Summary of Study ST001811

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.

Study ID	ST001811
Study Title	Evidence that class I glutamine amidotransferase, GAT1_2.1, acts as a glutaminase in roots of Arabidopsis thaliana
Study Type	Targeted Metabolite Quantification
Study Summary	In this study, we use a targeted metabolite quantification 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.
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:	SU001888
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	Raw file name	SampleType	Genotype
SA168210	AA-CS1-PRM	AA-CS1-PRM	Amino acid standard	Standard
SA168211	AA-CS2-PRM	AA-CS2-PRM	Amino acid standard	Standard
SA168212	AA-CS3-PRM	AA-CS3-PRM	Amino acid standard	Standard
SA168213	AA-CS4-PRM	AA-CS4-PRM	Amino acid standard	Standard
SA168214	AA-CS5-PRM	AA-CS5-PRM	Amino acid standard	Standard
SA168215	AA-CS6-PRM	AA-CS6-PRM	Amino acid standard	Standard
SA168216	GAT-GLN-1-neg-PRM	GAT-GLN-1-neg-PRM	Negative Control	gat1_2.1
SA168217	GAT-GLN-1-pos-PRM	GAT-GLN-1-pos-PRM	Negative Control	gat1_2.1
SA168218	GAT-GLN-2-neg-PRM	GAT-GLN-2-neg-PRM	Negative Control	gat1_2.1
SA168219	GAT-GLN-2-pos-PRM	GAT-GLN-2-pos-PRM	Negative Control	gat1_2.1
SA168220	GAT-GLN-3-neg-PRM	GAT-GLN-3-neg-PRM	Negative Control	gat1_2.1
SA168221	GAT-GLN-3-pos-PRM	GAT-GLN-3-pos-PRM	Negative Control	gat1_2.1
SA168222	GATp10GLN-1-neg-PRM	GATp10GLN-1-neg-PRM	Long Gln treatment	gat1_2.1
SA168223	GATp10GLN-1-pos-PRM	GATp10GLN-1-pos-PRM	Long Gln treatment	gat1_2.1
SA168224	GATp10GLN-2-neg-PRM	GATp10GLN-2-neg-PRM	Long Gln treatment	gat1_2.1
SA168225	GATp10GLN-2-pos-PRM	GATp10GLN-2-pos-PRM	Long Gln treatment	gat1_2.1
SA168226	GATp10GLN-3-neg-PRM	GATp10GLN-3-neg-PRM	Long Gln treatment	gat1_2.1
SA168227	GATp10GLN-3-pos-PRM	GATp10GLN-3-pos-PRM	Long Gln treatment	gat1_2.1
SA168228	GATp2GLN-1-neg-PRM	GATp2GLN-1-neg-PRM	Short Gln treatment	gat1_2.1
SA168229	GATp2GLN-1-pos-PRM	GATp2GLN-1-pos-PRM	Short Gln treatment	gat1_2.1
SA168230	GATp2GLN-2-neg-PRM	GATp2GLN-2-neg-PRM	Short Gln treatment	gat1_2.1
SA168231	GATp2GLN-2-pos-PRM	GATp2GLN-2-pos-PRM	Short Gln treatment	gat1_2.1
SA168232	GATp2GLN-3-neg-PRM	GATp2GLN-3-neg-PRM	Short Gln treatment	gat1_2.1
SA168233	GATp2GLN-3-pos-PRM	GATp2GLN-3-pos-PRM	Short Gln treatment	gat1_2.1
SA168234	GATp2GLU-1-neg-PRM	GATp2GLU-1-neg-PRM	Positive Control	gat1_2.1
SA168235	GATp2GLU-1-pos-PRM	GATp2GLU-1-pos-PRM	Positive Control	gat1_2.1
