#METABOLOMICS WORKBENCH skambhampati_20210601_103541 DATATRACK_ID:2664 STUDY_ID:ST001811 ANALYSIS_ID:AN002935 PROJECT_ID:PR001145
VERSION             	1
CREATED_ON             	June 1, 2021, 3:12 pm
#PROJECT
PR:PROJECT_TITLE                 	Evidence that class I glutamine amidotransferase, GAT1_2.1, acts as a
PR:PROJECT_TITLE                 	glutaminase in roots of Arabidopsis thaliana
PR:PROJECT_SUMMARY               	Carbon and Nitrogen balance in plant leaves, required for sustained growth, is
PR:PROJECT_SUMMARY               	achieved by inter-relationships between the processes of photosynthesis,
PR:PROJECT_SUMMARY               	respiration and amino acid metabolism in a photoperiod dependent manner. The
PR:PROJECT_SUMMARY               	GS/GOGAT cycle is one such mechanism and is highly elucidated in plants to serve
PR:PROJECT_SUMMARY               	as a crossroad between C and N metabolism. Non-photosynthetic tissues (e.g.,
PR:PROJECT_SUMMARY               	roots, germinating seeds), however, lack a sufficient supply of carbon skeletons
PR:PROJECT_SUMMARY               	under high N conditions and hence may resort to other mechanisms, along with
PR:PROJECT_SUMMARY               	GS/GOGAT cycle, to achieve the aforementioned C/N balance. Here, we propose a
PR:PROJECT_SUMMARY               	potential role of an enzyme, GAT1_2.1, in hydrolyzing excess glutamine to Glu,
PR:PROJECT_SUMMARY               	which channels carbon skeletons to the TCA cycle, under high N conditions, using
PR:PROJECT_SUMMARY               	Arabidopsis as a model. GAT1_2.1, a class I glutamine amidotrasferase of unknown
PR:PROJECT_SUMMARY               	substrate specificity, was shown to be highly responsive to N status, localized
PR:PROJECT_SUMMARY               	in mitochondria and is highly co-expressed with Glutamate Dehydrogenase 2
PR:PROJECT_SUMMARY               	(GDH2). Arabidopsis mutants lacking GAT1_2.1 have elevated GABA shunt pathway
PR:PROJECT_SUMMARY               	activity to replenish the depleted levels of Glu. This Glu may then be
PR:PROJECT_SUMMARY               	deaminated to 2-oxoglutarate by GDH2 and channeled into the TCA cycle thus
PR:PROJECT_SUMMARY               	providing a crossroad between C and N metabolism in root mitochondria. We use a
PR:PROJECT_SUMMARY               	metabolomics approach to demonstrate the difference in quantities of pathway
PR:PROJECT_SUMMARY               	intermediates between wild type Arabidopsis roots and gat1_2.1 mutants using
PR:PROJECT_SUMMARY               	glutamine as organic nitrogen treatment and KNO3 and Glu treatments as negative
PR:PROJECT_SUMMARY               	and positive controls, respectively. In addition, we used Arabidopsis root
PR:PROJECT_SUMMARY               	extracts, spiked with amide nitrogen labeled (15N1) Glutamine and a purified
PR:PROJECT_SUMMARY               	recombinant protein, both full length and glutaminase domain only versions, to
PR:PROJECT_SUMMARY               	determine the amido group acceptor, if any, in the glutamine amidotransferase
PR:PROJECT_SUMMARY               	reaction.
PR:INSTITUTE                     	Agriculture and Agri-Food Canada
PR:DEPARTMENT                    	London Research and Development Centre
PR:LABORATORY                    	Frederic Marsolais
PR:LAST_NAME                     	Kambhampati
PR:FIRST_NAME                    	Shrikaar
PR:ADDRESS                       	1391 Sandford St, London, ON N5V 4T3, Canada
PR:EMAIL                         	shrikaar.k@gmail.com
PR:PHONE                         	3144025550
PR:FUNDING_SOURCE                	Natural Sciences and Engineering Research Council of Canada
PR:CONTRIBUTORS                  	Shrikaar Kambhampati, Justin Renaud, Frederic Marsolais
#STUDY
ST:STUDY_TITLE                   	Evidence that class I glutamine amidotransferase, GAT1_2.1, acts as a
ST:STUDY_TITLE                   	glutaminase in roots of Arabidopsis thaliana
ST:STUDY_TYPE                    	Targeted Metabolite Quantification
ST:STUDY_SUMMARY                 	In this study, we use a targeted metabolite quantification approach to
ST:STUDY_SUMMARY                 	demonstrate the difference in quantities of pathway intermediates between wild
ST:STUDY_SUMMARY                 	type Arabidopsis roots and gat1_2.1 mutants using glutamine as organic nitrogen
ST:STUDY_SUMMARY                 	treatment and KNO3 and Glu treatments as negative and positive controls,
ST:STUDY_SUMMARY                 	respectively.
