#METABOLOMICS WORKBENCH skambhampati_20210601_114320_mwtab.txt DATATRACK_ID:2665 STUDY_ID:ST001812 ANALYSIS_ID:AN002938 PROJECT_ID:000000 VERSION 1 CREATED_ON June 1, 2021, 1:58 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 used Arabidopsis root extracts, spiked with amide nitrogen ST:STUDY_SUMMARY labeled (15N1) Glutamine and a purified recombinant protein, both full length ST:STUDY_SUMMARY and glutaminase domain only versions, to determine the amido group acceptor, if ST:STUDY_SUMMARY any, in the glutamine amidotransferase reaction. 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 - Extract_1_pos Rawfilename:Extract_1_pos RAW_FILE_NAME=Pos; Treatment=Extract SUBJECT_SAMPLE_FACTORS - Extract_1_neg Rawfilename:Extract_1_neg RAW_FILE_NAME=Neg; Treatment=Extract SUBJECT_SAMPLE_FACTORS - Extract_2_pos Rawfilename:Extract_2_pos RAW_FILE_NAME=Pos; Treatment=Extract SUBJECT_SAMPLE_FACTORS - Extract_2_neg Rawfilename:Extract_2_neg RAW_FILE_NAME=Neg; Treatment=Extract SUBJECT_SAMPLE_FACTORS - Extract_3_pos Rawfilename:Extract_3_pos RAW_FILE_NAME=Pos; Treatment=Extract SUBJECT_SAMPLE_FACTORS - Extract_3_neg Rawfilename:Extract_3_neg RAW_FILE_NAME=Neg; Treatment=Extract SUBJECT_SAMPLE_FACTORS - Extract_4_pos Rawfilename:Extract_4_pos RAW_FILE_NAME=Pos; Treatment=Extract SUBJECT_SAMPLE_FACTORS - Extract_4_neg Rawfilename:Extract_4_neg RAW_FILE_NAME=Neg; Treatment=Extract SUBJECT_SAMPLE_FACTORS - E_Gln_1_pos Rawfilename:E_Gln_1_pos RAW_FILE_NAME=Pos; Treatment=Extract_Gln SUBJECT_SAMPLE_FACTORS - E_Gln_1_neg Rawfilename:E_Gln_1_neg RAW_FILE_NAME=Neg; Treatment=Extract_Gln SUBJECT_SAMPLE_FACTORS - E_Gln_2_pos Rawfilename:E_Gln_2_pos RAW_FILE_NAME=Pos; Treatment=Extract_Gln SUBJECT_SAMPLE_FACTORS - E_Gln_2_neg Rawfilename:E_Gln_2_neg RAW_FILE_NAME=Neg; Treatment=Extract_Gln SUBJECT_SAMPLE_FACTORS - E_Gln_3_pos Rawfilename:E_Gln_3_pos RAW_FILE_NAME=Pos; Treatment=Extract_Gln SUBJECT_SAMPLE_FACTORS - E_Gln_3_neg Rawfilename:E_Gln_3_neg RAW_FILE_NAME=Neg; Treatment=Extract_Gln SUBJECT_SAMPLE_FACTORS - E_Gln_4_pos Rawfilename:E_Gln_4_pos RAW_FILE_NAME=Pos; Treatment=Extract_Gln SUBJECT_SAMPLE_FACTORS - E_Gln_4_neg Rawfilename:E_Gln_4_neg RAW_FILE_NAME=Neg; Treatment=Extract_Gln SUBJECT_SAMPLE_FACTORS - E_15NGln_1_pos Rawfilename:E_15NGln_1_pos RAW_FILE_NAME=Pos; Treatment=Extract_15N1Gln SUBJECT_SAMPLE_FACTORS - E_15NGln_1_neg Rawfilename:E_15NGln_1_neg RAW_FILE_NAME=Neg; Treatment=Extract_15N1Gln SUBJECT_SAMPLE_FACTORS - E_15NGln_2_pos Rawfilename:E_15NGln_2_pos RAW_FILE_NAME=Pos; Treatment=Extract_15N1Gln SUBJECT_SAMPLE_FACTORS - E_15NGln_2_neg Rawfilename:E_15NGln_2_neg RAW_FILE_NAME=Neg; Treatment=Extract_15N1Gln SUBJECT_SAMPLE_FACTORS - E_15NGln_3_pos Rawfilename:E_15NGln_3_pos RAW_FILE_NAME=Pos; Treatment=Extract_15N1Gln SUBJECT_SAMPLE_FACTORS - E_15NGln_3_neg Rawfilename:E_15NGln_3_neg RAW_FILE_NAME=Neg; Treatment=Extract_15N1Gln SUBJECT_SAMPLE_FACTORS - E_15NGln_4_pos Rawfilename:E_15NGln_4_pos RAW_FILE_NAME=Pos; Treatment=Extract_15N1Gln SUBJECT_SAMPLE_FACTORS - E_15NGln_4_neg