#METABOLOMICS WORKBENCH skambhampati_20220117_130218 DATATRACK_ID:3041 STUDY_ID:ST002060 ANALYSIS_ID:AN003355 PROJECT_ID:PR001304 VERSION 1 CREATED_ON January 18, 2022, 9:05 am #PROJECT PR:PROJECT_TITLE Pollen metabolomics using Arabidopsis thaliana: Comparison of pollen at mature, PR:PROJECT_TITLE hydration and germination stage PR:PROJECT_TYPE Untargeted Metabolomics PR:PROJECT_SUMMARY Pollen germination is an essential process for pollen tube growth, pollination, PR:PROJECT_SUMMARY and therefore seed production in flowing plants, which requires energy either PR:PROJECT_SUMMARY from remobilization of stored carbon sources, lipid and starch, or from secreted PR:PROJECT_SUMMARY exudates from stigma. Transcriptome analysis from in vitro pollen germination PR:PROJECT_SUMMARY previously showed 14 GO terms, including metabolism and energy, which are PR:PROJECT_SUMMARY overrepresented in Arabidopsis. However, the global changes of carbohydrate and PR:PROJECT_SUMMARY energy-related metabolites during the transition from mature pollen grain to PR:PROJECT_SUMMARY hydrated pollen, the prerequisite step to pollen germination, is poorly PR:PROJECT_SUMMARY understood in Arabidopsis. In this study, we investigated the differential PR:PROJECT_SUMMARY metabolic pathway enrichment among mature, hydrated, and germinated pollen using PR:PROJECT_SUMMARY untargeted metabolomics analysis. Integration of publicly available PR:PROJECT_SUMMARY transcriptome with presented metabolome data revealed starch and sucrose PR:PROJECT_SUMMARY metabolism was significantly increased during pollen hydration and germination. PR:PROJECT_SUMMARY The alterations in central metabolism focusing on sugar, fatty acids, and lipids PR:PROJECT_SUMMARY were analyzed in detail. Several metabolites, including palmitic acid, oleic PR:PROJECT_SUMMARY acid, linolenic acid, quercetin, luteolin/kaempferol, and γ-aminobutyric acid PR:PROJECT_SUMMARY (GABA), were elevated in the hydrated pollen, suggesting a potential role in PR:PROJECT_SUMMARY activating pollen tube emergence. The metabolite levels of mature, hydrated, and PR:PROJECT_SUMMARY germinated pollen, presented in this work provide insights on the molecular PR:PROJECT_SUMMARY basis of pollen germination. PR:INSTITUTE Donald Danforth Plant Science Center PR:DEPARTMENT Allen/USDA lab PR:LABORATORY Doug K. Allen PR:LAST_NAME Kambhampati PR:FIRST_NAME Shrikaar PR:ADDRESS 975 North Warson Road, St. Louis, MO 63132 PR:EMAIL shrikaar.k@gmail.com PR:PHONE 3144025550 PR:FUNDING_SOURCE NIH, USDA-ARS PR:CONTRIBUTORS Shrikaar Kambhampati, Jiang Wang, Doug Allen, Li-Qing Chen #STUDY ST:STUDY_TITLE Pollen metabolomics using Arabidopsis thaliana: Comparison