ST:INSTITUTE                     	Agriculture and Agri-Food Canada
ST:DEPARTMENT                    	London Research and Development Centre
ST:LABORATORY                    	Frederic Marsolais
ST:LAST_NAME                     	Kambhampati
ST:FIRST_NAME                    	Shrikaar
ST:ADDRESS                       	1391 Sandford St, London, ON N5V 4T3, Canada
ST:EMAIL                         	shrikaar.k@gmail.com
ST:PHONE                         	3144025550
#SUBJECT
SU:SUBJECT_TYPE                  	Plant
SU:SUBJECT_SPECIES               	Arabidopsis thaliana
SU:TAXONOMY_ID                   	3702
SU:GENOTYPE_STRAIN               	Col-0
SU:AGE_OR_AGE_RANGE              	10 day old seedlings
SU:GENDER                        	Not applicable
#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           	-	WT-GLN-1-pos-PRM	Raw file name:WT-GLN-1-pos-PRM | SampleType:Negative Control | Genotype:Wildtype	RAW_FILE_NAME=10 days in 5 mM KNO3; Polarity=Pos
SUBJECT_SAMPLE_FACTORS           	-	WT-GLN-1-neg-PRM	Raw file name:WT-GLN-1-neg-PRM | SampleType:Negative Control | Genotype:Wildtype	RAW_FILE_NAME=10 days in 5 mM KNO3; Polarity=Neg
SUBJECT_SAMPLE_FACTORS           	-	WT-GLN-2-pos-PRM	Raw file name:WT-GLN-2-pos-PRM | SampleType:Negative Control | Genotype:Wildtype	RAW_FILE_NAME=10 days in 5 mM KNO3; Polarity=Pos
SUBJECT_SAMPLE_FACTORS           	-	WT-GLN-2-neg-PRM	Raw file name:WT-GLN-2-neg-PRM | SampleType:Negative Control | Genotype:Wildtype	RAW_FILE_NAME=10 days in 5 mM KNO3; Polarity=Neg
SUBJECT_SAMPLE_FACTORS           	-	WT-GLN-3-pos-PRM	Raw file name:WT-GLN-3-pos-PRM | SampleType:Negative Control | Genotype:Wildtype	RAW_FILE_NAME=10 days in 5 mM KNO3; Polarity=Pos
SUBJECT_SAMPLE_FACTORS           	-	WT-GLN-3-neg-PRM	Raw file name:WT-GLN-3-neg-PRM | SampleType:Negative Control | Genotype:Wildtype	RAW_FILE_NAME=10 days in 5 mM KNO3; Polarity=Neg
SUBJECT_SAMPLE_FACTORS           	-	GAT-GLN-1-pos-PRM	Raw file name:GAT-GLN-1-pos-PRM | SampleType:Negative Control | Genotype:gat1_2.1	RAW_FILE_NAME=10 days in 5 mM KNO3; Polarity=Pos
SUBJECT_SAMPLE_FACTORS           	-	GAT-GLN-1-neg-PRM	Raw file name:GAT-GLN-1-neg-PRM | SampleType:Negative Control | Genotype:gat1_2.1	RAW_FILE_NAME=10 days in 5 mM KNO3; Polarity=Neg
SUBJECT_SAMPLE_FACTORS           	-	GAT-GLN-2-pos-PRM	Raw file name:GAT-GLN-2-pos-PRM | SampleType:Negative Control | Genotype:gat1_2.1	RAW_FILE_NAME=10 days in 5 mM KNO3; Polarity=Pos
SUBJECT_SAMPLE_FACTORS           	-	GAT-GLN-2-neg-PRM	Raw file name:GAT-GLN-2-neg-PRM | SampleType:Negative Control | Genotype:gat1_2.1	RAW_FILE_NAME=10 days in 5 mM KNO3; Polarity=Neg
SUBJECT_SAMPLE_FACTORS           	-	GAT-GLN-3-pos-PRM	Raw file name:GAT-GLN-3-pos-PRM | SampleType:Negative Control | Genotype:gat1_2.1	RAW_FILE_NAME=10 days in 5 mM KNO3; Polarity=Pos
