#METABOLOMICS WORKBENCH jrenaud86_20201217_070555 DATATRACK_ID:2362 STUDY_ID:ST001634 ANALYSIS_ID:AN002670 PROJECT_ID:PR001045 VERSION 1 CREATED_ON December 22, 2020, 10:10 am #PROJECT 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 #STUDY 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 #SUBJECT 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 #COLLECTION 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 #TREATMENT TR:TREATMENT_SUMMARY No treatment, only effects of genotype compared to wild-type was investigated #SAMPLEPREP 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 #CHROMATOGRAPHY 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:COLUMN_TEMPERATURE 35 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 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE 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_AN002670_Results.txt UNITS:peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END