#METABOLOMICS WORKBENCH AParrish_20210924_133329 DATATRACK_ID:2864 STUDY_ID:ST001934 ANALYSIS_ID:AN003144 PROJECT_ID:PR001223 VERSION 1 CREATED_ON September 24, 2021, 1:56 pm #PROJECT PR:PROJECT_TITLE Differential Accumulation of Metabolites and Transcripts Related to Flavonoid, PR:PROJECT_TITLE Styrylpyrone, and Galactolipid Biosynthesis in Equisetum Species and Tissue Types PR:PROJECT_SUMMARY Members of the genus Equisetum are often referred to as “living fossils”, PR:PROJECT_SUMMARY partly because they are the only extant representatives of the Equisetidae, a PR:PROJECT_SUMMARY subclass that was once prominent in late Paleozoic forests. Several classes of PR:PROJECT_SUMMARY specialized metabolites have been reported to occur in the genus Equisetum. PR:PROJECT_SUMMARY However, while steady progress is being made with identifying individual novel PR:PROJECT_SUMMARY metabolites of Equisetum, few if any analyses have focused on assessing the PR:PROJECT_SUMMARY chemical diversity across the genus. The present study focused on three species: PR:PROJECT_SUMMARY E. hyemale subsp. affine (rough horsetail or scouring rush), which is native to PR:PROJECT_SUMMARY the temperate to artic portions of North America; E. arvense (common horsetail), PR:PROJECT_SUMMARY which is endemic to the arctic and temperate regions of the northern hemisphere; PR:PROJECT_SUMMARY and Equisetum telmateia subsp. braunii (Milde) Hauke (giant horsetail), which is PR:PROJECT_SUMMARY native to western North America. Both below-ground rhizome and above-ground PR:PROJECT_SUMMARY shoot material was harvested from each species, extracted with aqueous methanol, PR:PROJECT_SUMMARY and subjected to non-targeted HPLC-QTOF-MS analysis. This research project was PR:PROJECT_SUMMARY designed to lay the foundation for continued research to capture the metabolic PR:PROJECT_SUMMARY capabilities in the ferns and fern allies. PR:INSTITUTE Washington State University PR:DEPARTMENT Institute of Biological Chemistry PR:LABORATORY Lange PR:LAST_NAME Lange PR:FIRST_NAME Mark PR:ADDRESS Plant Sciences Building, Pullman, Washington 99164 PR:EMAIL lange-m@wsu.edu PR:PHONE +1-509-335-3794 #STUDY ST:STUDY_TITLE Differential Accumulation of Metabolites and Transcripts Related to Flavonoid, ST:STUDY_TITLE Styrylpyrone, and Galactolipid Biosynthesis in Equisetum Species and Tissue Types ST:STUDY_SUMMARY Members of the genus Equisetum are often referred to as “living fossils”, ST:STUDY_SUMMARY partly because they are the only extant representatives of the Equisetidae, a ST:STUDY_SUMMARY subclass that was once prominent in late Paleozoic forests. Several classes of ST:STUDY_SUMMARY specialized metabolites have been reported to occur in the genus Equisetum. ST:STUDY_SUMMARY However, while steady progress is being made with identifying individual novel ST:STUDY_SUMMARY metabolites of Equisetum, few if any analyses have focused on assessing the ST:STUDY_SUMMARY chemical diversity across the genus. The present study focused on three species: ST:STUDY_SUMMARY E. hyemale subsp. affine (rough horsetail or scouring