Summary of Study ST001812

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 PR001145. The data can be accessed directly via it's Project DOI: 10.21228/M8Q12H This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

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 ID	ST001812
Study Title	Evidence that class I glutamine amidotransferase, GAT1_2.1, acts as a glutaminase in roots of Arabidopsis thaliana (part II)
Study Type	Targeted Metabolite Quantification
Study Summary	In this study, we used Arabidopsis root extracts, spiked with amide nitrogen labeled (15N1) Glutamine and a purified recombinant protein, both full length and glutaminase domain only versions, to determine the amido group acceptor, if any, in the glutamine amidotransferase reaction.
Institute	Agriculture and Agri-Food Canada
Department	London Research and Development Centre
Laboratory	Frederic Marsolais
Last Name	Kambhampati
First Name	Shrikaar
Address	1391 Sandford St, London, ON N5V 4T3, Canada
Email	shrikaar.k@gmail.com
Phone	3144025550
Submit Date	2021-06-01
Raw Data Available	Yes
Raw Data File Type(s)	raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2021-06-16
Release Version	1

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Project:

Project ID:	PR001145
Project DOI:	doi: 10.21228/M8Q12H
Project Title:	Evidence that class I glutamine amidotransferase, GAT1_2.1, acts as a glutaminase in roots of Arabidopsis thaliana
Project Summary:	Carbon and Nitrogen balance in plant leaves, required for sustained growth, is achieved by inter-relationships between the processes of photosynthesis, respiration and amino acid metabolism in a photoperiod dependent manner. The GS/GOGAT cycle is one such mechanism and is highly elucidated in plants to serve as a crossroad between C and N metabolism. Non-photosynthetic tissues (e.g., roots, germinating seeds), however, lack a sufficient supply of carbon skeletons under high N conditions and hence may resort to other mechanisms, along with GS/GOGAT cycle, to achieve the aforementioned C/N balance. Here, we propose a potential role of an enzyme, GAT1_2.1, in hydrolyzing excess glutamine to Glu, which channels carbon skeletons to the TCA cycle, under high N conditions, using Arabidopsis as a model. GAT1_2.1, a class I glutamine amidotrasferase of unknown substrate specificity, was shown to be highly responsive to N status, localized in mitochondria and is highly co-expressed with Glutamate Dehydrogenase 2 (GDH2). Arabidopsis mutants lacking GAT1_2.1 have elevated GABA shunt pathway activity to replenish the depleted levels of Glu. This Glu may then be deaminated to 2-oxoglutarate by GDH2 and channeled into the TCA cycle thus providing a crossroad between C and N metabolism in root mitochondria. We use a metabolomics approach to demonstrate the difference in quantities of pathway intermediates between wild type Arabidopsis roots and gat1_2.1 mutants using glutamine as organic nitrogen treatment and KNO3 and Glu treatments as negative and positive controls, respectively. In addition, we used Arabidopsis root extracts, spiked with amide nitrogen labeled (15N1) Glutamine and a purified recombinant protein, both full length and glutaminase domain only versions, to determine the amido group acceptor, if any, in the glutamine amidotransferase reaction.
Institute:	Agriculture and Agri-Food Canada
Department:	London Research and Development Centre
Laboratory:	Frederic Marsolais
Last Name:	Kambhampati
First Name:	Shrikaar
Address:	1391 Sandford St, London, ON N5V 4T3, Canada
Email:	shrikaar.k@gmail.com
Phone:	3144025550
Funding Source:	Natural Sciences and Engineering Research Council of Canada
Contributors:	Shrikaar Kambhampati, Justin Renaud, Frederic Marsolais

Subject:

Subject ID:	SU001889
Subject Type:	Plant
Subject Species:	Arabidopsis thaliana
Taxonomy ID:	3702
Genotype Strain:	Col-0
Age Or Age Range:	10 day old seedlings
Gender:	Not applicable

Factors:

