#METABOLOMICS WORKBENCH skambhampati_20220721_075626 DATATRACK_ID:3358 STUDY_ID:ST002239 ANALYSIS_ID:AN003655 VERSION 1 CREATED_ON 08-10-2022 #PROJECT PR:PROJECT_TITLE Using stable isotopes and mass spectrometry to elucidate the dynamics of PR:PROJECT_TITLE metabolic pathways PR:PROJECT_TYPE Stable Isotope Enriched Lipidomics PR:PROJECT_SUMMARY Data analysis and mass spectrometry tools have advanced significantly in the PR:PROJECT_SUMMARY last decade. This ongoing revolution has elevated the status of analytical PR:PROJECT_SUMMARY chemistry within the big-data omics era. High resolution mass spectrometers PR:PROJECT_SUMMARY (HRMS) can now distinguish different metabolites with mass to charge ratios PR:PROJECT_SUMMARY (i.e. m/z) that differ by 0.01 Da or less. This unprecedented level of PR:PROJECT_SUMMARY resolution not only enables identification of previously unknown compounds but PR:PROJECT_SUMMARY also presents an opportunity to establish active metabolic pathways through PR:PROJECT_SUMMARY quantification of isotope enrichment. Studies with stable isotope tracers PR:PROJECT_SUMMARY continue to contribute to our knowledge of biological pathways in human, plant PR:PROJECT_SUMMARY and bacterial species, however most current studies have been based on targeted PR:PROJECT_SUMMARY analyses. The capacity of HRMS to resolve near-overlapping isotopologues and PR:PROJECT_SUMMARY identify compounds with high mass precision presents a strategy to assess PR:PROJECT_SUMMARY ‘active’ pathways de novo from data generated in an untargeted way, that is PR:PROJECT_SUMMARY blind to the metabolic network and therefore unbiased. Currently, identifying PR:PROJECT_SUMMARY metabolic features, enriched with stable isotopes, at an ‘omics’ level PR:PROJECT_SUMMARY remains an experimental bottleneck, limiting our capacity to understand PR:PROJECT_SUMMARY biological network operation at the metabolic level. We developed data analysis PR:PROJECT_SUMMARY tools that: i) use labeling information and exact mass to determine the PR:PROJECT_SUMMARY elemental composition of each isotopically enriched ion, ii) apply PR:PROJECT_SUMMARY correlation-based approaches to cluster metabolite peaks with similar patterns PR:PROJECT_SUMMARY of isotopic labels and, iii) leverage this information to build directed PR:PROJECT_SUMMARY metabolic networks de novo. Using Camelina sativa, an emerging oilseed model, we PR:PROJECT_SUMMARY demonstrate the power of stable isotope labeling in combination with imaging and PR:PROJECT_SUMMARY HRMS to reconstruct lipid metabolic networks in developing seeds and are PR:PROJECT_SUMMARY currently addressing questions about lipid and central metabolism. Tools PR:PROJECT_SUMMARY developed in this study will have a broader application to assess context PR:PROJECT_SUMMARY specific operation of metabolic pathways. PR:INSTITUTE Donald Danforth Plant Science Center PR:DEPARTMENT Allen/USDA lab PR:LABORATORY Allen lab PR:LAST_NAME Shrikaar PR:FIRST_NAME Kambhampati PR:ADDRESS 975 North Warson road, St. Louis, MO 63132 PR:EMAIL skambhampati@danforthcenter.org PR:PHONE 3144025550 PR:FUNDING_SOURCE NIH, USDA-ARS PR:DOI http://dx.doi.org/10.21228/M80X3B #STUDY ST:STUDY_TITLE Insights into plant lipid metabolism using stable isotopes and high resolution ST:STUDY_TITLE mass spectrometry ST:STUDY_TYPE Stable isotope enriched lipidomics ST:STUDY_SUMMARY Data analysis and mass spectrometry tools have advanced significantly