SA168236	GATp2GLU-2-neg-PRM	GATp2GLU-2-neg-PRM	Positive Control	gat1_2.1
SA168237	GATp2GLU-2-pos-PRM	GATp2GLU-2-pos-PRM	Positive Control	gat1_2.1
SA168238	GATp2GLU-3-neg-PRM	GATp2GLU-3-neg-PRM	Positive Control	gat1_2.1
SA168239	GATp2GLU-3-pos-PRM	GATp2GLU-3-pos-PRM	Positive Control	gat1_2.1
SA168240	OA-CS1-PRM	OA-CS1-PRM	Organic acid standard	Standard
SA168241	OA-CS2-PRM	OA-CS2-PRM	Organic acid standard	Standard
SA168242	OA-CS3-PRM	OA-CS3-PRM	Organic acid standard	Standard
SA168243	OA-CS4-PRM	OA-CS4-PRM	Organic acid standard	Standard
SA168244	OA-CS5-PRM	OA-CS5-PRM	Organic acid standard	Standard
SA168245	OA-CS6-PRM	OA-CS6-PRM	Organic acid standard	Standard
SA168246	WT-GLN-1-neg-PRM	WT-GLN-1-neg-PRM	Negative Control	Wildtype
SA168247	WT-GLN-1-pos-PRM	WT-GLN-1-pos-PRM	Negative Control	Wildtype
SA168248	WT-GLN-2-neg-PRM	WT-GLN-2-neg-PRM	Negative Control	Wildtype
SA168249	WT-GLN-2-pos-PRM	WT-GLN-2-pos-PRM	Negative Control	Wildtype
SA168250	WT-GLN-3-neg-PRM	WT-GLN-3-neg-PRM	Negative Control	Wildtype
SA168251	WT-GLN-3-pos-PRM	WT-GLN-3-pos-PRM	Negative Control	Wildtype
SA168252	WTp10GLN-1-neg-PRM	WTp10GLN-1-neg-PRM	Long Gln treatment	Wildtype
SA168253	WTp10GLN-1-pos-PRM	WTp10GLN-1-pos-PRM	Long Gln treatment	Wildtype
SA168254	WTp10GLN-2-neg-PRM	WTp10GLN-2-neg-PRM	Long Gln treatment	Wildtype
SA168255	WTp10GLN-2-pos-PRM	WTp10GLN-2-pos-PRM	Long Gln treatment	Wildtype
SA168256	WTp10GLN-3-neg-PRM	WTp10GLN-3-neg-PRM	Long Gln treatment	Wildtype
SA168257	WTp10GLN-3-pos-PRM	WTp10GLN-3-pos-PRM	Long Gln treatment	Wildtype
SA168258	WTp2GLN-1-neg-PRM	WTp2GLN-1-neg-PRM	Short Gln treatment	Wildtype
SA168259	WTp2GLN-1-pos-PRM	WTp2GLN-1-pos-PRM	Short Gln treatment	Wildtype
SA168260	WTp2GLN-2-neg-PRM	WTp2GLN-2-neg-PRM	Short Gln treatment	Wildtype
SA168261	WTp2GLN-2-pos-PRM	WTp2GLN-2-pos-PRM	Short Gln treatment	Wildtype
SA168262	WTp2GLN-3-neg-PRM	WTp2GLN-3-neg-PRM	Short Gln treatment	Wildtype
SA168263	WTp2GLN-3-pos-PRM	WTp2GLN-3-pos-PRM	Short Gln treatment	Wildtype
SA168264	WTp2GLU-1-neg-PRM	WTp2GLU-1-neg-PRM	Positive Control	Wildtype
SA168265	WTp2GLU-1-pos-PRM	WTp2GLU-1-pos-PRM	Positive Control	Wildtype
SA168266	WTp2GLU-2-neg-PRM	WTp2GLU-2-neg-PRM	Positive Control	Wildtype
SA168267	WTp2GLU-2-pos-PRM	WTp2GLU-2-pos-PRM	Positive Control	Wildtype
SA168268	WTp2GLU-3-neg-PRM	WTp2GLU-3-neg-PRM	Positive Control	Wildtype
SA168269	WTp2GLU-3-pos-PRM	WTp2GLU-3-pos-PRM	Positive Control	Wildtype

