176-GmbZIP5-overexpressed	RAW_FILE_NAME=Sa_009_pos;Sa_009_neg
SUBJECT_SAMPLE_FACTORS           	-	Sa_010	Genotype:GmMYB176-GmbZIP5-overexpressed	RAW_FILE_NAME=Sa_010_pos;Sa_010_neg
CO:COLLECTION_SUMMARY            	Soybean harasoy63 seeds were planted and grown for 6 days. Cotyledons were
CO:COLLECTION_SUMMARY            	collected after 6 days for soybean hairy root transformation. After 21 days,
CO:COLLECTION_SUMMARY            	transgenic soybean hairy roots were collected for metabolomics
CO:SAMPLE_TYPE                   	Plant
TR:TREATMENT_SUMMARY             	No treatment, only effects of genotype compared to wild-type was investigated
SP:SAMPLEPREP_SUMMARY            	For metabolomics analysis, frozen hairy roots were ground with liquid nitrogen
SP:SAMPLEPREP_SUMMARY            	and extracted in methanol:water (80:20, v/v). The samples were sonicated on ice
SP:SAMPLEPREP_SUMMARY            	water bath for 15 min followed by centrifugation at 11,000×g for 10 min at
SP:SAMPLEPREP_SUMMARY            	ambient temperature. The supernatant (350 µL) was dried under nitrogen gas. The
SP:SAMPLEPREP_SUMMARY            	dried pellet was dissolved in 200 µL of 50% methanol containing 10 µg caffeine
SP:SAMPLEPREP_SUMMARY            	as an internal standard and filtered through a 0.45 µm syringe filter.
SP:SAMPLEPREP_PROTOCOL_ID        	jrenaud_SP_Sample_preparation.pdf
CH:CHROMATOGRAPHY_SUMMARY        	An Agilent 1290 HPLC using a Agilent Zorbax RRHD EclipsePlus (2.1 × 50 mm, 1.8
CH:CHROMATOGRAPHY_SUMMARY        	µm) was used to resolve the analytes.
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Agilent 1290
CH:COLUMN_NAME                   	Agilent Zorbax RRHD EclipsePlus (2.1 × 50 mm, 1.8 µm)
CH:FLOW_GRADIENT                 	0 min, 0% B; 0.5 min, 0% B; 3.5 min, 100% B; 6 min, 100% B; 6.5 min, 0% B
CH:FLOW_RATE                     	0.300 uL/min
CH:SOLVENT_A                     	Water + 0.1% Formic acid
CH:SOLVENT_B                     	Acetonitrile + 0.1% Formic acid
CH:SAMPLE_INJECTION              	5 uL
CH:CAPILLARY_VOLTAGE             	ESI+, 3.9kV; ESI-, 3.5 kV
AN:ANALYSIS_TYPE                 	MS
MS:INSTRUMENT_NAME               	Thermo Q Exactive Orbitrap
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	NEGATIVE
MS:MS_COMMENTS                   	Thermo.raw files were converted to .mzml format using Protewizard , with peak
MS:MS_COMMENTS                   	peaking filter applied. Features were detected using the XCMS package with the
MS:MS_COMMENTS                   	centWave method (ppm tolerance 3.0). The signal to noise threshold was set to 5,
MS:MS_COMMENTS                   	noise was set to 5×105 and pre-filter was set to five scans with a minimum
MS:MS_COMMENTS                   	5,000 intensity. Retention time correction was conducted using the obiwarp
MS:MS_COMMENTS                   	method.. Grouping of features was set to those present in at least 0.1% of all
MS:MS_COMMENTS                   	samples (retention time deviation 5 s; m/z width, 0.015). The ‘fillPeaks’
MS:MS_COMMENTS                   	function was used with default settings. Zero values were imputed by 2/3 the
MS:MS_COMMENTS                   	minimum peak area value of a specific feature across all samples. PCA plots were
MS:MS_COMMENTS                   	obtained by log transforming the imputed XCMS peak area values, and ‘pareto’
MS:MS_COMMENTS                   	scaling in Rstudio. Volcano plots were also generated using the imputed XCMS
MS:MS_COMMENTS                   	peak area values. Compounds were identified by accurate mass, comparison of
MS:MS_COMMENTS                   	retention times to authentic standards or by accurate mass and also comparison
MS:MS_COMMENTS                   	of fragmentation patterns to MS/MS databases.
MS:MS_RESULTS_FILE               	ST001634_AN002671_Results.txt	UNITS:peak area	Has m/z:Yes	Has RT:Yes	RT units:Minutes