#METABOLOMICS WORKBENCH skambhampati_20210601_103541 DATATRACK_ID:2664 STUDY_ID:ST001811 ANALYSIS_ID:AN002936 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
#MS
MS:INSTRUMENT_NAME               	Thermo Q Exactive Orbitrap
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	NEGATIVE
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-neg-PRM	WT-GLN-2-neg-PRM	WT-GLN-3-neg-PRM	WTp2GLN-1-neg-PRM	WTp2GLN-2-neg-PRM	WTp2GLN-3-neg-PRM	WTp10GLN-1-neg-PRM	WTp10GLN-2-neg-PRM	WTp10GLN-3-neg-PRM	GAT-GLN-1-neg-PRM	GAT-GLN-2-neg-PRM	GAT-GLN-3-neg-PRM	GATp2GLN-1-neg-PRM	GATp2GLN-2-neg-PRM	GATp2GLN-3-neg-PRM	GATp10GLN-1-neg-PRM	GATp10GLN-2-neg-PRM	GATp10GLN-3-neg-PRM	WTp2GLU-1-neg-PRM	WTp2GLU-2-neg-PRM	WTp2GLU-3-neg-PRM	GATp2GLU-1-neg-PRM	GATp2GLU-2-neg-PRM	GATp2GLU-3-neg-PRM
Factors	Raw file name:WT-GLN-1-neg-PRM | SampleType:Negative Control | Genotype:Wildtype	Raw file name:WT-GLN-2-neg-PRM | SampleType:Negative Control | Genotype:Wildtype	Raw file name:WT-GLN-3-neg-PRM | SampleType:Negative Control | Genotype:Wildtype	Raw file name:WTp2GLN-1-neg-PRM | SampleType:Short Gln treatment | Genotype:Wildtype	Raw file name:WTp2GLN-2-neg-PRM | SampleType:Short Gln treatment | Genotype:Wildtype	Raw file name:WTp2GLN-3-neg-PRM | SampleType:Short Gln treatment | Genotype:Wildtype	Raw file name:WTp10GLN-1-neg-PRM | SampleType:Long Gln treatment | Genotype:Wildtype	Raw file name:WTp10GLN-2-neg-PRM | SampleType:Long Gln treatment | Genotype:Wildtype	Raw file name:WTp10GLN-3-neg-PRM | SampleType:Long Gln treatment | Genotype:Wildtype	Raw file name:GAT-GLN-1-neg-PRM | SampleType:Negative Control | Genotype:gat1_2.1	Raw file name:GAT-GLN-2-neg-PRM | SampleType:Negative Control | Genotype:gat1_2.1	Raw file name:GAT-GLN-3-neg-PRM | SampleType:Negative Control | Genotype:gat1_2.1	Raw file name:GATp2GLN-1-neg-PRM | SampleType:Short Gln treatment | Genotype:gat1_2.1	Raw file name:GATp2GLN-2-neg-PRM | SampleType:Short Gln treatment | Genotype:gat1_2.1	Raw file name:GATp2GLN-3-neg-PRM | SampleType:Short Gln treatment | Genotype:gat1_2.1	Raw file name:GATp10GLN-1-neg-PRM | SampleType:Long Gln treatment | Genotype:gat1_2.1	Raw file name:GATp10GLN-2-neg-PRM | SampleType:Long Gln treatment | Genotype:gat1_2.1	Raw file name:GATp10GLN-3-neg-PRM | SampleType:Long Gln treatment | Genotype:gat1_2.1	Raw file name:WTp2GLU-1-neg-PRM | SampleType:Positive Control | Genotype:Wildtype	Raw file name:WTp2GLU-2-neg-PRM | SampleType:Positive Control | Genotype:Wildtype	Raw file name:WTp2GLU-3-neg-PRM | SampleType:Positive Control | Genotype:Wildtype	Raw file name:GATp2GLU-1-neg-PRM | SampleType:Positive Control | Genotype:gat1_2.1	Raw file name:GATp2GLU-2-neg-PRM | SampleType:Positive Control | Genotype:gat1_2.1	Raw file name:GATp2GLU-3-neg-PRM | SampleType:Positive Control | Genotype:gat1_2.1
Succinic acid	0.100683679	0.196015514	0.256142481	0.267706591	0.335361441	0.416502415	0.105905147	0.311336281	0.301918419	0.249831872	0.303744331	0.351282114	0.911420709	0.748914527	0.816056841	0.479095965	0.656049276	0.433992731	0.268219128	0.252939126	0.440559608	0.221514217	0.233847132	0.1790057
2-OG	-0.242165043	-0.189655323	0.091590581	2.623677428	2.295164038	2.982158927	0.435830676	0.596854659	0.520230658	0.410667948	0.317094054	0.708863746	1.41176008	1.820567035	1.121340265	1.437971255	1.525516579	1.383015159	5.357765061	4.823843432	6.088619982	5.667056922	5.901559565	5.35470709
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name
Succinic acid
2-OG
METABOLITES_END
#END