in the ST:STUDY_SUMMARY last decade. This ongoing revolution has elevated the status of analytical ST:STUDY_SUMMARY chemistry within the big-data omics era. High resolution mass spectrometers ST:STUDY_SUMMARY (HRMS) can now distinguish different metabolites with mass to charge ratios ST:STUDY_SUMMARY (i.e. m/z) that differ by 0.01 Da or less. This unprecedented level of ST:STUDY_SUMMARY resolution not only enables identification of previously unknown compounds but ST:STUDY_SUMMARY also presents an opportunity to establish active metabolic pathways through ST:STUDY_SUMMARY quantification of isotope enrichment. Studies with stable isotope tracers ST:STUDY_SUMMARY continue to contribute to our knowledge of biological pathways in human, plant ST:STUDY_SUMMARY and bacterial species, however most current studies have been based on targeted ST:STUDY_SUMMARY analyses. The capacity of HRMS to resolve near-overlapping isotopologues and ST:STUDY_SUMMARY identify compounds with high mass precision presents a strategy to assess ST:STUDY_SUMMARY ‘active’ pathways de novo from data generated in an untargeted way, that is ST:STUDY_SUMMARY blind to the metabolic network and therefore unbiased. Currently, identifying ST:STUDY_SUMMARY metabolic features, enriched with stable isotopes, at an ‘omics’ level ST:STUDY_SUMMARY remains an experimental bottleneck, limiting our capacity to understand ST:STUDY_SUMMARY biological network operation at the metabolic level. We developed data analysis ST:STUDY_SUMMARY tools that: i) use labeling information and exact mass to determine the ST:STUDY_SUMMARY elemental composition of each isotopically enriched ion, ii) apply ST:STUDY_SUMMARY correlation-based approaches to cluster metabolite peaks with similar patterns ST:STUDY_SUMMARY of isotopic labels and, iii) leverage this information to build directed ST:STUDY_SUMMARY metabolic networks de novo. Using Camelina sativa, an emerging oilseed model, we ST:STUDY_SUMMARY demonstrate the power of stable isotope labeling in combination with imaging and ST:STUDY_SUMMARY HRMS to reconstruct lipid metabolic networks in developing seeds and are ST:STUDY_SUMMARY currently addressing questions about lipid and central metabolism. Tools ST:STUDY_SUMMARY developed in this study will have a broader application to assess context ST:STUDY_SUMMARY specific operation of metabolic pathways. ST:INSTITUTE Donald Danforth Plant Science Center ST:DEPARTMENT Allen/USDA lab ST:LABORATORY Allen Lab ST:LAST_NAME Shrikaar ST:FIRST_NAME Kambhampati ST:ADDRESS 975 North Warson road ST:EMAIL skambhampati@danforthcenter.org ST:PHONE 3144025550 ST:SUBMIT_DATE 2022-07-21 #SUBJECT SU:SUBJECT_TYPE Plant SU:SUBJECT_SPECIES Camelina Sativa SU:AGE_OR_AGE_RANGE 10 days after fertilization SU:SPECIES_GROUP Developing seeds #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - 0_Cotyledon_1-neg Tissue type:Cotyledon | Time (hours):0 Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=0_Cotyledon_1-neg SUBJECT_SAMPLE_FACTORS - 0_Cotyledon_1-pos Tissue type:Cotyledon | Time (hours):0 Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=0_Cotyledon_1-pos SUBJECT_SAMPLE_FACTORS - 0_Cotyledon_2-neg Tissue type:Cotyledon | Time (hours):0 Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=0_Cotyledon_2-neg SUBJECT_SAMPLE_FACTORS - 0_Cotyledon_2-pos Tissue type:Cotyledon | Time (hours):0 Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=0_Cotyledon_2-pos