Rawfilename:E_15NGln_4_neg RAW_FILE_NAME=Neg; Treatment=Extract_15N1Gln SUBJECT_SAMPLE_FACTORS - E_FL_1_pos Rawfilename:E_FL_1_pos RAW_FILE_NAME=Pos; Treatment=Extract_FullLengthProtein SUBJECT_SAMPLE_FACTORS - E_FL_1_neg Rawfilename:E_FL_1_neg RAW_FILE_NAME=Neg; Treatment=Extract_FullLengthProtein SUBJECT_SAMPLE_FACTORS - E_FL_2_pos Rawfilename:E_FL_2_pos RAW_FILE_NAME=Pos; Treatment=Extract_FullLengthProtein SUBJECT_SAMPLE_FACTORS - E_FL_2_neg Rawfilename:E_FL_2_neg RAW_FILE_NAME=Neg; Treatment=Extract_FullLengthProtein SUBJECT_SAMPLE_FACTORS - E_FL_3_pos Rawfilename:E_FL_3_pos RAW_FILE_NAME=Pos; Treatment=Extract_FullLengthProtein SUBJECT_SAMPLE_FACTORS - E_FL_3_neg Rawfilename:E_FL_3_neg RAW_FILE_NAME=Neg; Treatment=Extract_FullLengthProtein SUBJECT_SAMPLE_FACTORS - E_FL_4_pos Rawfilename:E_FL_4_pos RAW_FILE_NAME=Pos; Treatment=Extract_FullLengthProtein SUBJECT_SAMPLE_FACTORS - E_FL_4_neg Rawfilename:E_FL_4_neg RAW_FILE_NAME=Neg; Treatment=Extract_FullLengthProtein SUBJECT_SAMPLE_FACTORS - E_15NGln_FL_1_pos Rawfilename:E_15NGln_FL_1_pos RAW_FILE_NAME=Pos; Treatment=Extract_15N1Gln_FullLengthProtein SUBJECT_SAMPLE_FACTORS - E_15NGln_FL_1_neg Rawfilename:E_15NGln_FL_1_neg RAW_FILE_NAME=Neg; Treatment=Extract_15N1Gln_FullLengthProtein SUBJECT_SAMPLE_FACTORS - E_15NGln_FL_2_pos Rawfilename:E_15NGln_FL_2_pos RAW_FILE_NAME=Pos; Treatment=Extract_15N1Gln_FullLengthProtein SUBJECT_SAMPLE_FACTORS - E_15NGln_FL_2_neg Rawfilename:E_15NGln_FL_2_neg RAW_FILE_NAME=Neg; Treatment=Extract_15N1Gln_FullLengthProtein SUBJECT_SAMPLE_FACTORS - E_15NGln_FL_3_pos Rawfilename:E_15NGln_FL_3_pos RAW_FILE_NAME=Pos; Treatment=Extract_15N1Gln_FullLengthProtein SUBJECT_SAMPLE_FACTORS - E_15NGln_FL_3_neg Rawfilename:E_15NGln_FL_3_neg RAW_FILE_NAME=Neg; Treatment=Extract_15N1Gln_FullLengthProtein SUBJECT_SAMPLE_FACTORS - E_15NGln_FL_4_pos Rawfilename:E_15NGln_FL_4_pos RAW_FILE_NAME=Pos; Treatment=Extract_15N1Gln_FullLengthProtein SUBJECT_SAMPLE_FACTORS - E_15NGln_FL_4_neg Rawfilename:E_15NGln_FL_4_neg RAW_FILE_NAME=Neg; Treatment=Extract_15N1Gln_FullLengthProtein SUBJECT_SAMPLE_FACTORS - E_15NGln_D_1_pos Rawfilename:E_15NGln_D_1_pos RAW_FILE_NAME=Pos; Treatment=Extract_15N1Gln_DomainonlyProtein SUBJECT_SAMPLE_FACTORS - E_15NGln_D_1_neg Rawfilename:E_15NGln_D_1_neg RAW_FILE_NAME=Neg; Treatment=Extract_15N1Gln_DomainonlyProtein SUBJECT_SAMPLE_FACTORS - E_15NGln_D_2_pos Rawfilename:E_15NGln_D_2_pos RAW_FILE_NAME=Pos; Treatment=Extract_15N1Gln_DomainonlyProtein SUBJECT_SAMPLE_FACTORS - E_15NGln_D_2_neg Rawfilename:E_15NGln_D_2_neg RAW_FILE_NAME=Neg; Treatment=Extract_15N1Gln_DomainonlyProtein SUBJECT_SAMPLE_FACTORS - E_15NGln_D_3_pos Rawfilename:E_15NGln_D_3_pos RAW_FILE_NAME=Pos; Treatment=Extract_15N1Gln_DomainonlyProtein SUBJECT_SAMPLE_FACTORS - E_15NGln_D_3_neg Rawfilename:E_15NGln_D_3_neg RAW_FILE_NAME=Neg; Treatment=Extract_15N1Gln_DomainonlyProtein SUBJECT_SAMPLE_FACTORS - E_15NGln_D_4_pos Rawfilename:E_15NGln_D_4_pos RAW_FILE_NAME=Pos; Treatment=Extract_15N1Gln_DomainonlyProtein SUBJECT_SAMPLE_FACTORS - E_15NGln_D_4_neg