{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST001812","ANALYSIS_ID":"AN002938","VERSION":"1","CREATED_ON":"June 1, 2021, 1:58 pm"},

"PROJECT":{"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"},

"STUDY":{"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 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"},

"SUBJECT":{"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"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"Extract_1_pos",
"Factors":{"Rawfilename":"Extract_1_pos"},
"Additional sample data":{"RAW_FILE_NAME":"Pos","Treatment":"Extract"}
},
{
"Subject ID":"-",
"Sample ID":"Extract_1_neg",
"Factors":{"Rawfilename":"Extract_1_neg"},
"Additional sample data":{"RAW_FILE_NAME":"Neg","Treatment":"Extract"}
},
{
"Subject ID":"-",
"Sample ID":"Extract_2_pos",
"Factors":{"Rawfilename":"Extract_2_pos"},
"Additional sample data":{"RAW_FILE_NAME":"Pos","Treatment":"Extract"}
},
{
"Subject ID":"-",
"Sample ID":"Extract_2_neg",
"Factors":{"Rawfilename":"Extract_2_neg"},
"Additional sample data":{"RAW_FILE_NAME":"Neg","Treatment":"Extract"}
},
{
"Subject ID":"-",
"Sample ID":"Extract_3_pos",
"Factors":{"Rawfilename":"Extract_3_pos"},
"Additional sample data":{"RAW_FILE_NAME":"Pos","Treatment":"Extract"}
},
{
"Subject ID":"-",
"Sample ID":"Extract_3_neg",
"Factors":{"Rawfilename":"Extract_3_neg"},
"Additional sample data":{"RAW_FILE_NAME":"Neg","Treatment":"Extract"}
},
{
"Subject ID":"-",
"Sample ID":"Extract_4_pos",
"Factors":{"Rawfilename":"Extract_4_pos"},
"Additional sample data":{"RAW_FILE_NAME":"Pos","Treatment":"Extract"}
},
{
"Subject ID":"-",
"Sample ID":"Extract_4_neg",
"Factors":{"Rawfilename":"Extract_4_neg"},
"Additional sample data":{"RAW_FILE_NAME":"Neg","Treatment":"Extract"}
},
{
"Subject ID":"-",
"Sample ID":"E_Gln_1_pos",
"Factors":{"Rawfilename":"E_Gln_1_pos"},
"Additional sample data":{"RAW_FILE_NAME":"Pos","Treatment":"Extract_Gln"}
},
{
"Subject ID":"-",
"Sample ID":"E_Gln_1_neg",
"Factors":{"Rawfilename":"E_Gln_1_neg"},
"Additional sample data":{"RAW_FILE_NAME":"Neg","Treatment":"Extract_Gln"}
},
{
"Subject ID":"-",
"Sample ID":"E_Gln_2_pos",
"Factors":{"Rawfilename":"E_Gln_2_pos"},
"Additional sample data":{"RAW_FILE_NAME":"Pos","Treatment":"Extract_Gln"}
},
{
"Subject ID":"-",
"Sample ID":"E_Gln_2_neg",
"Factors":{"Rawfilename":"E_Gln_2_neg"},
"Additional sample data":{"RAW_FILE_NAME":"Neg","Treatment":"Extract_Gln"}
},
{
"Subject ID":"-",
"Sample ID":"E_Gln_3_pos",
"Factors":{"Rawfilename":"E_Gln_3_pos"},
"Additional sample data":{"RAW_FILE_NAME":"Pos","Treatment":"Extract_Gln"}
},
{
"Subject ID":"-",
"Sample ID":"E_Gln_3_neg",
"Factors":{"Rawfilename":"E_Gln_3_neg"},
"Additional sample data":{"RAW_FILE_NAME":"Neg","Treatment":"Extract_Gln"}
},
{
"Subject ID":"-",
"Sample ID":"E_Gln_4_pos",
"Factors":{"Rawfilename":"E_Gln_4_pos"},
"Additional sample data":{"RAW_FILE_NAME":"Pos","Treatment":"Extract_Gln"}
},
{
"Subject ID":"-",
"Sample ID":"E_Gln_4_neg",
"Factors":{"Rawfilename":"E_Gln_4_neg"},
"Additional sample data":{"RAW_FILE_NAME":"Neg","Treatment":"Extract_Gln"}
},
{
"Subject ID":"-",
"Sample ID":"E_15NGln_1_pos",
"Factors":{"Rawfilename":"E_15NGln_1_pos"},
"Additional sample data":{"RAW_FILE_NAME":"Pos","Treatment":"Extract_15N1Gln"}
},
{
"Subject ID":"-",
"Sample ID":"E_15NGln_1_neg",
"Factors":{"Rawfilename":"E_15NGln_1_neg"},
"Additional sample data":{"RAW_FILE_NAME":"Neg","Treatment":"Extract_15N1Gln"}
},
{
"Subject ID":"-",
"Sample ID":"E_15NGln_2_pos",
"Factors":{"Rawfilename":"E_15NGln_2_pos"},
"Additional sample data":{"RAW_FILE_NAME":"Pos","Treatment":"Extract_15N1Gln"}
},
{
"Subject ID":"-",
"Sample ID":"E_15NGln_2_neg",
"Factors":{"Rawfilename":"E_15NGln_2_neg"},
"Additional sample data":{"RAW_FILE_NAME":"Neg","Treatment":"Extract_15N1Gln"}
},
{
"Subject ID":"-",
"Sample ID":"E_15NGln_3_pos",
"Factors":{"Rawfilename":"E_15NGln_3_pos"},
"Additional sample data":{"RAW_FILE_NAME":"Pos","Treatment":"Extract_15N1Gln"}
},
{
"Subject ID":"-",
"Sample ID":"E_15NGln_3_neg",
"Factors":{"Rawfilename":"E_15NGln_3_neg"},
"Additional sample data":{"RAW_FILE_NAME":"Neg","Treatment":"Extract_15N1Gln"}
},
{
"Subject ID":"-",
"Sample ID":"E_15NGln_4_pos",
"Factors":{"Rawfilename":"E_15NGln_4_pos"},
"Additional sample data":{"RAW_FILE_NAME":"Pos","Treatment":"Extract_15N1Gln"}
},
{
"Subject ID":"-",
"Sample ID":"E_15NGln_4_neg",
"Factors":{"Rawfilename":"E_15NGln_4_neg"},
"Additional sample data":{"RAW_FILE_NAME":"Neg","Treatment":"Extract_15N1Gln"}
},
{
"Subject ID":"-",
"Sample ID":"E_FL_1_pos",
"Factors":{"Rawfilename":"E_FL_1_pos"},