SUBJECT_SAMPLE_FACTORS - 0_Cotyledon_3-neg Tissue type:Cotyledon | Time (hours):0 Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=0_Cotyledon_3-neg SUBJECT_SAMPLE_FACTORS - 0_Cotyledon_3-pos Tissue type:Cotyledon | Time (hours):0 Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=0_Cotyledon_3-pos SUBJECT_SAMPLE_FACTORS - 16_Cotyledon_1-neg Tissue type:Cotyledon | Time (hours):16 Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=16_Cotyledon_1-neg SUBJECT_SAMPLE_FACTORS - 16_Cotyledon_1-pos Tissue type:Cotyledon | Time (hours):16 Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=16_Cotyledon_1-pos SUBJECT_SAMPLE_FACTORS - 16_Cotyledon_2-neg Tissue type:Cotyledon | Time (hours):16 Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=16_Cotyledon_2-neg SUBJECT_SAMPLE_FACTORS - 16_Cotyledon_2-pos Tissue type:Cotyledon | Time (hours):16 Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=16_Cotyledon_2-pos SUBJECT_SAMPLE_FACTORS - 16_Cotyledon_3-neg Tissue type:Cotyledon | Time (hours):16 Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=16_Cotyledon_3-neg SUBJECT_SAMPLE_FACTORS - 16_Cotyledon_3-pos Tissue type:Cotyledon | Time (hours):16 Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=16_Cotyledon_3-pos SUBJECT_SAMPLE_FACTORS - 2_Cotyledon_1-neg Tissue type:Cotyledon | Time (hours):2 Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=2_Cotyledon_1-neg SUBJECT_SAMPLE_FACTORS - 2_Cotyledon_1-pos Tissue type:Cotyledon | Time (hours):2 Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=2_Cotyledon_1-pos SUBJECT_SAMPLE_FACTORS - 2_Cotyledon_2-neg Tissue type:Cotyledon | Time (hours):2 Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=2_Cotyledon_2-neg SUBJECT_SAMPLE_FACTORS - 2_Cotyledon_2-pos Tissue type:Cotyledon | Time (hours):2 Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=2_Cotyledon_2-pos SUBJECT_SAMPLE_FACTORS - 2_Cotyledon_3-neg Tissue type:Cotyledon | Time (hours):2 Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=2_Cotyledon_3-neg SUBJECT_SAMPLE_FACTORS - 2_Cotyledon_3-pos Tissue type:Cotyledon | Time (hours):2 Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=2_Cotyledon_3-pos SUBJECT_SAMPLE_FACTORS - 32_Cotyledon_1-neg Tissue type:Cotyledon | Time (hours):32 Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=32_Cotyledon_1-neg SUBJECT_SAMPLE_FACTORS - 32_Cotyledon_1-pos Tissue type:Cotyledon | Time (hours):32 Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=32_Cotyledon_1-pos SUBJECT_SAMPLE_FACTORS - 32_Cotyledon_2-neg Tissue type:Cotyledon | Time (hours):32 Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=32_Cotyledon_2-neg SUBJECT_SAMPLE_FACTORS - 32_Cotyledon_2-pos Tissue type:Cotyledon | Time (hours):32 Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=32_Cotyledon_2-pos SUBJECT_SAMPLE_FACTORS - 32_Cotyledon_3-neg Tissue type:Cotyledon | Time (hours):32 Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=32_Cotyledon_3-neg SUBJECT_SAMPLE_FACTORS - 32_Cotyledon_3-pos Tissue type:Cotyledon | Time (hours):32 Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=32_Cotyledon_3-pos SUBJECT_SAMPLE_FACTORS - 4_Cotyledon_1-neg Tissue type:Cotyledon | Time (hours):4 Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=4_Cotyledon_1-neg SUBJECT_SAMPLE_FACTORS - 4_Cotyledon_1-pos Tissue type:Cotyledon | Time (hours):4 Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=4_Cotyledon_1-pos SUBJECT_SAMPLE_FACTORS - 4_Cotyledon_2-neg