rush), which is native to ST:STUDY_SUMMARY the temperate to artic portions of North America; E. arvense (common horsetail), ST:STUDY_SUMMARY which is endemic to the arctic and temperate regions of the northern hemisphere; ST:STUDY_SUMMARY and Equisetum telmateia subsp. braunii (Milde) Hauke (giant horsetail), which is ST:STUDY_SUMMARY native to western North America. Both below-ground rhizome and above-ground ST:STUDY_SUMMARY shoot material was harvested from each species, extracted with aqueous methanol, ST:STUDY_SUMMARY and subjected to non-targeted HPLC-QTOF-MS analysis. This research project was ST:STUDY_SUMMARY designed to lay the foundation for continued research to capture the metabolic ST:STUDY_SUMMARY capabilities in the ferns and fern allies. ST:INSTITUTE Washington State University ST:DEPARTMENT Institute of Biological Chemistry ST:LABORATORY Lange ST:LAST_NAME Lange ST:FIRST_NAME Mark ST:ADDRESS Plant Sciences Building, Pullman, Washington 99164 ST:EMAIL lange-m@wsu.edu ST:PHONE +1-509-335-3794 ST:NUM_GROUPS 6 ST:TOTAL_SUBJECTS 30 #SUBJECT SU:SUBJECT_TYPE Plant SU:SUBJECT_SPECIES Equisetum arvense;Equisetum hyemale;Equisetum telmateia #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 - Ea_rhiz_1 Species:E. arvense | Organ:rhizome RAW_FILE_NAME=EA.R1.d SUBJECT_SAMPLE_FACTORS - Ea_rhiz_2 Species:E. arvense | Organ:rhizome RAW_FILE_NAME=EA.R2.d SUBJECT_SAMPLE_FACTORS - Ea_rhiz_3 Species:E. arvense | Organ:rhizome RAW_FILE_NAME=EA.R3.d SUBJECT_SAMPLE_FACTORS - Ea_rhiz_4 Species:E. arvense | Organ:rhizome RAW_FILE_NAME=EA.R4.d SUBJECT_SAMPLE_FACTORS - Ea_rhiz_5 Species:E. arvense | Organ:rhizome RAW_FILE_NAME=EA.R5.d SUBJECT_SAMPLE_FACTORS - Ea_stem_1 Species:E. arvense | Organ:stem RAW_FILE_NAME=EA.S1.d SUBJECT_SAMPLE_FACTORS - Ea_stem_2 Species:E. arvense | Organ:stem RAW_FILE_NAME=EA.S2.d SUBJECT_SAMPLE_FACTORS - Ea_stem_3 Species:E. arvense | Organ:stem RAW_FILE_NAME=EA.S3.d SUBJECT_SAMPLE_FACTORS - Ea_stem_4 Species:E. arvense | Organ:stem RAW_FILE_NAME=EA.S4.d SUBJECT_SAMPLE_FACTORS - Ea_stem_5 Species:E. arvense | Organ:stem RAW_FILE_NAME=EA.S5.d SUBJECT_SAMPLE_FACTORS - Eh_rhiz_1 Species:E. hyemale | Organ:rhizome RAW_FILE_NAME=EH.R1.d SUBJECT_SAMPLE_FACTORS - Eh_rhiz_2 Species:E. hyemale | Organ:rhizome RAW_FILE_NAME=EH.R2.d SUBJECT_SAMPLE_FACTORS - Eh_rhiz_3 Species:E. hyemale | Organ:rhizome RAW_FILE_NAME=EH.R3.d SUBJECT_SAMPLE_FACTORS - Eh_rhiz_4 Species:E. hyemale | Organ:rhizome RAW_FILE_NAME=EH.R4.d SUBJECT_SAMPLE_FACTORS - Eh_rhiz_5 Species:E. hyemale | Organ:rhizome RAW_FILE_NAME=EH.R5.d SUBJECT_SAMPLE_FACTORS - Eh_stem_1 Species:E. hyemale | Organ:stem RAW_FILE_NAME=EH.S1.d SUBJECT_SAMPLE_FACTORS - Eh_stem_2 Species:E. hyemale | Organ:stem RAW_FILE_NAME=EH.S2.d SUBJECT_SAMPLE_FACTORS - Eh_stem_3 Species:E. hyemale | Organ:stem RAW_FILE_NAME=EH.S3.d SUBJECT_SAMPLE_FACTORS - Eh_stem_4 Species:E. hyemale | Organ:stem RAW_FILE_NAME=EH.S4.d SUBJECT_SAMPLE_FACTORS - Eh_stem_5 Species:E. hyemale | Organ:stem RAW_FILE_NAME=EH.S5.d SUBJECT_SAMPLE_FACTORS - Et_rhiz_1 Species:E. telmateia | Organ:rhizome RAW_FILE_NAME=ET.R1.d