#METABOLOMICS WORKBENCH ayansadhukhan_20240624_215513 DATATRACK_ID:4955 STUDY_ID:ST003861 ANALYSIS_ID:AN006347 PROJECT_ID:PR002418 VERSION 1 CREATED_ON April 14, 2025, 10:10 pm #PROJECT PR:PROJECT_TITLE Comparative effects of nano urea and equimolar urea on the metabolome profile of PR:PROJECT_TITLE Arabidopsis thaliana seedlings PR:PROJECT_TYPE GC MS PR:PROJECT_SUMMARY We compared the effects of Indian Farmers Fertilizer Cooperative (IFFCO) nano PR:PROJECT_SUMMARY urea formulation with equimolar conventional urea on the metabolome of PR:PROJECT_SUMMARY Arabidopsis thaliana seedlings. Seedlings were grown in hydroponics in 1/4th PR:PROJECT_SUMMARY strength Hoagland's solution for 12 days, then transferred to a nitrogen-free PR:PROJECT_SUMMARY modified Hoagland's solution and grown for 21 days. Thereafter whole seedlings PR:PROJECT_SUMMARY were crushed in lquid nitrogen to a fine powder. To 500 mg of fine powder, 5 mL PR:PROJECT_SUMMARY of HPLC-grade methanol was added, and the sample was shaken/vortexed for over 30 PR:PROJECT_SUMMARY minutes to extract analytes. The sample was spun at 8,000 rpm for 10 minutes to PR:PROJECT_SUMMARY separate the liquid phase, which was then transferred to a fresh tube and stored PR:PROJECT_SUMMARY at -20°C. Derivatization was not done, as it was not required for non-volatile PR:PROJECT_SUMMARY compounds. Gas Chromatography-Mass Spectrometry was conducted using GC-MS model PR:PROJECT_SUMMARY QP2010 (Shimadzu®) using a SH-I-5Sil MS Capillary (30 m x 0.25 mm x 0.25 µm) PR:PROJECT_SUMMARY column. 2 µL of samples were injected using a splitless injection mode. Helium PR:PROJECT_SUMMARY (1 mL/min) was used as a carrier gas. Ionization of 70 eV and a running time of PR:PROJECT_SUMMARY 9.10 min – 52.0 min was used. The oven Program included an initial temperature PR:PROJECT_SUMMARY of 45°C for 2 min, ramped to 140°C at 5°C/min, and then increased to 280°C, PR:PROJECT_SUMMARY held isothermally for 10 min. The NIST14.L library (2020) was used to identify PR:PROJECT_SUMMARY compounds based on retention times and mass spectra. PR:INSTITUTE Indian Institute of Technology Jodhpur PR:DEPARTMENT BIOSCIENCE AND BIOENGINEERING PR:LABORATORY Plant functional genomics PR:LAST_NAME SADHUKHAN PR:FIRST_NAME AYAN PR:ADDRESS NH62, NAGAUR ROAD PR:EMAIL ayansadhukhan@iitj.ac.in PR:PHONE 09038013015 PR:FUNDING_SOURCE IFFCO #STUDY ST:STUDY_TITLE Comparative effects of nano urea and equimolar urea on the metabolome profile of ST:STUDY_TITLE Arabidopsis thaliana seedlings ST:STUDY_SUMMARY We compared the effects of Indian Farmers Fertilizer Cooperative (IFFCO) nano ST:STUDY_SUMMARY urea formulation with equimolar conventional urea on the metabolome of ST:STUDY_SUMMARY Arabidopsis thaliana seedlings. Seedlings were grown in hydroponics in 1/4th ST:STUDY_SUMMARY strength Hoagland's solution for 12 days, then transferred to a nitrogen-free ST:STUDY_SUMMARY modified Hoagland's solution and grown for 21 days. Thereafter whole seedlings ST:STUDY_SUMMARY were crushed in lquid