of pollen at mature, ST:STUDY_TITLE hydration and germination stage ST:STUDY_TYPE Untargeted Metabolomics ST:STUDY_SUMMARY In this study, we investigated the differential metabolic pathway enrichment ST:STUDY_SUMMARY among mature, hydrated, and germinated pollen using untargeted metabolomics ST:STUDY_SUMMARY analysis. Integration of publicly available transcriptome with presented ST:STUDY_SUMMARY metabolome data revealed starch and sucrose metabolism was significantly ST:STUDY_SUMMARY increased during pollen hydration and germination. The alterations in central ST:STUDY_SUMMARY metabolism focusing on sugar, fatty acids, and lipids were analyzed in detail. ST:STUDY_SUMMARY Several metabolites, including palmitic acid, oleic acid, linolenic acid, ST:STUDY_SUMMARY quercetin, luteolin/kaempferol, and γ-aminobutyric acid (GABA), were elevated ST:STUDY_SUMMARY in the hydrated pollen, suggesting a potential role in activating pollen tube ST:STUDY_SUMMARY emergence. The metabolite levels of mature, hydrated, and germinated pollen, ST:STUDY_SUMMARY presented in this work provide insights on the molecular basis of pollen ST:STUDY_SUMMARY germination. ST:INSTITUTE University of Illinois, Urbana-Champaign ST:DEPARTMENT Department of Plant Biology ST:LABORATORY Li-Qing Chen Lab ST:LAST_NAME Kambhampati ST:FIRST_NAME Shrikaar ST:ADDRESS 975 North Warson Road, St. Louis, MO 63132 ST:EMAIL shrikaar.k@gmail.com ST:PHONE 3144025550 ST:NUM_GROUPS 3 ST:TOTAL_SUBJECTS 12 #SUBJECT SU:SUBJECT_TYPE Plant SU:SUBJECT_SPECIES Arabidopsis thaliana SU:TAXONOMY_ID 3702 SU:GENOTYPE_STRAIN Col-0 SU:AGE_OR_AGE_RANGE Mature pollen, Pollen hydration Stage (45 min), Pollen germination stage (4 SU:AGE_OR_AGE_RANGE hours) SU:SPECIES_GROUP Pollen #FACTORS #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 Control Mature_HILIC_Pos_1 Stage:Mature | Chromatography:HILIC | Polarity:Positive RAW_FILE_NAME=Col0_Mature_HILIC_Pos_1 SUBJECT_SAMPLE_FACTORS Control Mature_HILIC_Neg_1 Stage:Mature | Chromatography:HILIC | Polarity:Negative RAW_FILE_NAME=Col0_Mature_HILIC_Neg_1 SUBJECT_SAMPLE_FACTORS Control Mature_HILIC_Pos_2 Stage:Mature | Chromatography:HILIC | Polarity:Positive RAW_FILE_NAME=Col0_Mature_HILIC_Pos_2 SUBJECT_SAMPLE_FACTORS Control Mature_HILIC_Neg_2 Stage:Mature | Chromatography:HILIC | Polarity:Negative RAW_FILE_NAME=Col0_Mature_HILIC_Neg_2 SUBJECT_SAMPLE_FACTORS Control Mature_HILIC_Pos_3 Stage:Mature | Chromatography:HILIC | Polarity:Positive RAW_FILE_NAME=Col0_Mature_HILIC_Pos_3 SUBJECT_SAMPLE_FACTORS Control Mature_HILIC_Neg_3 Stage:Mature | Chromatography:HILIC | Polarity:Negative RAW_FILE_NAME=Col0_Mature_HILIC_Neg_3 SUBJECT_SAMPLE_FACTORS Control Mature_HILIC_Pos_4 