"Additional sample data":{"RAW_FILE_NAME":"Pos","Treatment":"Extract_FullLengthProtein"}
},
{
"Subject ID":"-",
"Sample ID":"E_FL_1_neg",
"Factors":{"Rawfilename":"E_FL_1_neg"},
"Additional sample data":{"RAW_FILE_NAME":"Neg","Treatment":"Extract_FullLengthProtein"}
},
{
"Subject ID":"-",
"Sample ID":"E_FL_2_pos",
"Factors":{"Rawfilename":"E_FL_2_pos"},
"Additional sample data":{"RAW_FILE_NAME":"Pos","Treatment":"Extract_FullLengthProtein"}
},
{
"Subject ID":"-",
"Sample ID":"E_FL_2_neg",
"Factors":{"Rawfilename":"E_FL_2_neg"},
"Additional sample data":{"RAW_FILE_NAME":"Neg","Treatment":"Extract_FullLengthProtein"}
},
{
"Subject ID":"-",
"Sample ID":"E_FL_3_pos",
"Factors":{"Rawfilename":"E_FL_3_pos"},
"Additional sample data":{"RAW_FILE_NAME":"Pos","Treatment":"Extract_FullLengthProtein"}
},
{
"Subject ID":"-",
"Sample ID":"E_FL_3_neg",
"Factors":{"Rawfilename":"E_FL_3_neg"},
"Additional sample data":{"RAW_FILE_NAME":"Neg","Treatment":"Extract_FullLengthProtein"}
},
{
"Subject ID":"-",
"Sample ID":"E_FL_4_pos",
"Factors":{"Rawfilename":"E_FL_4_pos"},
"Additional sample data":{"RAW_FILE_NAME":"Pos","Treatment":"Extract_FullLengthProtein"}
},
{
"Subject ID":"-",
"Sample ID":"E_FL_4_neg",
"Factors":{"Rawfilename":"E_FL_4_neg"},
"Additional sample data":{"RAW_FILE_NAME":"Neg","Treatment":"Extract_FullLengthProtein"}
},
{
"Subject ID":"-",
"Sample ID":"E_15NGln_FL_1_pos",
"Factors":{"Rawfilename":"E_15NGln_FL_1_pos"},
"Additional sample data":{"RAW_FILE_NAME":"Pos","Treatment":"Extract_15N1Gln_FullLengthProtein"}
},
{
"Subject ID":"-",
"Sample ID":"E_15NGln_FL_1_neg",
"Factors":{"Rawfilename":"E_15NGln_FL_1_neg"},
"Additional sample data":{"RAW_FILE_NAME":"Neg","Treatment":"Extract_15N1Gln_FullLengthProtein"}
},
{
"Subject ID":"-",
"Sample ID":"E_15NGln_FL_2_pos",
"Factors":{"Rawfilename":"E_15NGln_FL_2_pos"},
"Additional sample data":{"RAW_FILE_NAME":"Pos","Treatment":"Extract_15N1Gln_FullLengthProtein"}
},
{
"Subject ID":"-",
"Sample ID":"E_15NGln_FL_2_neg",
"Factors":{"Rawfilename":"E_15NGln_FL_2_neg"},
"Additional sample data":{"RAW_FILE_NAME":"Neg","Treatment":"Extract_15N1Gln_FullLengthProtein"}
},
{
"Subject ID":"-",
"Sample ID":"E_15NGln_FL_3_pos",
"Factors":{"Rawfilename":"E_15NGln_FL_3_pos"},
"Additional sample data":{"RAW_FILE_NAME":"Pos","Treatment":"Extract_15N1Gln_FullLengthProtein"}
},
{
"Subject ID":"-",
"Sample ID":"E_15NGln_FL_3_neg",
"Factors":{"Rawfilename":"E_15NGln_FL_3_neg"},
"Additional sample data":{"RAW_FILE_NAME":"Neg","Treatment":"Extract_15N1Gln_FullLengthProtein"}
},
{
"Subject ID":"-",
"Sample ID":"E_15NGln_FL_4_pos",
"Factors":{"Rawfilename":"E_15NGln_FL_4_pos"},
"Additional sample data":{"RAW_FILE_NAME":"Pos","Treatment":"Extract_15N1Gln_FullLengthProtein"}
},
{
"Subject ID":"-",
"Sample ID":"E_15NGln_FL_4_neg",
"Factors":{"Rawfilename":"E_15NGln_FL_4_neg"},
"Additional sample data":{"RAW_FILE_NAME":"Neg","Treatment":"Extract_15N1Gln_FullLengthProtein"}
},
{
"Subject ID":"-",
"Sample ID":"E_15NGln_D_1_pos",
"Factors":{"Rawfilename":"E_15NGln_D_1_pos"},
"Additional sample data":{"RAW_FILE_NAME":"Pos","Treatment":"Extract_15N1Gln_DomainonlyProtein"}
},
{
"Subject ID":"-",
"Sample ID":"E_15NGln_D_1_neg",
"Factors":{"Rawfilename":"E_15NGln_D_1_neg"},
"Additional sample data":{"RAW_FILE_NAME":"Neg","Treatment":"Extract_15N1Gln_DomainonlyProtein"}
},
{
"Subject ID":"-",
"Sample ID":"E_15NGln_D_2_pos",
"Factors":{"Rawfilename":"E_15NGln_D_2_pos"},
"Additional sample data":{"RAW_FILE_NAME":"Pos","Treatment":"Extract_15N1Gln_DomainonlyProtein"}
},
{
"Subject ID":"-",
"Sample ID":"E_15NGln_D_2_neg",
"Factors":{"Rawfilename":"E_15NGln_D_2_neg"},
"Additional sample data":{"RAW_FILE_NAME":"Neg","Treatment":"Extract_15N1Gln_DomainonlyProtein"}
},
{
"Subject ID":"-",
"Sample ID":"E_15NGln_D_3_pos",
"Factors":{"Rawfilename":"E_15NGln_D_3_pos"},
"Additional sample data":{"RAW_FILE_NAME":"Pos","Treatment":"Extract_15N1Gln_DomainonlyProtein"}
},
{
"Subject ID":"-",
"Sample ID":"E_15NGln_D_3_neg",
"Factors":{"Rawfilename":"E_15NGln_D_3_neg"},
"Additional sample data":{"RAW_FILE_NAME":"Neg","Treatment":"Extract_15N1Gln_DomainonlyProtein"}
},
{
"Subject ID":"-",
"Sample ID":"E_15NGln_D_4_pos",
"Factors":{"Rawfilename":"E_15NGln_D_4_pos"},
"Additional sample data":{"RAW_FILE_NAME":"Pos","Treatment":"Extract_15N1Gln_DomainonlyProtein"}
},
{
"Subject ID":"-",
"Sample ID":"E_15NGln_D_4_neg",
"Factors":{"Rawfilename":"E_15NGln_D_4_neg"},
"Additional sample data":{"RAW_FILE_NAME":"Neg","Treatment":"Extract_15N1Gln_DomainonlyProtein"}
}
],
"COLLECTION":{"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_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]."},