Tissue type:Cotyledon | Time (hours):4 Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=4_Cotyledon_2-neg SUBJECT_SAMPLE_FACTORS - 4_Cotyledon_2-pos Tissue type:Cotyledon | Time (hours):4 Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=4_Cotyledon_2-pos SUBJECT_SAMPLE_FACTORS - 4_Cotyledon_3-neg Tissue type:Cotyledon | Time (hours):4 Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=4_Cotyledon_3-neg SUBJECT_SAMPLE_FACTORS - 4_Cotyledon_3-pos Tissue type:Cotyledon | Time (hours):4 Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=4_Cotyledon_3-pos SUBJECT_SAMPLE_FACTORS - 8_Cotyledon_1-neg Tissue type:Cotyledon | Time (hours):8 Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=8_Cotyledon_1-neg SUBJECT_SAMPLE_FACTORS - 8_Cotyledon_1-pos Tissue type:Cotyledon | Time (hours):8 Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=8_Cotyledon_1-pos SUBJECT_SAMPLE_FACTORS - 8_Cotyledon_2-neg Tissue type:Cotyledon | Time (hours):8 Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=8_Cotyledon_2-neg SUBJECT_SAMPLE_FACTORS - 8_Cotyledon_2-pos Tissue type:Cotyledon | Time (hours):8 Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=8_Cotyledon_2-pos SUBJECT_SAMPLE_FACTORS - 8_Cotyledon_3-neg Tissue type:Cotyledon | Time (hours):8 Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=8_Cotyledon_3-neg SUBJECT_SAMPLE_FACTORS - 8_Cotyledon_3-pos Tissue type:Cotyledon | Time (hours):8 Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=8_Cotyledon_3-pos SUBJECT_SAMPLE_FACTORS - 0_EA_1-neg Tissue type:Embyo axis | Time (hours):0 Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=0_EA_1-neg SUBJECT_SAMPLE_FACTORS - 0_EA_1-pos Tissue type:Embyo axis | Time (hours):0 Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=0_EA_1-pos SUBJECT_SAMPLE_FACTORS - 0_EA_2-neg Tissue type:Embyo axis | Time (hours):0 Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=0_EA_2-neg SUBJECT_SAMPLE_FACTORS - 0_EA_2-pos Tissue type:Embyo axis | Time (hours):0 Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=0_EA_2-pos SUBJECT_SAMPLE_FACTORS - 0_EA_3-neg Tissue type:Embyo axis | Time (hours):0 Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=0_EA_3-neg SUBJECT_SAMPLE_FACTORS - 0_EA_3-pos Tissue type:Embyo axis | Time (hours):0 Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=0_EA_3-pos SUBJECT_SAMPLE_FACTORS - 16_EA_1-neg Tissue type:Embyo axis | Time (hours):16 Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=16_EA_1-neg SUBJECT_SAMPLE_FACTORS - 16_EA_1-pos Tissue type:Embyo axis | Time (hours):16 Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=16_EA_1-pos SUBJECT_SAMPLE_FACTORS - 16_EA_2-neg Tissue type:Embyo axis | Time (hours):16 Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=16_EA_2-neg SUBJECT_SAMPLE_FACTORS - 16_EA_2-pos Tissue type:Embyo axis | Time (hours):16 Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=16_EA_2-pos SUBJECT_SAMPLE_FACTORS - 16_EA_3-neg Tissue type:Embyo axis | Time (hours):16 Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=16_EA_3-neg SUBJECT_SAMPLE_FACTORS - 16_EA_3-pos Tissue type:Embyo axis | Time (hours):16 Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=16_EA_3-pos SUBJECT_SAMPLE_FACTORS - 2_EA_1-neg Tissue type:Embyo axis | Time (hours):2 Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=2_EA_1-neg SUBJECT_SAMPLE_FACTORS - 2_EA_1-pos Tissue type:Embyo axis | Time (hours):2 Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=2_EA_1-pos SUBJECT_SAMPLE_FACTORS - 2_EA_2-neg Tissue type:Embyo axis | Time (hours):2 Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=2_EA_2-neg SUBJECT_SAMPLE_FACTORS - 2_EA_2-pos Tissue type:Embyo axis | Time (hours):2 Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=2_EA_2-pos SUBJECT_SAMPLE_FACTORS - 2_EA_3-neg Tissue type:Embyo axis | Time (hours):2 Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=2_EA_3-neg SUBJECT_SAMPLE_FACTORS - 2_EA_3-pos Tissue type:Embyo axis | Time (hours):2 Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=2_EA_3-pos SUBJECT_SAMPLE_FACTORS - 32_EA_1-neg Tissue type:Embyo axis | Time (hours):32 Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=32_EA_1-neg SUBJECT_SAMPLE_FACTORS - 32_EA_1-pos Tissue type:Embyo axis | Time (hours):32 Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=32_EA_1-pos SUBJECT_SAMPLE_FACTORS - 32_EA_2-neg Tissue type:Embyo axis | Time (hours):32 Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=32_EA_2-neg SUBJECT_SAMPLE_FACTORS - 32_EA_2-pos Tissue type:Embyo axis | Time (hours):32 Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=32_EA_2-pos SUBJECT_SAMPLE_FACTORS - 32_EA_3-neg Tissue type:Embyo axis | Time (hours):32 Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=32_EA_3-neg SUBJECT_SAMPLE_FACTORS - 32_EA_3-pos Tissue type:Embyo axis | Time (hours):32 Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=32_EA_3-pos SUBJECT_SAMPLE_FACTORS - 4_EA_1-neg Tissue type:Embyo axis | Time (hours):4 Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=4_EA_1-neg SUBJECT_SAMPLE_FACTORS - 4_EA_1-pos Tissue type:Embyo axis | Time (hours):4 Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=4_EA_1-pos SUBJECT_SAMPLE_FACTORS - 4_EA_2-neg Tissue type:Embyo axis | Time (hours):4 Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=4_EA_2-neg SUBJECT_SAMPLE_FACTORS - 4_EA_2-pos Tissue type:Embyo axis | Time (hours):4 Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=4_EA_2-pos SUBJECT_SAMPLE_FACTORS - 4_EA_3-neg Tissue type:Embyo axis | Time (hours):4 Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=4_EA_3-neg SUBJECT_SAMPLE_FACTORS - 4_EA_3-pos Tissue type:Embyo axis | Time (hours):4 Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=4_EA_3-pos SUBJECT_SAMPLE_FACTORS - 8_EA_1-neg Tissue type:Embyo axis | Time (hours):8 Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=8_EA_1-neg SUBJECT_SAMPLE_FACTORS - 8_EA_1-pos Tissue type:Embyo axis | Time (hours):8 Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=8_EA_1-pos SUBJECT_SAMPLE_FACTORS - 8_EA_2-neg Tissue type:Embyo axis | Time (hours):8 Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=8_EA_2-neg SUBJECT_SAMPLE_FACTORS - 8_EA_2-pos Tissue type:Embyo axis | Time (hours):8 Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=8_EA_2-pos SUBJECT_SAMPLE_FACTORS - 8_EA_3-neg Tissue type:Embyo axis | Time (hours):8 Chromatography=C8; Polarity=Negative; RAW_FILE_NAME=8_EA_3-neg SUBJECT_SAMPLE_FACTORS - 8_EA_3-pos Tissue type:Embyo axis | Time (hours):8 Chromatography=C8; Polarity=Positive; RAW_FILE_NAME=8_EA_3-pos #COLLECTION CO:COLLECTION_SUMMARY Plant growth and culture conditions: Plants were grown in greenhouses with CO:COLLECTION_SUMMARY day/night temperature maintained at 22/20°C, 40-50% relative humidity, and 16h CO:COLLECTION_SUMMARY day/8h night photoperiod. Intact siliques during the seed