Stage:Mature | Chromatography:HILIC | Polarity:Positive RAW_FILE_NAME=Col0_Mature_HILIC_Pos_4 SUBJECT_SAMPLE_FACTORS Control Mature_HILIC_Neg_4 Stage:Mature | Chromatography:HILIC | Polarity:Negative RAW_FILE_NAME=Col0_Mature_HILIC_Neg_4 SUBJECT_SAMPLE_FACTORS Control Mature_RPLC_Pos_1 Stage:Mature | Chromatography:RPLC (C18) | Polarity:Positive RAW_FILE_NAME=Col0_Mature_RPLC_Pos_1 SUBJECT_SAMPLE_FACTORS Control Mature_RPLC_Neg_1 Stage:Mature | Chromatography:RPLC (C18) | Polarity:Negative RAW_FILE_NAME=Col0_Mature_RPLC_Neg_1 SUBJECT_SAMPLE_FACTORS Control Mature_RPLC_Pos_2 Stage:Mature | Chromatography:RPLC (C18) | Polarity:Positive RAW_FILE_NAME=Col0_Mature_RPLC_Pos_2 SUBJECT_SAMPLE_FACTORS Control Mature_RPLC_Neg_2 Stage:Mature | Chromatography:RPLC (C18) | Polarity:Negative RAW_FILE_NAME=Col0_Mature_RPLC_Neg_2 SUBJECT_SAMPLE_FACTORS Control Mature_RPLC_Pos_3 Stage:Mature | Chromatography:RPLC (C18) | Polarity:Positive RAW_FILE_NAME=Col0_Mature_RPLC_Pos_3 SUBJECT_SAMPLE_FACTORS Control Mature_RPLC_Neg_3 Stage:Mature | Chromatography:RPLC (C18) | Polarity:Negative RAW_FILE_NAME=Col0_Mature_RPLC_Neg_3 SUBJECT_SAMPLE_FACTORS Control Mature_RPLC_Pos_4 Stage:Mature | Chromatography:RPLC (C18) | Polarity:Positive RAW_FILE_NAME=Col0_Mature_RPLC_Pos_4 SUBJECT_SAMPLE_FACTORS Control Mature_RPLC_Neg_4 Stage:Mature | Chromatography:RPLC (C18) | Polarity:Negative RAW_FILE_NAME=Col0_Mature_RPLC_Neg_4 SUBJECT_SAMPLE_FACTORS Control Mature_Lipids_Pos_1 Stage:Mature | Chromatography:Lipidomics (C8) | Polarity:Positive RAW_FILE_NAME=Col0_Mature_Lipid_Pos_1 SUBJECT_SAMPLE_FACTORS Control Mature_Lipids_Neg_1 Stage:Mature | Chromatography:Lipidomics (C8) | Polarity:Negative RAW_FILE_NAME=Col0_Mature_Lipid_Neg_1 SUBJECT_SAMPLE_FACTORS Control Mature_Lipids_Pos_2 Stage:Mature | Chromatography:Lipidomics (C8) | Polarity:Positive RAW_FILE_NAME=Col0_Mature_Lipid_Pos_2 SUBJECT_SAMPLE_FACTORS Control Mature_Lipids_Neg_2 Stage:Mature | Chromatography:Lipidomics (C8) | Polarity:Negative RAW_FILE_NAME=Col0_Mature_Lipid_Neg_2 SUBJECT_SAMPLE_FACTORS Control Mature_Lipids_Pos_3 Stage:Mature | Chromatography:Lipidomics (C8) | Polarity:Positive RAW_FILE_NAME=Col0_Mature_Lipid_Pos_3 SUBJECT_SAMPLE_FACTORS Control Mature_Lipids_Neg_3 Stage:Mature | Chromatography:Lipidomics (C8) | Polarity:Negative RAW_FILE_NAME=Col0_Mature_Lipid_Neg_3 SUBJECT_SAMPLE_FACTORS Control Mature_Lipids_Pos_4 Stage:Mature | Chromatography:Lipidomics (C8) | Polarity:Positive RAW_FILE_NAME=Col0_Mature_Lipid_Pos_4 SUBJECT_SAMPLE_FACTORS Control Mature_Lipids_Neg_4 Stage:Mature | Chromatography:Lipidomics (C8) | Polarity:Negative RAW_FILE_NAME=Col0_Mature_Lipid_Neg_4 SUBJECT_SAMPLE_FACTORS 45 min