SUBJECT_SAMPLE_FACTORS           	-	GAT-GLN-3-neg-PRM	Raw file name:GAT-GLN-3-neg-PRM | SampleType:Negative Control | Genotype:gat1_2.1	RAW_FILE_NAME=10 days in 5 mM KNO3; Polarity=Neg
SUBJECT_SAMPLE_FACTORS           	-	WTp2GLN-1-pos-PRM	Raw file name:WTp2GLN-1-pos-PRM | SampleType:Short Gln treatment | Genotype:Wildtype	RAW_FILE_NAME=10 days 5 mM KNO3 + 2 hrs 10 mM Gln; Polarity=Pos
SUBJECT_SAMPLE_FACTORS           	-	WTp2GLN-1-neg-PRM	Raw file name:WTp2GLN-1-neg-PRM | SampleType:Short Gln treatment | Genotype:Wildtype	RAW_FILE_NAME=10 days 5 mM KNO3 + 2 hrs 10 mM Gln; Polarity=Neg
SUBJECT_SAMPLE_FACTORS           	-	WTp2GLN-2-pos-PRM	Raw file name:WTp2GLN-2-pos-PRM | SampleType:Short Gln treatment | Genotype:Wildtype	RAW_FILE_NAME=10 days 5 mM KNO3 + 2 hrs 10 mM Gln; Polarity=Pos
SUBJECT_SAMPLE_FACTORS           	-	WTp2GLN-2-neg-PRM	Raw file name:WTp2GLN-2-neg-PRM | SampleType:Short Gln treatment | Genotype:Wildtype	RAW_FILE_NAME=10 days 5 mM KNO3 + 2 hrs 10 mM Gln; Polarity=Neg
SUBJECT_SAMPLE_FACTORS           	-	WTp2GLN-3-pos-PRM	Raw file name:WTp2GLN-3-pos-PRM | SampleType:Short Gln treatment | Genotype:Wildtype	RAW_FILE_NAME=10 days 5 mM KNO3 + 2 hrs 10 mM Gln; Polarity=Pos
SUBJECT_SAMPLE_FACTORS           	-	WTp2GLN-3-neg-PRM	Raw file name:WTp2GLN-3-neg-PRM | SampleType:Short Gln treatment | Genotype:Wildtype	RAW_FILE_NAME=10 days 5 mM KNO3 + 2 hrs 10 mM Gln; Polarity=Neg
SUBJECT_SAMPLE_FACTORS           	-	GATp2GLN-1-pos-PRM	Raw file name:GATp2GLN-1-pos-PRM | SampleType:Short Gln treatment | Genotype:gat1_2.1	RAW_FILE_NAME=10 days 5 mM KNO3 + 2 hrs 10 mM Gln; Polarity=Pos
SUBJECT_SAMPLE_FACTORS           	-	GATp2GLN-1-neg-PRM	Raw file name:GATp2GLN-1-neg-PRM | SampleType:Short Gln treatment | Genotype:gat1_2.1	RAW_FILE_NAME=10 days 5 mM KNO3 + 2 hrs 10 mM Gln; Polarity=Neg
SUBJECT_SAMPLE_FACTORS           	-	GATp2GLN-2-pos-PRM	Raw file name:GATp2GLN-2-pos-PRM | SampleType:Short Gln treatment | Genotype:gat1_2.1	RAW_FILE_NAME=10 days 5 mM KNO3 + 2 hrs 10 mM Gln; Polarity=Pos
SUBJECT_SAMPLE_FACTORS           	-	GATp2GLN-2-neg-PRM	Raw file name:GATp2GLN-2-neg-PRM | SampleType:Short Gln treatment | Genotype:gat1_2.1	RAW_FILE_NAME=10 days 5 mM KNO3 + 2 hrs 10 mM Gln; Polarity=Neg
SUBJECT_SAMPLE_FACTORS           	-	GATp2GLN-3-pos-PRM	Raw file name:GATp2GLN-3-pos-PRM | SampleType:Short Gln treatment | Genotype:gat1_2.1	RAW_FILE_NAME=10 days 5 mM KNO3 + 2 hrs 10 mM Gln; Polarity=Pos
SUBJECT_SAMPLE_FACTORS           	-	GATp2GLN-3-neg-PRM	Raw file name:GATp2GLN-3-neg-PRM | SampleType:Short Gln treatment | Genotype:gat1_2.1	RAW_FILE_NAME=10 days 5 mM KNO3 + 2 hrs 10 mM Gln; Polarity=Neg
SUBJECT_SAMPLE_FACTORS           	-	WTp10GLN-1-pos-PRM	Raw file name:WTp10GLN-1-pos-PRM | SampleType:Long Gln treatment | Genotype:Wildtype	RAW_FILE_NAME=10 days in 2 mM Gln; Polarity=Pos
SUBJECT_SAMPLE_FACTORS           	-	WTp10GLN-1-neg-PRM	Raw file name:WTp10GLN-1-neg-PRM | SampleType:Long Gln treatment | Genotype:Wildtype	RAW_FILE_NAME=10 days in 2 mM Gln; Polarity=Neg