Rawfilename:E_15NGln_D_4_neg RAW_FILE_NAME=Neg; Treatment=Extract_15N1Gln_DomainonlyProtein #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 growth. Plants were grown on vertical plates at 22 °C under continuous TR:TREATMENT_SUMMARY light (ca. 70 μmol m-2 s-2), as previously described by Ivanov et al. (2012) on TR:TREATMENT_SUMMARY a defined nutrient medium containing a final concentration of 10 mM potassium TR:TREATMENT_SUMMARY phosphate (pH 6.5), 5 mM KNO3, 2 mM MgSO4, 1 mM CaCl2, 125 μg FeNaEDTA, TR:TREATMENT_SUMMARY micronutrients (50 mM H3BO3, 12 mM MnSO4, 1 mM ZnCl2, 1 mM CuSO4 and 0.2 mM TR:TREATMENT_SUMMARY Na2MoO4), 1% sucrose and 1% agar [28]. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Fifty mg of root tissue was excised from 10 day old seedlings of Arabidopsis SP:SAMPLEPREP_SUMMARY grown with 5 mM KNO3, collected in 2 mL Eppendorf tubes and flash frozen in SP:SAMPLEPREP_SUMMARY liquid N2. Frozen tissue was homogenized using a tissue lyser and metabolites SP:SAMPLEPREP_SUMMARY were isolated using 1 mL of methanol:water (4:1) with incubation in an SP:SAMPLEPREP_SUMMARY ultra-sonication bath for 20 min followed by shaking for 30 min at 4 °C. The SP:SAMPLEPREP_SUMMARY mixture was centrifuged at 12,000 × g for 10 min at 4 °C and 700 µl of the SP:SAMPLEPREP_SUMMARY supernatant was transferred into fresh tubes and evaporated to dryness using a SP:SAMPLEPREP_SUMMARY Vacufuge at ambient temperature. Dried metabolite extracts were re-suspended in SP:SAMPLEPREP_SUMMARY HEPES buffer pH 7.5 instead of 1:1 methanol:water. Samples were spiked with a SP:SAMPLEPREP_SUMMARY final concentration of 1 µM 15N Gln and 5 µg of the full length or glutaminase SP:SAMPLEPREP_SUMMARY domain versions of recombinant GAT1_2.1 protein along with 2 mM DTT and 5 mM SP:SAMPLEPREP_SUMMARY MgCl2. Following this, samples were incubated at 37 °C for 2 hours and then SP:SAMPLEPREP_SUMMARY filtered through a 3K micro centrifuge filter (Sigma-Aldrich) to remove the SP:SAMPLEPREP_SUMMARY protein. Samples were then evaporated to dryness using a vacufuge at ambient SP:SAMPLEPREP_SUMMARY temperature and the residue was re-dissolved in 1:1 methanol:water, filtered SP:SAMPLEPREP_SUMMARY with a 0.2 µm PTFE microfuge filters (Whatman) and subjected to LC-MS analysis SP:SAMPLEPREP_SUMMARY and ammonium quantification. #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 Enzyme_spike_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 8/27/2017 AN:ANALYSIS_PROTOCOL_FILE Enzyme_spike_analysis.pdf AN:DATA_FORMAT .RAW #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Full MS measurements were collected from mass ranges of 75-1100 m/z and 65-900 MS:MS_COMMENTS m/z in positive and negative ionization modes respectively at 140,000 MS:MS_COMMENTS resolutions. The AGC target and maximum IT was set to 3 e6 and 524 ms MS:MS_COMMENTS respectively. MS:CAPILLARY_TEMPERATURE 250 MS:CAPILLARY_VOLTAGE 3.5 MS:MS_RESULTS_FILE ST001812_AN002938_Results.txt UNITS:Intensity Has m/z:Yes Has RT:Yes RT units:Minutes #END