Summary of Study ST002060

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001304. The data can be accessed directly via it's Project DOI: 10.21228/M8570V This work is supported by NIH grant, U2C- DK119886.

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This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST002060
Study TitlePollen metabolomics using Arabidopsis thaliana: Comparison of pollen at mature, hydration and germination stage
Study TypeUntargeted Metabolomics
Study SummaryIn this study, we investigated the differential metabolic pathway enrichment among mature, hydrated, and germinated pollen using untargeted metabolomics analysis. Integration of publicly available transcriptome with presented metabolome data revealed starch and sucrose metabolism was significantly increased during pollen hydration and germination. The alterations in central metabolism focusing on sugar, fatty acids, and lipids were analyzed in detail. Several metabolites, including palmitic acid, oleic acid, linolenic acid, quercetin, luteolin/kaempferol, and γ-aminobutyric acid (GABA), were elevated in the hydrated pollen, suggesting a potential role in activating pollen tube emergence. The metabolite levels of mature, hydrated, and germinated pollen, presented in this work provide insights on the molecular basis of pollen germination.
Institute
University of Illinois Urbana-Champaign
DepartmentDepartment of Plant Biology
LaboratoryLi-Qing Chen Lab
Last NameKambhampati
First NameShrikaar
Address975 North Warson Road, St. Louis, MO 63132
Emailshrikaar.k@gmail.com
Phone3144025550
Submit Date2022-01-17
Num Groups3
Total Subjects12
Raw Data AvailableYes
Raw Data File Type(s)mzML
Analysis Type DetailLC-MS
Release Date2022-02-07
Release Version1
Shrikaar Kambhampati Shrikaar Kambhampati
https://dx.doi.org/10.21228/M8570V
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR001304
Project DOI:doi: 10.21228/M8570V
Project Title:Pollen metabolomics using Arabidopsis thaliana: Comparison of pollen at mature, hydration and germination stage
Project Type:Untargeted Metabolomics
Project Summary:Pollen germination is an essential process for pollen tube growth, pollination, and therefore seed production in flowing plants, which requires energy either from remobilization of stored carbon sources, lipid and starch, or from secreted exudates from stigma. Transcriptome analysis from in vitro pollen germination previously showed 14 GO terms, including metabolism and energy, which are overrepresented in Arabidopsis. However, the global changes of carbohydrate and energy-related metabolites during the transition from mature pollen grain to hydrated pollen, the prerequisite step to pollen germination, is poorly understood in Arabidopsis. In this study, we investigated the differential metabolic pathway enrichment among mature, hydrated, and germinated pollen using untargeted metabolomics analysis. Integration of publicly available transcriptome with presented metabolome data revealed starch and sucrose metabolism was significantly increased during pollen hydration and germination. The alterations in central metabolism focusing on sugar, fatty acids, and lipids were analyzed in detail. Several metabolites, including palmitic acid, oleic acid, linolenic acid, quercetin, luteolin/kaempferol, and γ-aminobutyric acid (GABA), were elevated in the hydrated pollen, suggesting a potential role in activating pollen tube emergence. The metabolite levels of mature, hydrated, and germinated pollen, presented in this work provide insights on the molecular basis of pollen germination.
Institute:Donald Danforth Plant Science Center
Department:Allen/USDA lab
Laboratory:Doug K. Allen
Last Name:Kambhampati
First Name:Shrikaar
Address:975 North Warson Road, St. Louis, MO 63132
Email:shrikaar.k@gmail.com
Phone:3144025550
Funding Source:NIH, USDA-ARS
Contributors:Shrikaar Kambhampati, Jiang Wang, Doug Allen, Li-Qing Chen

Subject:

Subject ID:SU002142
Subject Type:Plant
Subject Species:Arabidopsis thaliana
Taxonomy ID:3702
Genotype Strain:Col-0
Age Or Age Range:Mature pollen, Pollen hydration Stage (45 min), Pollen germination stage (4 hours)
Species Group:Pollen

Factors:

Subject type: Plant; Subject species: Arabidopsis thaliana (Factor headings shown in green)