SUBJECT_SAMPLE_FACTORS - Et_rhiz_2 Species:E. telmateia | Organ:rhizome RAW_FILE_NAME=ET.R2.d SUBJECT_SAMPLE_FACTORS - Et_rhiz_3 Species:E. telmateia | Organ:rhizome RAW_FILE_NAME=ET.R3.d SUBJECT_SAMPLE_FACTORS - Et_rhiz_4 Species:E. telmateia | Organ:rhizome RAW_FILE_NAME=ET.R4.d SUBJECT_SAMPLE_FACTORS - Et_rhiz_5 Species:E. telmateia | Organ:rhizome RAW_FILE_NAME=ET.R5.d SUBJECT_SAMPLE_FACTORS - Et_stem_1 Species:E. telmateia | Organ:stem RAW_FILE_NAME=ET.S1.d SUBJECT_SAMPLE_FACTORS - Et_stem_2 Species:E. telmateia | Organ:stem RAW_FILE_NAME=ET.S2.d SUBJECT_SAMPLE_FACTORS - Et_stem_3 Species:E. telmateia | Organ:stem RAW_FILE_NAME=ET.S3.d SUBJECT_SAMPLE_FACTORS - Et_stem_4 Species:E. telmateia | Organ:stem RAW_FILE_NAME=ET.S4.d SUBJECT_SAMPLE_FACTORS - Et_stem_5 Species:E. telmateia | Organ:stem RAW_FILE_NAME=ET.S5.d #COLLECTION CO:COLLECTION_SUMMARY E. arvense, E. hyemale and E. telmateia (voucher specimens deposited with the CO:COLLECTION_SUMMARY John G. Searle Herbarium of the Field Museum, Chicago, IL, USA) were maintained CO:COLLECTION_SUMMARY in a greenhouse under ambient lighting, with supplemental lighting from sodium- CO:COLLECTION_SUMMARY vapor lamps. The photosynthetically active radiation varied from 15 to 25 mol CO:COLLECTION_SUMMARY m-2 d-1. Temperatures ranged between 22 and 27 °C and the humidity was set to 70 CO:COLLECTION_SUMMARY ± 10 %. Five biological replicates (separate plants) were harvested at the same CO:COLLECTION_SUMMARY time of day for below-ground rhizomes and above-ground stems of vegetative CO:COLLECTION_SUMMARY shoots. Samples were snap-frozen in liquid nitrogen and freeze-dried (aerial CO:COLLECTION_SUMMARY parts for 5 days, rhizomes for 7 days). Lyophilized material was submerged in CO:COLLECTION_SUMMARY liquid nitrogen, homogenized using mortar and pestle. CO:SAMPLE_TYPE Tissue homogenate CO:STORAGE_CONDITIONS -80 °C #TREATMENT TR:TREATMENT_SUMMARY No Treatment #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Five biological replicates (separate plants) were harvested at the same SP:SAMPLEPREP_SUMMARY time of day for below-ground rhizomes and above-ground stems of vegetative SP:SAMPLEPREP_SUMMARY shoots. Samples were snap-frozen in liquid nitrogen and freeze-dried (aerial SP:SAMPLEPREP_SUMMARY parts for 5 days, rhizomes for 7 days). Lyophilized material was submerged in SP:SAMPLEPREP_SUMMARY liquid nitrogen, homogenized using mortar and pestle. SP:EXTRACTION_METHOD Frozen tissue homogenate (30 mg per sample) was transferred to a 2 ml reaction SP:EXTRACTION_METHOD tube and extracted with 1 ml of 80 % aqueous methanol (containing 10 mg/l SP:EXTRACTION_METHOD anthracene-9-carboxylic acid as internal standard) by vigorous shaking (VX-2500 SP:EXTRACTION_METHOD multi-tube vortexer, VWR Scientific, South Plainfield, NY, USA) for 10 min and SP:EXTRACTION_METHOD subsequent sonication for 20 min (FS30 ultrasonic cleaner, Fisher Scientific, SP:EXTRACTION_METHOD Hampton, NY, USA). Following centrifugation for 10 min at 13,000 × g (5415 SP:EXTRACTION_METHOD microfuge, Eppendorf, Enfield, CT, USA), the supernatant was filtered through SP:EXTRACTION_METHOD 0.22 µm polypropylene syringe filter tips, and the flow-through collected in SP:EXTRACTION_METHOD plastic inserts for 2 ml reaction vials. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HPLC CH:INSTRUMENT_NAME Agilent 1290 HPLC CH:COLUMN_NAME HD Zorbax SB-Aq (100 × 2.1 × mm, 1.8 µm) CH:FLOW_GRADIENT 5 % B to 10 % B at 5 min, 20 % B at 10 min, 80 % B at 35 min, 95 % B at 45 min CH:FLOW_RATE 0.6 ml/min CH:COLUMN_TEMPERATURE 60 °C CH:SOLVENT_A 0.1 % formic acid in water CH:SOLVENT_B 0.1 % formic acid in acetonitrile CH:INTERNAL_STANDARD 10 mg/l Anthracene-9-carboxylic acid CH:SAMPLE_INJECTION 10 ul CH:TARGET_SAMPLE_TEMPERATURE Autosampler set to 4 °C #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME Lange AN:OPERATOR_NAME Parrish AN:SOFTWARE_VERSION MassHunter Qualitative Analysis software version B.07.00 Service Pack 1 build AN:SOFTWARE_VERSION 7.0.7024.29, Profinder B.06.00 build 6.0.0625.0 #MS MS:INSTRUMENT_NAME Agilent 6530 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS HPLC–QTOF–MS with electrospray ion source (positive ion mode) Raw data sets MS:MS_COMMENTS were opened in the Profinder B.06.00 build 6.0.0625.0 software package (Agilent MS:MS_COMMENTS Technologies, Santa Clara, CA, USA) and molecular feature elements (MFEs) MS:MS_COMMENTS obtained using the Batch Recursive Feature Extraction algorithm. Binning and MS:MS_COMMENTS alignment tolerances were set to 10 % + 20 s for the retention time and 10 ppm + MS:MS_COMMENTS 2 mDa for the mass accuracy, and 0.0025 m/z + 5.0 ppm for the isotope grouping MS:MS_COMMENTS space tolerance. Additional parameters that were considered for feature MS:MS_COMMENTS extraction were quasi-molecular ions and adducts ([M+H]+, [M+Na]+, [M+K]+, MS:MS_COMMENTS [M+NH4]+), dimers, neutral losses (H2O, H3PO4, C6H10O5 (glucose), C12H20O9 MS:MS_COMMENTS (rutinose), C12H20O10 (sophorose), C6H10O4 (rhamnose), and C5H8O4 (xylose)), MS:MS_COMMENTS absolute peak height = 2000 counts, and occurrence required in a minimum of MS:MS_COMMENTS four of the five replicates of each sample type. These pre-processing steps MS:MS_COMMENTS generated 848 MFEs (849 including ISTD), and exported into an Excel spreadsheet. MS:MS_COMMENTS Additional exclusion criteria for MFEs were: relative standard deviation of mass MS:MS_COMMENTS accuracy > 5.0 ppm; percent relative standard deviation returned as ”NaN” MS:MS_COMMENTS (Not a Number) or an empty cell; an unacceptably close accurate mass and MS:MS_COMMENTS retention time (± 0.010 m/z and ± 0.02 min.; screened as duplicates); or if it MS:MS_COMMENTS was a fragment. This additional filtering returned 544 remaining MFEs. Peak MS:MS_COMMENTS areas of MFEs for each sample were normalized based on sample weight and the MS:MS_COMMENTS peak area of the internal standard (MFEs without a peak area were filled in with MS:MS_COMMENTS a nominal value of two). MS:SOURCE_TEMPERATURE 325 °C MS:DATAFORMAT .d MS:NEBULIZER 2.4 bar MS:MS_RESULTS_FILE ST001934_AN003144_Results.txt UNITS:Peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END