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

mb_sample_id	local_sample_id	Rawfilename
SA168270	E_15NGln_1_neg	E_15NGln_1_neg
SA168271	E_15NGln_1_pos	E_15NGln_1_pos
SA168272	E_15NGln_2_neg	E_15NGln_2_neg
SA168273	E_15NGln_2_pos	E_15NGln_2_pos
SA168274	E_15NGln_3_neg	E_15NGln_3_neg
SA168275	E_15NGln_3_pos	E_15NGln_3_pos
SA168276	E_15NGln_4_neg	E_15NGln_4_neg
SA168277	E_15NGln_4_pos	E_15NGln_4_pos
SA168278	E_15NGln_D_1_neg	E_15NGln_D_1_neg
SA168279	E_15NGln_D_1_pos	E_15NGln_D_1_pos
SA168280	E_15NGln_D_2_neg	E_15NGln_D_2_neg
SA168281	E_15NGln_D_2_pos	E_15NGln_D_2_pos
SA168282	E_15NGln_D_3_neg	E_15NGln_D_3_neg
SA168283	E_15NGln_D_3_pos	E_15NGln_D_3_pos
SA168284	E_15NGln_D_4_neg	E_15NGln_D_4_neg
SA168285	E_15NGln_D_4_pos	E_15NGln_D_4_pos
SA168286	E_15NGln_FL_1_neg	E_15NGln_FL_1_neg
SA168287	E_15NGln_FL_1_pos	E_15NGln_FL_1_pos
SA168288	E_15NGln_FL_2_neg	E_15NGln_FL_2_neg
SA168289	E_15NGln_FL_2_pos	E_15NGln_FL_2_pos
SA168290	E_15NGln_FL_3_neg	E_15NGln_FL_3_neg
SA168291	E_15NGln_FL_3_pos	E_15NGln_FL_3_pos
SA168292	E_15NGln_FL_4_neg	E_15NGln_FL_4_neg
SA168293	E_15NGln_FL_4_pos	E_15NGln_FL_4_pos
SA168294	E_FL_1_neg	E_FL_1_neg
SA168295	E_FL_1_pos	E_FL_1_pos
SA168296	E_FL_2_neg	E_FL_2_neg
SA168297	E_FL_2_pos	E_FL_2_pos
SA168298	E_FL_3_neg	E_FL_3_neg
SA168299	E_FL_3_pos	E_FL_3_pos
SA168300	E_FL_4_neg	E_FL_4_neg
SA168301	E_FL_4_pos	E_FL_4_pos
SA168302	E_Gln_1_neg	E_Gln_1_neg
SA168303	E_Gln_1_pos	E_Gln_1_pos
SA168304	E_Gln_2_neg	E_Gln_2_neg
SA168305	E_Gln_2_pos	E_Gln_2_pos
SA168306	E_Gln_3_neg	E_Gln_3_neg
SA168307	E_Gln_3_pos	E_Gln_3_pos
SA168308	E_Gln_4_neg	E_Gln_4_neg
SA168309	E_Gln_4_pos	E_Gln_4_pos
SA168310	Extract_1_neg	Extract_1_neg
SA168311	Extract_1_pos	Extract_1_pos
SA168312	Extract_2_neg	Extract_2_neg
SA168313	Extract_2_pos	Extract_2_pos
SA168314	Extract_3_neg	Extract_3_neg
SA168315	Extract_3_pos	Extract_3_pos
SA168316	Extract_4_neg	Extract_4_neg
SA168317	Extract_4_pos	Extract_4_pos

Showing results 1 to 48 of 48

Showing results 1 to 48 of 48

Collection:

Collection ID:	CO001882
Collection Summary:	Roots from 50 seedlings grown in plates containing required treatment were collected and processed as single replicate.
Collection Protocol ID:	001
Sample Type:	Plant
Collection Method:	50 mg collected and flash frozen in Liquid N2
Collection Location:	London Research and Development Center
Storage Conditions:	-80℃

Treatment:

Treatment ID:	TR001902
Treatment Summary:	Wild-type Arabidopsis ecotype Columbia and gat1_2.1 T-DNA insertion lines were used for growth. Plants were grown on vertical plates at 22 °C under continuous light (ca. 70 μmol m-2 s-2), as previously described by Ivanov et al. (2012) on a defined nutrient medium containing a final concentration of 10 mM potassium phosphate (pH 6.5), 5 mM KNO3, 2 mM MgSO4, 1 mM CaCl2, 125 μg FeNaEDTA, micronutrients (50 mM H3BO3, 12 mM MnSO4, 1 mM ZnCl2, 1 mM CuSO4 and 0.2 mM Na2MoO4), 1% sucrose and 1% agar [28].

Treatment ID:

TR001902

Treatment Summary:

Wild-type Arabidopsis ecotype Columbia and gat1_2.1 T-DNA insertion lines were used for growth. Plants were grown on vertical plates at 22 °C under continuous light (ca. 70 μmol m-2 s-2), as previously described by Ivanov et al. (2012) on a defined nutrient medium containing a final concentration of 10 mM potassium phosphate (pH 6.5), 5 mM KNO3, 2 mM MgSO4, 1 mM CaCl2, 125 μg FeNaEDTA, micronutrients (50 mM H3BO3, 12 mM MnSO4, 1 mM ZnCl2, 1 mM CuSO4 and 0.2 mM Na2MoO4), 1% sucrose and 1% agar [28].