SUBJECT_SAMPLE_FACTORS           	-	WTp10GLN-2-pos-PRM	Raw file name:WTp10GLN-2-pos-PRM | SampleType:Long Gln treatment | Genotype:Wildtype	RAW_FILE_NAME=10 days in 2 mM Gln; Polarity=Pos
SUBJECT_SAMPLE_FACTORS           	-	WTp10GLN-2-neg-PRM	Raw file name:WTp10GLN-2-neg-PRM | SampleType:Long Gln treatment | Genotype:Wildtype	RAW_FILE_NAME=10 days in 2 mM Gln; Polarity=Neg
SUBJECT_SAMPLE_FACTORS           	-	WTp10GLN-3-pos-PRM	Raw file name:WTp10GLN-3-pos-PRM | SampleType:Long Gln treatment | Genotype:Wildtype	RAW_FILE_NAME=10 days in 2 mM Gln; Polarity=Pos
SUBJECT_SAMPLE_FACTORS           	-	WTp10GLN-3-neg-PRM	Raw file name:WTp10GLN-3-neg-PRM | SampleType:Long Gln treatment | Genotype:Wildtype	RAW_FILE_NAME=10 days in 2 mM Gln; Polarity=Neg
SUBJECT_SAMPLE_FACTORS           	-	GATp10GLN-1-pos-PRM	Raw file name:GATp10GLN-1-pos-PRM | SampleType:Long Gln treatment | Genotype:gat1_2.1	RAW_FILE_NAME=10 days in 2 mM Gln; Polarity=Pos
SUBJECT_SAMPLE_FACTORS           	-	GATp10GLN-1-neg-PRM	Raw file name:GATp10GLN-1-neg-PRM | SampleType:Long Gln treatment | Genotype:gat1_2.1	RAW_FILE_NAME=10 days in 2 mM Gln; Polarity=Neg
SUBJECT_SAMPLE_FACTORS           	-	GATp10GLN-2-pos-PRM	Raw file name:GATp10GLN-2-pos-PRM | SampleType:Long Gln treatment | Genotype:gat1_2.1	RAW_FILE_NAME=10 days in 2 mM Gln; Polarity=Pos
SUBJECT_SAMPLE_FACTORS           	-	GATp10GLN-2-neg-PRM	Raw file name:GATp10GLN-2-neg-PRM | SampleType:Long Gln treatment | Genotype:gat1_2.1	RAW_FILE_NAME=10 days in 2 mM Gln; Polarity=Neg
SUBJECT_SAMPLE_FACTORS           	-	GATp10GLN-3-pos-PRM	Raw file name:GATp10GLN-3-pos-PRM | SampleType:Long Gln treatment | Genotype:gat1_2.1	RAW_FILE_NAME=10 days in 2 mM Gln; Polarity=Pos
SUBJECT_SAMPLE_FACTORS           	-	GATp10GLN-3-neg-PRM	Raw file name:GATp10GLN-3-neg-PRM | SampleType:Long Gln treatment | Genotype:gat1_2.1	RAW_FILE_NAME=10 days in 2 mM Gln; Polarity=Neg
SUBJECT_SAMPLE_FACTORS           	-	WTp2GLU-1-pos-PRM	Raw file name:WTp2GLU-1-pos-PRM | SampleType:Positive Control | Genotype:Wildtype	RAW_FILE_NAME=10 days in 2 mM Glu; Polarity=Pos
SUBJECT_SAMPLE_FACTORS           	-	WTp2GLU-1-neg-PRM	Raw file name:WTp2GLU-1-neg-PRM | SampleType:Positive Control | Genotype:Wildtype	RAW_FILE_NAME=10 days in 2 mM Glu; Polarity=Neg
SUBJECT_SAMPLE_FACTORS           	-	WTp2GLU-2-pos-PRM	Raw file name:WTp2GLU-2-pos-PRM | SampleType:Positive Control | Genotype:Wildtype	RAW_FILE_NAME=10 days in 2 mM Glu; Polarity=Pos
SUBJECT_SAMPLE_FACTORS           	-	WTp2GLU-2-neg-PRM	Raw file name:WTp2GLU-2-neg-PRM | SampleType:Positive Control | Genotype:Wildtype	RAW_FILE_NAME=11 days in 2 mM Gln; Polarity=Neg
SUBJECT_SAMPLE_FACTORS           	-	WTp2GLU-3-pos-PRM	Raw file name:WTp2GLU-3-pos-PRM | SampleType:Positive Control | Genotype:Wildtype	RAW_FILE_NAME=10 days in 2 mM Glu; Polarity=Pos