mb_sample_id local_sample_id Stage Chromatography Polarity
SA193945Germinated_HILIC_Neg_3Germination HILIC Negative
SA193946Germinated_HILIC_Neg_1Germination HILIC Negative
SA193947Germinated_HILIC_Neg_4Germination HILIC Negative
SA193948Germinated_HILIC_Neg_2Germination HILIC Negative
SA193949Germinated_HILIC_Pos_4Germination HILIC Positive
SA193950Germinated_HILIC_Pos_1Germination HILIC Positive
SA193951Germinated_HILIC_Pos_3Germination HILIC Positive
SA193952Germinated_HILIC_Pos_2Germination HILIC Positive
SA193953Germinated_Lipids_Neg_4Germination Lipidomics (C8) Negative
SA193954Germinated_Lipids_Neg_1Germination Lipidomics (C8) Negative
SA193955Germinated_Lipids_Neg_2Germination Lipidomics (C8) Negative
SA193956Germinated_Lipids_Neg_3Germination Lipidomics (C8) Negative
SA193957Germinated_Lipids_Pos_4Germination Lipidomics (C8) Positive
SA193958Germinated_Lipids_Pos_2Germination Lipidomics (C8) Positive
SA193959Germinated_Lipids_Pos_1Germination Lipidomics (C8) Positive
SA193960Germinated_Lipids_Pos_3Germination Lipidomics (C8) Positive
SA193961Germinated_RPLC_Neg_3Germination RPLC (C18) Negative
SA193962Germinated_RPLC_Neg_4Germination RPLC (C18) Negative
SA193963Germinated_RPLC_Neg_2Germination RPLC (C18) Negative
SA193964Germinated_RPLC_Neg_1Germination RPLC (C18) Negative
SA193965Germinated_RPLC_Pos_1Germination RPLC (C18) Positive
SA193966Germinated_RPLC_Pos_4Germination RPLC (C18) Positive
SA193967Germinated_RPLC_Pos_3Germination RPLC (C18) Positive
SA193968Germinated_RPLC_Pos_2Germination RPLC (C18) Positive
SA193969Hydrated_HILIC_Neg_3Hydration HILIC Negative
SA193970Hydrated_HILIC_Neg_4Hydration HILIC Negative
SA193971Hydrated_HILIC_Neg_1Hydration HILIC Negative
SA193972Hydrated_HILIC_Neg_2Hydration HILIC Negative
SA193973Hydrated_HILIC_Pos_4Hydration HILIC Positive
SA193974Hydrated_HILIC_Pos_1Hydration HILIC Positive
SA193975Hydrated_HILIC_Pos_3Hydration HILIC Positive
SA193976Hydrated_HILIC_Pos_2Hydration HILIC Positive
SA193977Hydrated_Lipids_Neg_4Hydration Lipidomics (C8) Negative
SA193978Hydrated_Lipids_Neg_1Hydration Lipidomics (C8) Negative
SA193979Hydrated_Lipids_Neg_2Hydration Lipidomics (C8) Negative
SA193980Hydrated_Lipids_Neg_3Hydration Lipidomics (C8) Negative
SA193981Hydrated_Lipids_Pos_1Hydration Lipidomics (C8) Positive
SA193982Hydrated_Lipids_Pos_2Hydration Lipidomics (C8) Positive
SA193983Hydrated_Lipids_Pos_4Hydration Lipidomics (C8) Positive
SA193984Hydrated_Lipids_Pos_3Hydration Lipidomics (C8) Positive
SA193985Hydrated_RPLC_Neg_3Hydration RPLC (C18) Negative
SA193986Hydrated_RPLC_Neg_1Hydration RPLC (C18) Negative
SA193987Hydrated_RPLC_Neg_2Hydration RPLC (C18) Negative
SA193988Hydrated_RPLC_Neg_4Hydration RPLC (C18) Negative
SA193989Hydrated_RPLC_Pos_3Hydration RPLC (C18) Positive
SA193990Hydrated_RPLC_Pos_1Hydration RPLC (C18) Positive
SA193991Hydrated_RPLC_Pos_2Hydration RPLC (C18) Positive
SA193992Hydrated_RPLC_Pos_4Hydration RPLC (C18) Positive
SA193993Mature_HILIC_Neg_1Mature HILIC Negative
SA193994Mature_HILIC_Neg_2Mature HILIC Negative
SA193995Mature_HILIC_Neg_3Mature HILIC Negative
SA193996Mature_HILIC_Neg_4Mature HILIC Negative
SA193997Mature_HILIC_Pos_3Mature HILIC Positive
SA193998Mature_HILIC_Pos_2Mature HILIC Positive
SA193999Mature_HILIC_Pos_4Mature HILIC Positive
SA194000Mature_HILIC_Pos_1Mature HILIC Positive
SA194001Mature_Lipids_Neg_4Mature Lipidomics (C8) Negative
SA194002Mature_Lipids_Neg_3Mature Lipidomics (C8) Negative
SA194003Mature_Lipids_Neg_2Mature Lipidomics (C8) Negative
SA194004Mature_Lipids_Neg_1Mature Lipidomics (C8) Negative
SA194005Mature_Lipids_Pos_1Mature Lipidomics (C8) Positive
SA194006Mature_Lipids_Pos_3Mature Lipidomics (C8) Positive
SA194007Mature_Lipids_Pos_4Mature Lipidomics (C8) Positive
SA194008Mature_Lipids_Pos_2Mature Lipidomics (C8) Positive
SA194009Mature_RPLC_Neg_4Mature RPLC (C18) Negative
SA194010Mature_RPLC_Neg_3Mature RPLC (C18) Negative
SA194011Mature_RPLC_Neg_1Mature RPLC (C18) Negative
SA194012Mature_RPLC_Neg_2Mature RPLC (C18) Negative
SA194013Mature_RPLC_Pos_4Mature RPLC (C18) Positive
SA194014Mature_RPLC_Pos_3Mature RPLC (C18) Positive
SA194015Mature_RPLC_Pos_2Mature RPLC (C18) Positive
SA194016Mature_RPLC_Pos_1Mature RPLC (C18) Positive
Showing results 1 to 72 of 72