filling growth stage CO:COLLECTION_SUMMARY (15 days after fertilization) were excised and placed in sterile media CO:COLLECTION_SUMMARY containing a modified Linsmaier and Skoog medium23,24 with Gamborg’s vitamins CO:COLLECTION_SUMMARY (Sigma) and 5 mM MES buffer adjusted to pH 5.8. Fifty mM [U-13C6]glucose was CO:COLLECTION_SUMMARY used as labeled substrate, and the composition of the remaining carbon and CO:COLLECTION_SUMMARY nitrogen sources represented maternal phloem composition to minimize metabolic CO:COLLECTION_SUMMARY perturbation and to maintain pseudo in vivo conditions as previously CO:COLLECTION_SUMMARY described25. Silique culturing was performed in a 96-well plate with 0.3 mL of CO:COLLECTION_SUMMARY medium and a single silique per well, under continuous light (250 µmol m-2 CO:COLLECTION_SUMMARY s-1). Tissue was collected and flash frozen immediately after each time point CO:COLLECTION_SUMMARY (2, 4, 8, 16 and 32h). Uncultured siliques excised from the maternal plant were CO:COLLECTION_SUMMARY used as unlabeled (0h) controls. Frozen tissue was sectioned, on top of dry ice, CO:COLLECTION_SUMMARY to excise embryo from the siliques and to separate cotyledons from the embryo CO:COLLECTION_SUMMARY axis. Cotyledon samples were extracted and analyzed for lipids in triplicates. CO:COLLECTION_PROTOCOL_FILENAME 13CLipids_CamelinaSeeds_Methods.docx CO:SAMPLE_TYPE Seeds CO:COLLECTION_LOCATION Donald Danforth Plant Science Center CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY Plants were grown in greenhouses with day/night temperature maintained at TR:TREATMENT_SUMMARY 22/20°C, 40-50% relative humidity, and 16h day/8h night photoperiod. Intact TR:TREATMENT_SUMMARY siliques during the seed filling growth stage (15 days after fertilization) were TR:TREATMENT_SUMMARY excised and placed in sterile media containing a modified Linsmaier and Skoog TR:TREATMENT_SUMMARY medium23,24 with Gamborg’s vitamins (Sigma) and 5 mM MES buffer adjusted to pH TR:TREATMENT_SUMMARY 5.8. Fifty mM [U-13C6]glucose was used as labeled substrate, and the composition TR:TREATMENT_SUMMARY of the remaining carbon and nitrogen sources represented maternal phloem TR:TREATMENT_SUMMARY composition to minimize metabolic perturbation and to maintain pseudo in vivo TR:TREATMENT_SUMMARY conditions as previously described25. Silique culturing was performed in a TR:TREATMENT_SUMMARY 96-well plate with 0.3 mL of medium and a single silique per well, under TR:TREATMENT_SUMMARY continuous light (250 µmol m-2 s-1). Tissue was collected and flash frozen TR:TREATMENT_SUMMARY immediately after each time point (2, 4, 8, 16 and 32h). Uncultured siliques TR:TREATMENT_SUMMARY excised from the maternal plant were used as unlabeled (0h) controls. Frozen TR:TREATMENT_SUMMARY tissue was sectioned, on top of dry ice, to excise embryo from the siliques and TR:TREATMENT_SUMMARY to separate cotyledons from the embryo axis. Cotyledon samples were extracted TR:TREATMENT_SUMMARY and analyzed for lipids in triplicates. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Frozen cotyledon samples from Camelina were homogenized using a tissue lyser and SP:SAMPLEPREP_SUMMARY the extraction of lipids was carried out using a phase separation method SP:SAMPLEPREP_SUMMARY previously described26. Briefly, 1 mL 7:3 methanol:chloroform (-20°C) SP:SAMPLEPREP_SUMMARY containing the ultimateSPLASHTM ONE lipid mix (Avanti Polar lipids, Alabaster, SP:SAMPLEPREP_SUMMARY AL) as internal standard (1:20 dilution) was added to the samples, vortexed SP:SAMPLEPREP_SUMMARY vigorously and incubated on a rotary shaker for 2 hours at 4°C. After SP:SAMPLEPREP_SUMMARY incubation, 500 µL of ice-cold water was added to the samples, vortexed and SP:SAMPLEPREP_SUMMARY centrifuged at 14,000 rpm at 4°C for 10 min to achieve phase separation. The SP:SAMPLEPREP_SUMMARY upper aqueous phase was carefully removed, 200 µL of methanol was added to the SP:SAMPLEPREP_SUMMARY remaining organic phase containing lipids and centrifuged at 14,000 rpm for 5 SP:SAMPLEPREP_SUMMARY min to pellet the debris. The organic phase (supernatant) was transferred to a SP:SAMPLEPREP_SUMMARY glass tube and dried using a speedvac centrifuge. Samples were re-suspended in SP:SAMPLEPREP_SUMMARY 200 µL of 49:49:2 acetonitrile: methanol: chloroform, filtered using 0.2 µm SP:SAMPLEPREP_SUMMARY PTFE microcentrifuge filters and transferred to a glass vial for RPLC-HRMS SP:SAMPLEPREP_SUMMARY analysis. SP:PROCESSING_STORAGE_CONDITIONS -80℃ SP:EXTRACTION_METHOD methanol:chloroform:water #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Separations for lipidomics were achieved using the loading pump of a Dionex CH:CHROMATOGRAPHY_SUMMARY UltiMate 3000 RSLCnano system (Thermo Fisher Scientific) operating at a flow CH:CHROMATOGRAPHY_SUMMARY rate of 40 µL min-1 equipped with a custom-made C8 column (100 x 0.5 x 5 µm) CH:CHROMATOGRAPHY_SUMMARY from Higgins Analytical Inc. (Mountain view, CA) re-packed from a nucleodur C8 CH:CHROMATOGRAPHY_SUMMARY Gravity column (Macherey-Nagel, Allentown, PA). Mobile phases comprised of 1% 1 CH:CHROMATOGRAPHY_SUMMARY M ammonium acetate, 0.1 % acetic acid in water (A) and 1% 1 M ammonium acetate, CH:CHROMATOGRAPHY_SUMMARY 0.1% acetic acid in 7:3 (v/v) acetonitrile: isopropanol (B). The following CH:CHROMATOGRAPHY_SUMMARY gradient modified from a previously described method27 to adapt to micro flow CH:CHROMATOGRAPHY_SUMMARY was used; 0-1 min at 55% B, 4 min at 75% B, 12 min at 89% B, 15 min at 99% B, 18 CH:CHROMATOGRAPHY_SUMMARY min at 99% B and 20 min at 55% B followed by equilibration up to 30 min. CH:INSTRUMENT_NAME Dionex UltiMate 3000 RSLCnano CH:COLUMN_NAME Custom C8 - Higgins Analytical CH:COLUMN_TEMPERATURE 40 CH:FLOW_RATE 0.04 mL min-1 CH:INTERNAL_STANDARD Equisplash CH:SOLVENT_A 1% 1 M ammonium acetate, 0.1 % acetic acid in water CH:SOLVENT_B 1% 1 M ammonium acetate, 0.1% acetic acid in 7:3 (v/v) acetonitrile: isopropanol CH:CHROMATOGRAPHY_TYPE Reversed phase #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Fusion Tribrid Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:MS_COMMENTS The eluent was sprayed on to the HESI source of an Orbitrap Fusion Lumos Tribrid MS:MS_COMMENTS MS, operated with sheath gas, 25 arbitrary units; auxiliary gas, 5 arbitrary MS:MS_COMMENTS units; ion transfer tube temperature, 300oC; vaporizer temperature, 100oC; and MS:MS_COMMENTS S-lens RF level, 60. The spray voltage was 4 kV in both positive and negative MS:MS_COMMENTS modes. Full MS data were collected for mass ranges 450-1200 m/z at 240,000 MS:MS_COMMENTS resolution from both positive and negative modes simultaneously, using polarity MS:MS_COMMENTS switch. The AGC target was set to “Standard” and the maximum IT was set to MS:MS_COMMENTS 100 ms. MS:ION_MODE POSITIVE MS:MS_RESULTS_FILE ST002239_AN003655_Results.txt UNITS:Intensity Has m/z:Yes Has RT:Yes RT units:Minutes #END