Sucr Hydrated_HILIC_Pos_1 Stage:Hydration | Chromatography:HILIC | Polarity:Positive RAW_FILE_NAME=Col0_Hydr_HILIC_Pos_1 SUBJECT_SAMPLE_FACTORS 45 min Sucr Hydrated_HILIC_Neg_1 Stage:Hydration | Chromatography:HILIC | Polarity:Negative RAW_FILE_NAME=Col0_Hydr_HILIC_Neg_1 SUBJECT_SAMPLE_FACTORS 45 min Sucr Hydrated_HILIC_Pos_2 Stage:Hydration | Chromatography:HILIC | Polarity:Positive RAW_FILE_NAME=Col0_Hydr_HILIC_Pos_2 SUBJECT_SAMPLE_FACTORS 45 min Sucr Hydrated_HILIC_Neg_2 Stage:Hydration | Chromatography:HILIC | Polarity:Negative RAW_FILE_NAME=Col0_Hydr_HILIC_Neg_2 SUBJECT_SAMPLE_FACTORS 45 min Sucr Hydrated_HILIC_Pos_3 Stage:Hydration | Chromatography:HILIC | Polarity:Positive RAW_FILE_NAME=Col0_Hydr_HILIC_Pos_3 SUBJECT_SAMPLE_FACTORS 45 min Sucr Hydrated_HILIC_Neg_3 Stage:Hydration | Chromatography:HILIC | Polarity:Negative RAW_FILE_NAME=Col0_Hydr_HILIC_Neg_3 SUBJECT_SAMPLE_FACTORS 45 min Sucr Hydrated_HILIC_Pos_4 Stage:Hydration | Chromatography:HILIC | Polarity:Positive RAW_FILE_NAME=Col0_Hydr_HILIC_Pos_4 SUBJECT_SAMPLE_FACTORS 45 min Sucr Hydrated_HILIC_Neg_4 Stage:Hydration | Chromatography:HILIC | Polarity:Negative RAW_FILE_NAME=Col0_Hydr_HILIC_Neg_4 SUBJECT_SAMPLE_FACTORS 45 min Sucr Hydrated_RPLC_Pos_1 Stage:Hydration | Chromatography:RPLC (C18) | Polarity:Positive RAW_FILE_NAME=Col0_Hydr_RPLC_Pos_1 SUBJECT_SAMPLE_FACTORS 45 min Sucr Hydrated_RPLC_Neg_1 Stage:Hydration | Chromatography:RPLC (C18) | Polarity:Negative RAW_FILE_NAME=Col0_Hydr_RPLC_Neg_1 SUBJECT_SAMPLE_FACTORS 45 min Sucr Hydrated_RPLC_Pos_2 Stage:Hydration | Chromatography:RPLC (C18) | Polarity:Positive RAW_FILE_NAME=Col0_Hydr_RPLC_Pos_2 SUBJECT_SAMPLE_FACTORS 45 min Sucr Hydrated_RPLC_Neg_2 Stage:Hydration | Chromatography:RPLC (C18) | Polarity:Negative RAW_FILE_NAME=Col0_Hydr_RPLC_Neg_2 SUBJECT_SAMPLE_FACTORS 45 min Sucr Hydrated_RPLC_Pos_3 Stage:Hydration | Chromatography:RPLC (C18) | Polarity:Positive RAW_FILE_NAME=Col0_Hydr_RPLC_Pos_3 SUBJECT_SAMPLE_FACTORS 45 min Sucr Hydrated_RPLC_Neg_3 Stage:Hydration | Chromatography:RPLC (C18) | Polarity:Negative RAW_FILE_NAME=Col0_Hydr_RPLC_Neg_3 SUBJECT_SAMPLE_FACTORS 45 min Sucr Hydrated_RPLC_Pos_4 Stage:Hydration | Chromatography:RPLC (C18) | Polarity:Positive RAW_FILE_NAME=Col0_Hydr_RPLC_Pos_4 SUBJECT_SAMPLE_FACTORS 45 min Sucr Hydrated_RPLC_Neg_4 Stage:Hydration | Chromatography:RPLC (C18) | Polarity:Negative RAW_FILE_NAME=Col0_Hydr_RPLC_Neg_4 SUBJECT_SAMPLE_FACTORS 45 min Sucr Hydrated_Lipids_Pos_1 Stage:Hydration | Chromatography:Lipidomics (C8) | Polarity:Positive RAW_FILE_NAME=Col0_Hydr_Lipid_Pos_1 SUBJECT_SAMPLE_FACTORS 45 min Sucr Hydrated_Lipids_Neg_1 Stage:Hydration | Chromatography:Lipidomics (C8) | Polarity:Negative