Collection:

Collection ID:CO002135
Collection Summary:The Arabidopsis Col-0 plants were grown under controlled temperature (22°C) with a 16-h light (100-150 µmol m-2 s-1)/ 8-h dark photoperiod. Mature pollen grains from the fully opened flowers were collected from more than 1000 plants using a vacuum cleaner method (Johnson-Brousseau and McCormick, 2004) at around 5 hours into the light period. For mature pollen samples, collected pollen grains were resuspended in 2 ml of ice-cold Pollen Isolation Buffer (PIB, composed of 100 mM NaPO4, pH 7.5, 1 mM EDTA, and 0.1% (v/v) Triton X-100) right after collection followed by centrifuging at 15,000 g for 1 min (4°C). For hydrated pollen and germinated pollen samples, mature pollen grains were germinated in vitro according to a previously described pollen transcriptome study (Wang et al., 2008). In brief, pollen pellets were washed with 1 ml of liquid Pollen Germination Medium (PGM, composed of 15% (w/v) sucrose, 1.5 mM boric acid, 0.8 mM MgSO4, 1 mM KCl, 5 mM MES, 0.05% (w/v) lactalbumin hydrolysate, 10 µM myo-inositol, 5 mM CaCl2) before they were resuspended in 30 µl of liquid PGM and subsequently cultured in Petri dishes (35 mm in diameter). A 70 µm mesh was used to cover the pollen droplet to create a thin layer for optimal germination for each Petri dish. The Petri dishes were covered and placed in the dark for 45 min or 4 h and collected as hydrated pollen or germinated pollen, respectively. All pollen samples were washed by 1 ml ice-cold ddH2O three times before being stored in a -80 °C freezer.
Collection Protocol Filename:PlantGrowth_PollenCollection.docx
Sample Type:Plant
Collection Method:Flash frozen in Liquid N2
Collection Location:University of Illinois, Urbana-Champain
Storage Conditions:-80℃

Treatment:

Treatment ID:TR002154
Treatment Summary:For hydrated pollen and germinated pollen samples, mature pollen grains were germinated in vitro according to a previously described pollen transcriptome study (Wang et al., 2008). In brief, pollen pellets were washed with 1 ml of liquid Pollen Germination Medium (PGM, composed of 15% (w/v) sucrose, 1.5 mM boric acid, 0.8 mM MgSO4, 1 mM KCl, 5 mM MES, 0.05% (w/v) lactalbumin hydrolysate, 10 µM myo-inositol, 5 mM CaCl2) before they were resuspended in 30 µl of liquid PGM and subsequently cultured in Petri dishes (35 mm in diameter). A 70 µm mesh was used to cover the pollen droplet to create a thin layer for optimal germination for each Petri dish. The Petri dishes were covered and placed in the dark for 45 min or 4 h and collected as hydrated pollen or germinated pollen, respectively.

Sample Preparation:

Sampleprep ID:SP002148
Sampleprep Summary:Total metabolites from pollen were extracted using a phase separation method previously described (Kambhampati et al., 2021) with slight modifications. Briefly, 20-30 mg pollen tissue, collected in Eppendorf tubes, was extracted using 700 µL of chilled 7:3 (v/v) methanol: chloroform spiked with 50 µM each of 1.4-piperazinediethanesulfonic acid (PEPES), ribitol, and norvaline as internal standards. After two metal beads were also added to the samples, they were homogenized using a Tissue-Lyser for 5 min at 30 Hz. The samples were incubated on a rotary shaker at 4°C for 2 hours after which 300 µL ddH2O was added. The samples were then centrifuged at 14,000 rpm for 10 min to achieve phase separation and the upper aqueous phase, as well as the lower organic phase, were collected separately. The aqueous phases containing polar and nonpolar metabolites were split into two equal parts and dried using a speed vacuum centrifuge (Labconco®, Kansas City, USA). The two dried parts were re-suspended in 50 µL 80% (v/v) methanol, and 30 % (v/v) methanol for metabolomics analyses using hydrophilic interaction (HILIC) and reverse phase chromatography (RPLC), respectively. The organic phase was also dried using a speed vacuum centrifuge and re-suspended in 50 µL of 49:49:2 (v/v/v) mixture of acetonitrile: methanol: chloroform. All samples were filtered using a 0.8 µM PES membrane centrifuge filter (Sartorius, Goettingen, Germany) and transferred to a glass vial for injection into an LC-MS/MS system.
Sampleprep Protocol Filename:Total_Metabolite_Extraction.docx
Processing Storage Conditions:-80℃