SUBJECT_SAMPLE_FACTORS           	-	WTp2GLU-3-neg-PRM	Raw file name:WTp2GLU-3-neg-PRM | SampleType:Positive Control | Genotype:Wildtype	RAW_FILE_NAME=10 days in 2 mM Glu; Polarity=Neg
SUBJECT_SAMPLE_FACTORS           	-	GATp2GLU-1-pos-PRM	Raw file name:GATp2GLU-1-pos-PRM | SampleType:Positive Control | Genotype:gat1_2.1	RAW_FILE_NAME=10 days in 2 mM Glu; Polarity=Pos
SUBJECT_SAMPLE_FACTORS           	-	GATp2GLU-1-neg-PRM	Raw file name:GATp2GLU-1-neg-PRM | SampleType:Positive Control | Genotype:gat1_2.1	RAW_FILE_NAME=12 days in 2 mM Gln; Polarity=Neg
SUBJECT_SAMPLE_FACTORS           	-	GATp2GLU-2-pos-PRM	Raw file name:GATp2GLU-2-pos-PRM | SampleType:Positive Control | Genotype:gat1_2.1	RAW_FILE_NAME=10 days in 2 mM Glu; Polarity=Pos
SUBJECT_SAMPLE_FACTORS           	-	GATp2GLU-2-neg-PRM	Raw file name:GATp2GLU-2-neg-PRM | SampleType:Positive Control | Genotype:gat1_2.1	RAW_FILE_NAME=10 days in 2 mM Glu; Polarity=Neg
SUBJECT_SAMPLE_FACTORS           	-	GATp2GLU-3-pos-PRM	Raw file name:GATp2GLU-3-pos-PRM | SampleType:Positive Control | Genotype:gat1_2.1	RAW_FILE_NAME=10 days in 2 mM Glu; Polarity=Pos
SUBJECT_SAMPLE_FACTORS           	-	GATp2GLU-3-neg-PRM	Raw file name:GATp2GLU-3-neg-PRM | SampleType:Positive Control | Genotype:gat1_2.1	RAW_FILE_NAME=13 days in 2 mM Gln; Polarity=Neg
SUBJECT_SAMPLE_FACTORS           	-	AA-CS1-PRM	Raw file name:AA-CS1-PRM | SampleType:Amino acid standard | Genotype:Standard	RAW_FILE_NAME=NA; Polarity=Pos
SUBJECT_SAMPLE_FACTORS           	-	AA-CS2-PRM	Raw file name:AA-CS2-PRM | SampleType:Amino acid standard | Genotype:Standard	RAW_FILE_NAME=NA; Polarity=Pos
SUBJECT_SAMPLE_FACTORS           	-	AA-CS3-PRM	Raw file name:AA-CS3-PRM | SampleType:Amino acid standard | Genotype:Standard	RAW_FILE_NAME=NA; Polarity=Pos
SUBJECT_SAMPLE_FACTORS           	-	AA-CS4-PRM	Raw file name:AA-CS4-PRM | SampleType:Amino acid standard | Genotype:Standard	RAW_FILE_NAME=NA; Polarity=Pos
SUBJECT_SAMPLE_FACTORS           	-	AA-CS5-PRM	Raw file name:AA-CS5-PRM | SampleType:Amino acid standard | Genotype:Standard	RAW_FILE_NAME=NA; Polarity=Pos
SUBJECT_SAMPLE_FACTORS           	-	AA-CS6-PRM	Raw file name:AA-CS6-PRM | SampleType:Amino acid standard | Genotype:Standard	RAW_FILE_NAME=NA; Polarity=Pos
SUBJECT_SAMPLE_FACTORS           	-	OA-CS1-PRM	Raw file name:OA-CS1-PRM | SampleType:Organic acid standard | Genotype:Standard	RAW_FILE_NAME=NA; Polarity=Neg
SUBJECT_SAMPLE_FACTORS           	-	OA-CS2-PRM	Raw file name:OA-CS2-PRM | SampleType:Organic acid standard | Genotype:Standard	RAW_FILE_NAME=NA; Polarity=Neg
SUBJECT_SAMPLE_FACTORS           	-	OA-CS3-PRM	Raw file name:OA-CS3-PRM | SampleType:Organic acid standard | Genotype:Standard	RAW_FILE_NAME=NA; Polarity=Neg
SUBJECT_SAMPLE_FACTORS           	-	OA-CS4-PRM	Raw file name:OA-CS4-PRM | SampleType:Organic acid standard | Genotype:Standard	RAW_FILE_NAME=NA; Polarity=Neg