RAW_FILE_NAME=Col0_Hydr_Lipid_Neg_1 SUBJECT_SAMPLE_FACTORS 45 min Sucr Hydrated_Lipids_Pos_2 Stage:Hydration | Chromatography:Lipidomics (C8) | Polarity:Positive RAW_FILE_NAME=Col0_Hydr_Lipid_Pos_2 SUBJECT_SAMPLE_FACTORS 45 min Sucr Hydrated_Lipids_Neg_2 Stage:Hydration | Chromatography:Lipidomics (C8) | Polarity:Negative RAW_FILE_NAME=Col0_Hydr_Lipid_Neg_2 SUBJECT_SAMPLE_FACTORS 45 min Sucr Hydrated_Lipids_Pos_3 Stage:Hydration | Chromatography:Lipidomics (C8) | Polarity:Positive RAW_FILE_NAME=Col0_Hydr_Lipid_Pos_3 SUBJECT_SAMPLE_FACTORS 45 min Sucr Hydrated_Lipids_Neg_3 Stage:Hydration | Chromatography:Lipidomics (C8) | Polarity:Negative RAW_FILE_NAME=Col0_Hydr_Lipid_Neg_3 SUBJECT_SAMPLE_FACTORS 45 min Sucr Hydrated_Lipids_Pos_4 Stage:Hydration | Chromatography:Lipidomics (C8) | Polarity:Positive RAW_FILE_NAME=Col0_Hydr_Lipid_Pos_4 SUBJECT_SAMPLE_FACTORS 45 min Sucr Hydrated_Lipids_Neg_4 Stage:Hydration | Chromatography:Lipidomics (C8) | Polarity:Negative RAW_FILE_NAME=Col0_Hydr_Lipid_Neg_4 SUBJECT_SAMPLE_FACTORS 4 Hr Sucr Germinated_HILIC_Pos_1 Stage:Germination | Chromatography:HILIC | Polarity:Positive RAW_FILE_NAME=Col0_Germ_HILIC_Pos_1 SUBJECT_SAMPLE_FACTORS 4 Hr Sucr Germinated_HILIC_Neg_1 Stage:Germination | Chromatography:HILIC | Polarity:Negative RAW_FILE_NAME=Col0_Germ_HILIC_Neg_1 SUBJECT_SAMPLE_FACTORS 4 Hr Sucr Germinated_HILIC_Pos_2 Stage:Germination | Chromatography:HILIC | Polarity:Positive RAW_FILE_NAME=Col0_Germ_HILIC_Pos_2 SUBJECT_SAMPLE_FACTORS 4 Hr Sucr Germinated_HILIC_Neg_2 Stage:Germination | Chromatography:HILIC | Polarity:Negative RAW_FILE_NAME=Col0_Germ_HILIC_Neg_2 SUBJECT_SAMPLE_FACTORS 4 Hr Sucr Germinated_HILIC_Pos_3 Stage:Germination | Chromatography:HILIC | Polarity:Positive RAW_FILE_NAME=Col0_Germ_HILIC_Pos_3 SUBJECT_SAMPLE_FACTORS 4 Hr Sucr Germinated_HILIC_Neg_3 Stage:Germination | Chromatography:HILIC | Polarity:Negative RAW_FILE_NAME=Col0_Germ_HILIC_Neg_3 SUBJECT_SAMPLE_FACTORS 4 Hr Sucr Germinated_HILIC_Pos_4 Stage:Germination | Chromatography:HILIC | Polarity:Positive RAW_FILE_NAME=Col0_Germ_HILIC_Pos_4 SUBJECT_SAMPLE_FACTORS 4 Hr Sucr Germinated_HILIC_Neg_4 Stage:Germination | Chromatography:HILIC | Polarity:Negative RAW_FILE_NAME=Col0_Germ_HILIC_Neg_4 SUBJECT_SAMPLE_FACTORS 4 Hr Sucr Germinated_RPLC_Pos_1 Stage:Germination | Chromatography:RPLC (C18) | Polarity:Positive RAW_FILE_NAME=Col0_Germ_RPLC_Pos_1 SUBJECT_SAMPLE_FACTORS 4 Hr Sucr Germinated_RPLC_Neg_1 Stage:Germination | Chromatography:RPLC (C18) | Polarity:Negative RAW_FILE_NAME=Col0_Germ_RPLC_Neg_1 SUBJECT_SAMPLE_FACTORS 4 Hr Sucr Germinated_RPLC_Pos_2 Stage:Germination | Chromatography:RPLC (C18) | Polarity:Positive RAW_FILE_NAME=Col0_Germ_RPLC_Pos_2 