#METABOLOMICS WORKBENCH skambhampati_20220117_130218 DATATRACK_ID:3041 STUDY_ID:ST002060 ANALYSIS_ID:AN003359 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           	Reversed phase
CH:INSTRUMENT_NAME               	Eksigent Ekspert microLC 200
CH:COLUMN_NAME                   	Machery-Nagel Nucleodur C8 (150 x 0.5mm x 5um)
CH:FLOW_RATE                     	0.04
CH:COLUMN_TEMPERATURE            	35
CH:METHODS_FILENAME              	Chromatography_PollenMetabolomics
CH:SOLVENT_A                     	1% 1 M ammonium acetate, 0.1% acetic acid in ddH2O
CH:SOLVENT_B                     	1% 1 M ammonium acetate, 0.1% acetic acid in 7:3 (v/v) acetonitrile: isopropanol
#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_AN003359_Results.txt	UNITS:Intensity	Has m/z:Yes	Has RT:Yes	RT units:Minutes
#END