SUBJECT_SAMPLE_FACTORS           	-	OA-CS5-PRM	Raw file name:OA-CS5-PRM | SampleType:Organic acid standard | Genotype:Standard	RAW_FILE_NAME=NA; Polarity=Neg
SUBJECT_SAMPLE_FACTORS           	-	OA-CS6-PRM	Raw file name:OA-CS6-PRM | SampleType:Organic acid standard | Genotype:Standard	RAW_FILE_NAME=NA; Polarity=Neg
#COLLECTION
CO:COLLECTION_SUMMARY            	Roots from 50 seedlings grown in plates containing required treatment were
CO:COLLECTION_SUMMARY            	collected and processed as single replicate.
CO:COLLECTION_PROTOCOL_ID        	001
CO:SAMPLE_TYPE                   	Plant
CO:COLLECTION_METHOD             	50 mg collected and flash frozen in Liquid N2
CO:COLLECTION_LOCATION           	London Research and Development Center
CO:STORAGE_CONDITIONS            	-80℃
#TREATMENT
TR:TREATMENT_SUMMARY             	Wild-type Arabidopsis ecotype Columbia and gat1_2.1 T-DNA insertion lines were
TR:TREATMENT_SUMMARY             	used for Gln and Glu treatments. Plants were grown on vertical plates at 22 °C
TR:TREATMENT_SUMMARY             	under continuous light (ca. 70 μmol m-2 s-2), as previously described by Ivanov
TR:TREATMENT_SUMMARY             	et al. (2012) on a defined nutrient medium containing a final concentration of
TR:TREATMENT_SUMMARY             	10 mM potassium phosphate (pH 6.5), 5 mM KNO3, 2 mM MgSO4, 1 mM CaCl2, 125 μg
TR:TREATMENT_SUMMARY             	FeNaEDTA, micronutrients (50 mM H3BO3, 12 mM MnSO4, 1 mM ZnCl2, 1 mM CuSO4 and
TR:TREATMENT_SUMMARY             	0.2 mM Na2MoO4), 1% sucrose and 1% agar [28]. Ten-day old seedlings were
TR:TREATMENT_SUMMARY             	transferred to plates containing the same medium without nitrogen as control or
TR:TREATMENT_SUMMARY             	10 mM Gln as sole N source. After 2 h, root tissue was harvested, frozen in
TR:TREATMENT_SUMMARY             	liquid N2 and stored at -80 °C until total metabolite extractions was carried
TR:TREATMENT_SUMMARY             	out. For growth in Gln and Glu, the same media and growth conditions were used
TR:TREATMENT_SUMMARY             	with the exception of 5 mM KNO3 being substituted with either 2 mM Gln or 2 mM
TR:TREATMENT_SUMMARY             	Glu and tissue was collected after 10 days.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Fifty mg of root tissue was excised from 10 day old seedlings of WT or gat1_2.1
SP:SAMPLEPREP_SUMMARY            	grown under conditions described above, collected in 2 mL Eppendorf tubes and
SP:SAMPLEPREP_SUMMARY            	flash frozen in liquid N2. Frozen tissue was homogenized using a tissue lyser
SP:SAMPLEPREP_SUMMARY            	and metabolites were isolated using 1 mL of methanol:water (4:1) with incubation
SP:SAMPLEPREP_SUMMARY            	in an ultra-sonication bath for 20 min followed by shaking for 30 min at 4 °C.