Combined analysis:

Analysis ID AN003354 AN003355 AN003356 AN003357 AN003358 AN003359
Analysis type MS MS MS MS MS MS
Chromatography type HILIC HILIC Reversed phase Reversed phase Reversed phase Reversed phase
Chromatography system Eksigent Ekspert microLC 200 Eksigent Ekspert microLC 200 Eksigent Ekspert microLC 200 Eksigent Ekspert microLC 200 Eksigent Ekspert microLC 200 Eksigent Ekspert microLC 200
Column SeQuant Zic-HILIC (150 x 0.5mm,5um) SeQuant Zic-HILIC (150 x 0.5mm,5um) Higgins Analytica Targa C18 (150 x 0.3mm,3.5um) Higgins Analytica Targa C18 (150 x 0.3mm,3.5um) Machery-Nagel Nucleodur C8 (150 x 0.5mm,5um) Machery-Nagel Nucleodur C8 (150 x 0.5mm,5um)
MS Type ESI ESI ESI ESI ESI ESI
MS instrument type Orbitrap Orbitrap Orbitrap Orbitrap Orbitrap Orbitrap
MS instrument name Thermo Q Exactive Orbitrap Thermo Q Exactive Orbitrap Thermo Q Exactive Orbitrap Thermo Q Exactive Orbitrap Thermo Q Exactive Orbitrap Thermo Q Exactive Orbitrap
Ion Mode POSITIVE NEGATIVE POSITIVE NEGATIVE POSITIVE NEGATIVE
Units Intensity Intensity Intensity Intensity Intensity Intensity

Chromatography:

Chromatography ID:CH002483
Methods Filename:Chromatography_PollentMetabolomics.docx
Instrument Name:Eksigent Ekspert microLC 200
Column Name:SeQuant Zic-HILIC (150 x 0.5mm,5um)
Column Temperature:35
Flow Rate:0.015
Solvent A:100% water; 10 mM ammonium bicarbonate
Solvent B:95% acetonitrile/5% water; 10 mM ammonium bicarbonate
Chromatography Type:HILIC
  
Chromatography ID:CH002484
Methods Filename:Chromatography_PollentMetabolomics.docx
Instrument Name:Eksigent Ekspert microLC 200
Column Name:Higgins Analytica Targa C18 (150 x 0.3mm,3.5um)
Column Temperature:35
Flow Rate:0.015
Solvent A:100% water; 0.1% formic acid
Solvent B:100% acetonitrile; 0.1% formic acid
Chromatography Type:Reversed phase
  
Chromatography ID:CH002485
Methods Filename:Chromatography_PollenMetabolomics
Instrument Name:Eksigent Ekspert microLC 200
Column Name:Machery-Nagel Nucleodur C8 (150 x 0.5mm,5um)
Column Temperature:35
Flow Rate:0.04
Solvent A:100% water; 0.1% acetic acid; 10 mM ammonium acetate
Solvent B:70% acetonitrile/30% isopropanol; 0.1% acetic acid; 10 mM ammonium acetate
Chromatography Type:Reversed phase

MS:

MS ID:MS003123
Analysis ID:AN003354
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:All parameters listed in the attached document
Ion Mode:POSITIVE
Acquisition Parameters File:MS_DataAcquisition.docx
  
MS ID:MS003124
Analysis ID:AN003355
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:All parameters listed in the attached document
Ion Mode:NEGATIVE
Acquisition Parameters File:MS_DataAcquisition.docx
  
MS ID:MS003125
Analysis ID:AN003356
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:All parameters listed in the attached document
Ion Mode:POSITIVE
Acquisition Parameters File:MS_DataAcquisition.docx
  
MS ID:MS003126
Analysis ID:AN003357
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:All parameters listed in the attached document
Ion Mode:NEGATIVE
Acquisition Parameters File:MS_DataAcquisition.docx
  
MS ID:MS003127
Analysis ID:AN003358
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:All parameters listed in the attached document
Ion Mode:POSITIVE
Acquisition Parameters File:MS_DataAcquisition.docx
  
MS ID:MS003128
Analysis ID:AN003359
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:All parameters listed in the attached document
Ion Mode:NEGATIVE
Acquisition Parameters File:MS_DataAcquisition.docx
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