Showing results 1 to 60 of 60

Showing results 1 to 60 of 60

Collection:

Collection ID:	CO001881
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:	TR001901
Treatment Summary:	Wild-type Arabidopsis ecotype Columbia and gat1_2.1 T-DNA insertion lines were used for Gln and Glu treatments. 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]. Ten-day old seedlings were transferred to plates containing the same medium without nitrogen as control or 10 mM Gln as sole N source. After 2 h, root tissue was harvested, frozen in liquid N2 and stored at -80 °C until total metabolite extractions was carried out. For growth in Gln and Glu, the same media and growth conditions were used with the exception of 5 mM KNO3 being substituted with either 2 mM Gln or 2 mM Glu and tissue was collected after 10 days.

Treatment ID:

TR001901

Treatment Summary:

Wild-type Arabidopsis ecotype Columbia and gat1_2.1 T-DNA insertion lines were used for Gln and Glu treatments. 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]. Ten-day old seedlings were transferred to plates containing the same medium without nitrogen as control or 10 mM Gln as sole N source. After 2 h, root tissue was harvested, frozen in liquid N2 and stored at -80 °C until total metabolite extractions was carried out. For growth in Gln and Glu, the same media and growth conditions were used with the exception of 5 mM KNO3 being substituted with either 2 mM Gln or 2 mM Glu and tissue was collected after 10 days.

Sample Preparation:

Sampleprep ID:	SP001894
Sampleprep Summary:	Fifty mg of root tissue was excised from 10 day old seedlings of WT or gat1_2.1 grown under conditions described above, 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. The residue was re-dissolved in 500 µl of 1:1 methanol:water and the samples were filtered using a 0.2 µm PTFE microfuge filter (Cytiva Whatman). Five µl of 1 µg/mL 13C6 Phe was added to the samples for monitoring the quality of LC-MS runs.

Sampleprep ID:

SP001894

Sampleprep Summary:

Fifty mg of root tissue was excised from 10 day old seedlings of WT or gat1_2.1 grown under conditions described above, 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. The residue was re-dissolved in 500 µl of 1:1 methanol:water and the samples were filtered using a 0.2 µm PTFE microfuge filter (Cytiva Whatman). Five µl of 1 µg/mL 13C6 Phe was added to the samples for monitoring the quality of LC-MS runs.

Combined analysis:

Analysis ID	AN002935	AN002936
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	umol g FW-1	umol g FW-1

Chromatography:

Chromatography ID:	CH002175
Methods Filename:	Targeted Metabolite Analysis
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:	MS002726
Analysis ID:	AN002935
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	The following heated electrospray ionization (HESI) conditions were optimized for the analysis of amino and organic acids: spray voltage, 3.9 kV (ESI+), 3.5 kV (ESI-); capillary temperature, 250 °C; probe heater temperature, 450 °C; sheath gas, 30 arbitrary units; auxiliary gas, 8 arbitrary units; and S-Lens RF level, 60%. Injections of 5 μl were used with a flow rate of 0.3 mL min-1. Compounds were detected and monitored using targeted MS/MS, spectra were collected at 17,500 resolution, AGC target 1e6, maximum IT 65 ms, isolation window of 1 m/z, normalized collision energy of 30, intensity threshold of 1.6e5 and 10s dynamic exclusion.
Ion Mode:	POSITIVE
Analysis Protocol File:	Targeted_Metabolite_Analysis.pdf

MS ID:	MS002727
Analysis ID:	AN002936
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	The following heated electrospray ionization (HESI) conditions were optimized for the analysis of amino and organic acids: spray voltage, 3.9 kV (ESI+), 3.5 kV (ESI-); capillary temperature, 250 °C; probe heater temperature, 450 °C; sheath gas, 30 arbitrary units; auxiliary gas, 8 arbitrary units; and S-Lens RF level, 60%. Injections of 5 μl were used with a flow rate of 0.3 mL min-1. Compounds were detected and monitored using targeted MS/MS, spectra were collected at 17,500 resolution, AGC target 1e6, maximum IT 65 ms, isolation window of 1 m/z, normalized collision energy of 30, intensity threshold of 1.6e5 and 10s dynamic exclusion.
Ion Mode:	NEGATIVE
Analysis Protocol File:	Targeted_Metabolite_Analysis