"SAMPLEPREP":{"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."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_TYPE":"HILIC","INSTRUMENT_NAME":"Agilent 1290 Infinity II","COLUMN_NAME":"SeQuant ZIC-HILIC (100 x 2.1mm, 3.5um)","FLOW_RATE":"0.3 mL min-1","COLUMN_TEMPERATURE":"35","METHODS_FILENAME":"Enzyme_spike_analysis","SOLVENT_A":"5 mM Ammonium Acetate pH 4.0","SOLVENT_B":"90% ACN 0.1% Acetic acid","INTERNAL_STANDARD":"13C6 Phenylalanine"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS","OPERATOR_NAME":"Shrikaar Kambhampati","DETECTOR_TYPE":"Orbitrap","ACQUISITION_DATE":"8/27/2017","ANALYSIS_PROTOCOL_FILE":"Enzyme_spike_analysis.pdf","DATA_FORMAT":".RAW"},

"MS":{"INSTRUMENT_NAME":"Thermo Q Exactive Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"NEGATIVE","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.","CAPILLARY_TEMPERATURE":"250","CAPILLARY_VOLTAGE":"3.5","MS_RESULTS_FILE":"ST001812_AN002938_Results.txt UNITS:Intensity Has m/z:Yes Has RT:Yes RT units:Minutes"}

}