SUBJECT_SAMPLE_FACTORS 4 Hr Sucr Germinated_RPLC_Neg_2 Stage:Germination | Chromatography:RPLC (C18) | Polarity:Negative RAW_FILE_NAME=Col0_Germ_RPLC_Neg_2 SUBJECT_SAMPLE_FACTORS 4 Hr Sucr Germinated_RPLC_Pos_3 Stage:Germination | Chromatography:RPLC (C18) | Polarity:Positive RAW_FILE_NAME=Col0_Germ_RPLC_Pos_3 SUBJECT_SAMPLE_FACTORS 4 Hr Sucr Germinated_RPLC_Neg_3 Stage:Germination | Chromatography:RPLC (C18) | Polarity:Negative RAW_FILE_NAME=Col0_Germ_RPLC_Neg_3 SUBJECT_SAMPLE_FACTORS 4 Hr Sucr Germinated_RPLC_Pos_4 Stage:Germination | Chromatography:RPLC (C18) | Polarity:Positive RAW_FILE_NAME=Col0_Germ_RPLC_Pos_4 SUBJECT_SAMPLE_FACTORS 4 Hr Sucr Germinated_RPLC_Neg_4 Stage:Germination | Chromatography:RPLC (C18) | Polarity:Negative RAW_FILE_NAME=Col0_Germ_RPLC_Neg_4 SUBJECT_SAMPLE_FACTORS 4 Hr Sucr Germinated_Lipids_Pos_1 Stage:Germination | Chromatography:Lipidomics (C8) | Polarity:Positive RAW_FILE_NAME=Col0_Germ_Lipid_Pos_1 SUBJECT_SAMPLE_FACTORS 4 Hr Sucr Germinated_Lipids_Neg_1 Stage:Germination | Chromatography:Lipidomics (C8) | Polarity:Negative RAW_FILE_NAME=Col0_Germ_Lipid_Neg_1 SUBJECT_SAMPLE_FACTORS 4 Hr Sucr Germinated_Lipids_Pos_2 Stage:Germination | Chromatography:Lipidomics (C8) | Polarity:Positive RAW_FILE_NAME=Col0_Germ_Lipid_Pos_2 SUBJECT_SAMPLE_FACTORS 4 Hr Sucr Germinated_Lipids_Neg_2 Stage:Germination | Chromatography:Lipidomics (C8) | Polarity:Negative RAW_FILE_NAME=Col0_Germ_Lipid_Neg_2 SUBJECT_SAMPLE_FACTORS 4 Hr Sucr Germinated_Lipids_Pos_3 Stage:Germination | Chromatography:Lipidomics (C8) | Polarity:Positive RAW_FILE_NAME=Col0_Germ_Lipid_Pos_3 SUBJECT_SAMPLE_FACTORS 4 Hr Sucr Germinated_Lipids_Neg_3 Stage:Germination | Chromatography:Lipidomics (C8) | Polarity:Negative RAW_FILE_NAME=Col0_Germ_Lipid_Neg_3 SUBJECT_SAMPLE_FACTORS 4 Hr Sucr Germinated_Lipids_Pos_4 Stage:Germination | Chromatography:Lipidomics (C8) | Polarity:Positive RAW_FILE_NAME=Col0_Germ_Lipid_Pos_4 SUBJECT_SAMPLE_FACTORS 4 Hr Sucr Germinated_Lipids_Neg_4 Stage:Germination | Chromatography:Lipidomics (C8) | Polarity:Negative RAW_FILE_NAME=Col0_Germ_Lipid_Neg_4 #COLLECTION CO:COLLECTION_SUMMARY The Arabidopsis Col-0 plants were grown under controlled temperature (22°C) CO:COLLECTION_SUMMARY with a 16-h light (100-150 µmol m-2 s-1)/ 8-h dark photoperiod. Mature pollen CO:COLLECTION_SUMMARY grains from the fully opened flowers were collected from more than 1000 plants CO:COLLECTION_SUMMARY using a vacuum cleaner method (Johnson-Brousseau and McCormick, 2004) at around CO:COLLECTION_SUMMARY 5 hours into the light period. For mature pollen samples, collected pollen CO:COLLECTION_SUMMARY grains were resuspended in 2 ml of ice-cold Pollen