Sample Preparation:

Sampleprep ID:	SP001895
Sampleprep Summary:	Fifty mg of root tissue was excised from 10 day old seedlings of Arabidopsis grown with 5 mM KNO3, collected in 2 mL Eppendorf tubes and flash frozen in liquid N2. Frozen tissue was homogenized using a tissue lyser and metabolites were isolated using 1 mL of methanol:water (4:1) with incubation in an ultra-sonication bath for 20 min followed by shaking for 30 min at 4 °C. The mixture was centrifuged at 12,000 × g for 10 min at 4 °C and 700 µl of the supernatant was transferred into fresh tubes and evaporated to dryness using a Vacufuge at ambient temperature. Dried metabolite extracts were re-suspended in HEPES buffer pH 7.5 instead of 1:1 methanol:water. Samples were spiked with a final concentration of 1 µM 15N Gln and 5 µg of the full length or glutaminase domain versions of recombinant GAT1_2.1 protein along with 2 mM DTT and 5 mM MgCl2. Following this, samples were incubated at 37 °C for 2 hours and then filtered through a 3K micro centrifuge filter (Sigma-Aldrich) to remove the protein. Samples were then evaporated to dryness using a vacufuge at ambient temperature and the residue was re-dissolved in 1:1 methanol:water, filtered with a 0.2 µm PTFE microfuge filters (Whatman) and subjected to LC-MS analysis and ammonium quantification.

Sampleprep ID:

SP001895

Sampleprep Summary:

Fifty mg of root tissue was excised from 10 day old seedlings of Arabidopsis grown with 5 mM KNO3, collected in 2 mL Eppendorf tubes and flash frozen in liquid N2. Frozen tissue was homogenized using a tissue lyser and metabolites were isolated using 1 mL of methanol:water (4:1) with incubation in an ultra-sonication bath for 20 min followed by shaking for 30 min at 4 °C. The mixture was centrifuged at 12,000 × g for 10 min at 4 °C and 700 µl of the supernatant was transferred into fresh tubes and evaporated to dryness using a Vacufuge at ambient temperature. Dried metabolite extracts were re-suspended in HEPES buffer pH 7.5 instead of 1:1 methanol:water. Samples were spiked with a final concentration of 1 µM 15N Gln and 5 µg of the full length or glutaminase domain versions of recombinant GAT1_2.1 protein along with 2 mM DTT and 5 mM MgCl2. Following this, samples were incubated at 37 °C for 2 hours and then filtered through a 3K micro centrifuge filter (Sigma-Aldrich) to remove the protein. Samples were then evaporated to dryness using a vacufuge at ambient temperature and the residue was re-dissolved in 1:1 methanol:water, filtered with a 0.2 µm PTFE microfuge filters (Whatman) and subjected to LC-MS analysis and ammonium quantification.

Combined analysis:

Analysis ID	AN002937	AN002938
Analysis type	MS	MS
Chromatography type	HILIC	HILIC
Chromatography system	Agilent 1290 Infinity II	Agilent 1290 Infinity II
Column	SeQuant ZIC-HILIC (100 x 2.1mm,3.5um)	SeQuant ZIC-HILIC (100 x 2.1mm,3.5um)
MS Type	ESI	ESI
MS instrument type	Orbitrap	Orbitrap
MS instrument name	Thermo Q Exactive Orbitrap	Thermo Q Exactive Orbitrap
Ion Mode	POSITIVE	NEGATIVE
Units	Intensity	Intensity

Chromatography:

Chromatography ID:	CH002176
Methods Filename:	Enzyme_spike_analysis.pdf
Instrument Name:	Agilent 1290 Infinity II
Column Name:	SeQuant ZIC-HILIC (100 x 2.1mm,3.5um)
Column Temperature:	35
Flow Rate:	0.3 mL min-1
Internal Standard:	13C6 Phenylalanine
Solvent A:	100% water; 5 mM ammonium acetate, pH 4
Solvent B:	90% acetonitrile/10% water; 0.1% acetic acid
Chromatography Type:	HILIC

MS:

MS ID:	MS002728
Analysis ID:	AN002937
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Full MS measurements were collected from mass ranges of 75-1100 m/z and 65-900 m/z in positive and negative ionization modes respectively at 140,000 resolutions. The AGC target and maximum IT was set to 3 e6 and 524 ms respectively.
Ion Mode:	POSITIVE
Capillary Temperature:	250
Capillary Voltage:	3.9
Analysis Protocol File:	Enzyme_spike_analysis.pdf

MS ID:	MS002729
Analysis ID:	AN002938
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Full MS measurements were collected from mass ranges of 75-1100 m/z and 65-900 m/z in positive and negative ionization modes respectively at 140,000 resolutions. The AGC target and maximum IT was set to 3 e6 and 524 ms respectively.
Ion Mode:	NEGATIVE
Capillary Temperature:	250
Capillary Voltage:	3.5
Analysis Protocol File:	Enzyme_spike_analysis.pdf