SP:SAMPLEPREP_SUMMARY            	The mixture was centrifuged at 12,000 × g for 10 min at 4 °C and 700 µl of
SP:SAMPLEPREP_SUMMARY            	the supernatant was transferred into fresh tubes and evaporated to dryness using
SP:SAMPLEPREP_SUMMARY            	a Vacufuge at ambient temperature. The residue was re-dissolved in 500 µl of
SP:SAMPLEPREP_SUMMARY            	1:1 methanol:water and the samples were filtered using a 0.2 µm PTFE microfuge
SP:SAMPLEPREP_SUMMARY            	filter (Cytiva Whatman). Five µl of 1 µg/mL 13C6 Phe was added to the samples
SP:SAMPLEPREP_SUMMARY            	for monitoring the quality of LC-MS runs.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	HILIC
CH:INSTRUMENT_NAME               	Agilent 1290 Infinity II
CH:COLUMN_NAME                   	SeQuant ZIC-HILIC (100 x 2.1mm, 3.5um)
CH:FLOW_RATE                     	0.3 mL min-1
CH:COLUMN_TEMPERATURE            	35
CH:METHODS_FILENAME              	Targeted Metabolite Analysis
CH:SOLVENT_A                     	5 mM Ammonium Acetate pH 4.0
CH:SOLVENT_B                     	90% ACN 0.1% Acetic acid
CH:INTERNAL_STANDARD             	13C6 Phenylalanine
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
AN:OPERATOR_NAME                 	Shrikaar Kambhampati
AN:DETECTOR_TYPE                 	Orbitrap
AN:ACQUISITION_DATE              	3/11/2016
AN:ANALYSIS_PROTOCOL_FILE        	Targeted_Metabolite_Analysis.pdf
#MS
MS:INSTRUMENT_NAME               	Thermo Q Exactive Orbitrap
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	POSITIVE
MS:MS_COMMENTS                   	The following heated electrospray ionization (HESI) conditions were optimized
MS:MS_COMMENTS                   	for the analysis of amino and organic acids: spray voltage, 3.9 kV (ESI+), 3.5
MS:MS_COMMENTS                   	kV (ESI-); capillary temperature, 250 °C; probe heater temperature, 450 °C;
MS:MS_COMMENTS                   	sheath gas, 30 arbitrary units; auxiliary gas, 8 arbitrary units; and S-Lens RF
MS:MS_COMMENTS                   	level, 60%. Injections of 5 μl were used with a flow rate of 0.3 mL min-1.
MS:MS_COMMENTS                   	Compounds were detected and monitored using targeted MS/MS, spectra were
MS:MS_COMMENTS                   	collected at 17,500 resolution, AGC target 1e6, maximum IT 65 ms, isolation
MS:MS_COMMENTS                   	window of 1 m/z, normalized collision energy of 30, intensity threshold of 1.6e5
MS:MS_COMMENTS                   	and 10s dynamic exclusion.