Isolation Buffer (PIB, CO:COLLECTION_SUMMARY composed of 100 mM NaPO4, pH 7.5, 1 mM EDTA, and 0.1% (v/v) Triton X-100) right CO:COLLECTION_SUMMARY after collection followed by centrifuging at 15,000 g for 1 min (4°C). For CO:COLLECTION_SUMMARY hydrated pollen and germinated pollen samples, mature pollen grains were CO:COLLECTION_SUMMARY germinated in vitro according to a previously described pollen transcriptome CO:COLLECTION_SUMMARY study (Wang et al., 2008). In brief, pollen pellets were washed with 1 ml of CO:COLLECTION_SUMMARY liquid Pollen Germination Medium (PGM, composed of 15% (w/v) sucrose, 1.5 mM CO:COLLECTION_SUMMARY boric acid, 0.8 mM MgSO4, 1 mM KCl, 5 mM MES, 0.05% (w/v) lactalbumin CO:COLLECTION_SUMMARY hydrolysate, 10 µM myo-inositol, 5 mM CaCl2) before they were resuspended in 30 CO:COLLECTION_SUMMARY µl of liquid PGM and subsequently cultured in Petri dishes (35 mm in diameter). CO:COLLECTION_SUMMARY A 70 µm mesh was used to cover the pollen droplet to create a thin layer for CO:COLLECTION_SUMMARY optimal germination for each Petri dish. The Petri dishes were covered and CO:COLLECTION_SUMMARY placed in the dark for 45 min or 4 h and collected as hydrated pollen or CO:COLLECTION_SUMMARY germinated pollen, respectively. All pollen samples were washed by 1 ml ice-cold CO:COLLECTION_SUMMARY ddH2O three times before being stored in a -80 °C freezer. CO:COLLECTION_PROTOCOL_FILENAME PlantGrowth_PollenCollection.docx CO:SAMPLE_TYPE Plant CO:COLLECTION_METHOD Flash frozen in Liquid N2 CO:COLLECTION_LOCATION University of Illinois, Urbana-Champain CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY For hydrated pollen and germinated pollen samples, mature pollen grains were TR:TREATMENT_SUMMARY germinated in vitro according to a previously described pollen transcriptome TR:TREATMENT_SUMMARY study (Wang et al., 2008). In brief, pollen pellets were washed with 1 ml of TR:TREATMENT_SUMMARY liquid Pollen Germination Medium (PGM, composed of 15% (w/v) sucrose, 1.5 mM TR:TREATMENT_SUMMARY boric acid, 0.8 mM MgSO4, 1 mM KCl, 5 mM MES, 0.05% (w/v) lactalbumin TR:TREATMENT_SUMMARY hydrolysate, 10 µM myo-inositol, 5 mM CaCl2) before they were resuspended in 30 TR:TREATMENT_SUMMARY µl of liquid PGM and subsequently cultured in Petri dishes (35 mm in diameter). TR:TREATMENT_SUMMARY A 70 µm mesh was used to cover the pollen droplet to create a thin layer for TR:TREATMENT_SUMMARY optimal germination for each Petri dish. The Petri dishes were covered and TR:TREATMENT_SUMMARY placed in the dark for 45 min or 4 h and collected as hydrated pollen or TR:TREATMENT_SUMMARY germinated pollen, respectively. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Total metabolites from pollen were extracted using a phase separation method SP:SAMPLEPREP_SUMMARY previously described (Kambhampati et al., 2021) with slight modifications. SP:SAMPLEPREP_SUMMARY Briefly, 20-30 mg pollen tissue, collected in Eppendorf tubes, was extracted SP:SAMPLEPREP_SUMMARY using 700 µL of chilled 7:3 (v/v) methanol: chloroform spiked with 50 µM each SP:SAMPLEPREP_SUMMARY of 1.4-piperazinediethanesulfonic acid (PEPES), ribitol, and norvaline as SP:SAMPLEPREP_SUMMARY internal standards. After two metal beads were also added to the samples, they SP:SAMPLEPREP_SUMMARY were homogenized using a Tissue-Lyser for 5 min at 30 Hz. The samples were SP:SAMPLEPREP_SUMMARY incubated on a rotary shaker at 4°C for 2 hours after which 300 µL ddH2O was SP:SAMPLEPREP_SUMMARY added. The samples were then centrifuged at 14,000 rpm for 10 min to achieve SP:SAMPLEPREP_SUMMARY phase separation and the upper aqueous phase, as well as the lower organic SP:SAMPLEPREP_SUMMARY phase, were collected separately. The aqueous phases containing polar and SP:SAMPLEPREP_SUMMARY nonpolar metabolites were split into two equal parts and dried using a speed SP:SAMPLEPREP_SUMMARY vacuum centrifuge (Labconco®, Kansas City, USA). The two dried parts were SP:SAMPLEPREP_SUMMARY re-suspended in 50 µL 80% (v/v) methanol, and 30 % (v/v) methanol for SP:SAMPLEPREP_SUMMARY metabolomics analyses using hydrophilic interaction (HILIC) and reverse phase SP:SAMPLEPREP_SUMMARY chromatography (RPLC), respectively. The organic phase was also dried using a SP:SAMPLEPREP_SUMMARY speed vacuum centrifuge and re-suspended in 50 µL of 49:49:2 (v/v/v) mixture of SP:SAMPLEPREP_SUMMARY acetonitrile: methanol: chloroform. All samples were filtered using a 0.8 µM SP:SAMPLEPREP_SUMMARY PES membrane centrifuge filter (Sartorius, Goettingen, Germany) and transferred SP:SAMPLEPREP_SUMMARY to a glass vial for injection into an LC-MS/MS system. SP:SAMPLEPREP_PROTOCOL_FILENAME Total_Metabolite_Extraction.docx SP:PROCESSING_STORAGE_CONDITIONS -80℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Eksigent Ekspert microLC 200 CH:COLUMN_NAME SeQuant Zic-HILIC (150 x 0.5mm x 5um) CH:FLOW_RATE 0.015 CH:COLUMN_TEMPERATURE 35 CH:METHODS_FILENAME Chromatography_PollentMetabolomics.docx CH:SOLVENT_A 10 mM ammonium bicarbonate in ddH2O CH:SOLVENT_B 10 mM ammonium bicarbonate in 95:5 (v/v) Acetonitrile: ddH2O #ANALYSIS AN:ANALYSIS_TYPE MS AN:ACQUISITION_PARAMETERS_FILE MS_DataAcquisition.docx #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS All parameters listed in the attached document MS:MS_RESULTS_FILE ST002060_AN003355_Results.txt UNITS:Intensity Has m/z:Yes Has RT:Yes RT units:Minutes #END