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	umol g FW-1
MS_METABOLITE_DATA_START
Samples	WT-GLN-1-pos-PRM	WT-GLN-2-pos-PRM	WT-GLN-3-pos-PRM	WTp2GLN-1-pos-PRM	WTp2GLN-2-pos-PRM	WTp2GLN-3-pos-PRM	WTp10GLN-1-pos-PRM	WTp10GLN-2-pos-PRM	WTp10GLN-3-pos-PRM	GAT-GLN-1-pos-PRM	GAT-GLN-2-pos-PRM	GAT-GLN-3-pos-PRM	GATp2GLN-1-pos-PRM	GATp2GLN-2-pos-PRM	GATp2GLN-3-pos-PRM	GATp10GLN-1-pos-PRM	GATp10GLN-2-pos-PRM	GATp10GLN-3-pos-PRM	WTp2GLU-1-pos-PRM	WTp2GLU-2-pos-PRM	WTp2GLU-3-pos-PRM	GATp2GLU-1-pos-PRM	GATp2GLU-2-pos-PRM	GATp2GLU-3-pos-PRM
Factors	Raw file name:WT-GLN-1-pos-PRM | SampleType:Negative Control | Genotype:Wildtype	Raw file name:WT-GLN-2-pos-PRM | SampleType:Negative Control | Genotype:Wildtype	Raw file name:WT-GLN-3-pos-PRM | SampleType:Negative Control | Genotype:Wildtype	Raw file name:WTp2GLN-1-pos-PRM | SampleType:Short Gln treatment | Genotype:Wildtype	Raw file name:WTp2GLN-2-pos-PRM | SampleType:Short Gln treatment | Genotype:Wildtype	Raw file name:WTp2GLN-3-pos-PRM | SampleType:Short Gln treatment | Genotype:Wildtype	Raw file name:WTp10GLN-1-pos-PRM | SampleType:Long Gln treatment | Genotype:Wildtype	Raw file name:WTp10GLN-2-pos-PRM | SampleType:Long Gln treatment | Genotype:Wildtype	Raw file name:WTp10GLN-3-pos-PRM | SampleType:Long Gln treatment | Genotype:Wildtype	Raw file name:GAT-GLN-1-pos-PRM | SampleType:Negative Control | Genotype:gat1_2.1	Raw file name:GAT-GLN-2-pos-PRM | SampleType:Negative Control | Genotype:gat1_2.1	Raw file name:GAT-GLN-3-pos-PRM | SampleType:Negative Control | Genotype:gat1_2.1	Raw file name:GATp2GLN-1-pos-PRM | SampleType:Short Gln treatment | Genotype:gat1_2.1	Raw file name:GATp2GLN-2-pos-PRM | SampleType:Short Gln treatment | Genotype:gat1_2.1	Raw file name:GATp2GLN-3-pos-PRM | SampleType:Short Gln treatment | Genotype:gat1_2.1	Raw file name:GATp10GLN-1-pos-PRM | SampleType:Long Gln treatment | Genotype:gat1_2.1	Raw file name:GATp10GLN-2-pos-PRM | SampleType:Long Gln treatment | Genotype:gat1_2.1	Raw file name:GATp10GLN-3-pos-PRM | SampleType:Long Gln treatment | Genotype:gat1_2.1	Raw file name:WTp2GLU-1-pos-PRM | SampleType:Positive Control | Genotype:Wildtype	Raw file name:WTp2GLU-2-pos-PRM | SampleType:Positive Control | Genotype:Wildtype	Raw file name:WTp2GLU-3-pos-PRM | SampleType:Positive Control | Genotype:Wildtype	Raw file name:GATp2GLU-1-pos-PRM | SampleType:Positive Control | Genotype:gat1_2.1	Raw file name:GATp2GLU-2-pos-PRM | SampleType:Positive Control | Genotype:gat1_2.1	Raw file name:GATp2GLU-3-pos-PRM | SampleType:Positive Control | Genotype:gat1_2.1
gamma-aminobutyric acid	0.254114562	0.306378192	0.244204279	0.310491373	0.337413389	0.31847619	0.20000138	0.219400966	0.232002761	0.142202899	0.165439614	0.181554175	0.197917184	0.214266391	0.230222222	0.09410766	0.071519669	0.075432712	0.147185645	0.149884058	0.182879227	0.171029676	0.157868875	0.144507937
Glutamine	14.90781983	13.56566464	13.1814641	181.9444722	197.5057425	174.2909218	25.63896934	24.85828423	22.67552182	11.38390093	6.905922301	16.38509937	222.756916	228.4565065	189.839209	27.43972835	28.91960451	21.91121542	9.585039449	12.8031559	12.40936782	7.896434635	11.30610207	10.51123539
Glutamate	9.998529202	11.2373203	8.653461119	28.66152678	32.71096456	30.13042653	21.0926128	22.72282583	23.7718366	14.44698012	15.98420079	12.93680315	27.1456564	25.27489681	24.70128576	13.74906296	15.28201357	14.59405988	335.3897139	373.6446838	455.0011387	403.5455236	336.2845282	474.3454476
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name
gamma-aminobutyric acid
Glutamine
Glutamate
METABOLITES_END
#END