nitrogen to a fine powder. To 500 mg of fine powder, 5 mL ST:STUDY_SUMMARY of HPLC-grade methanol was added, and the sample was shaken/vortexed for over 30 ST:STUDY_SUMMARY minutes to extract analytes. The sample was spun at 8,000 rpm for 10 minutes to ST:STUDY_SUMMARY separate the liquid phase, which was then transferred to a fresh tube and stored ST:STUDY_SUMMARY at -20°C. Derivatization was not done, as it was not required for non-volatile ST:STUDY_SUMMARY compounds. Gas Chromatography-Mass Spectrometry was conducted using GC-MS model ST:STUDY_SUMMARY QP2010 (Shimadzu®) using a SH-I-5Sil MS Capillary (30 m x 0.25 mm x 0.25 µm) ST:STUDY_SUMMARY column. 2 µL of samples were injected using a splitless injection mode. Helium ST:STUDY_SUMMARY (1 mL/min) was used as a carrier gas. Ionization of 70 eV and a running time of ST:STUDY_SUMMARY 9.10 min – 52.0 min was used. The oven Program included an initial temperature ST:STUDY_SUMMARY of 45°C for 2 min, ramped to 140°C at 5°C/min, and then increased to 280°C, ST:STUDY_SUMMARY held isothermally for 10 min. The NIST14.L library (2020) was used to identify ST:STUDY_SUMMARY compounds based on retention times and mass spectra. ST:INSTITUTE Indian Institute of Technology Jodhpur ST:LAST_NAME SADHUKHAN ST:FIRST_NAME AYAN ST:ADDRESS NH62, NAGAUR ROAD ST:EMAIL ayansadhukhan@iitj.ac.in ST:PHONE 09038013015 #SUBJECT SU:SUBJECT_TYPE Plant SU:SUBJECT_SPECIES Arabidopsis thaliana SU:TAXONOMY_ID 3702 #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 - 15d-c1-1 Sample source:Wild-type seedlings | Treatment:Control RAW_FILE_NAME(Raw file name)=15d-C1_1.qgd SUBJECT_SAMPLE_FACTORS - 15d-c2-2 Sample source:Wild-type seedlings | Treatment:Control RAW_FILE_NAME(Raw file name)=15d-C2_2.qgd SUBJECT_SAMPLE_FACTORS - 15d-c3-3 Sample source:Wild-type seedlings | Treatment:Control RAW_FILE_NAME(Raw file name)=15d-C3_3.qgd SUBJECT_SAMPLE_FACTORS - 15d-u1-4 Sample source:Wild-type seedlings | Treatment:urea RAW_FILE_NAME(Raw file name)=15d-u1-4.qgd SUBJECT_SAMPLE_FACTORS - 15d-u2-5 Sample source:Wild-type seedlings | Treatment:urea RAW_FILE_NAME(Raw file name)=15d-u2-5.qgd SUBJECT_SAMPLE_FACTORS - 15d-u3-6 Sample source:Wild-type seedlings | Treatment:urea RAW_FILE_NAME(Raw file name)=15d-u3-6.qgd SUBJECT_SAMPLE_FACTORS - 15d-nu1-7 Sample source:Wild-type seedlings | Treatment:nanourea RAW_FILE_NAME(Raw file name)=15d-nu1-7.qgd SUBJECT_SAMPLE_FACTORS - 15d-nu2-8 Sample source:Wild-type seedlings | Treatment:nanourea RAW_FILE_NAME(Raw file name)=15d-nu2-8.qgd SUBJECT_SAMPLE_FACTORS - 15d-nu3-9 Sample source:Wild-type seedlings | Treatment:nanourea RAW_FILE_NAME(Raw file name)=15d-nu3-9.qgd #COLLECTION CO:COLLECTION_SUMMARY Seedlings were grown in hydroponics in 1/4th strength Hoagland's solution for 12 CO:COLLECTION_SUMMARY days, then transferred to a nitrogen-free modified Hoagland's solution and grown CO:COLLECTION_SUMMARY for 21 days. Thereafter whole seedlings were crushed in liquid nitrogen to a CO:COLLECTION_SUMMARY fine powder. CO:SAMPLE_TYPE Plant CO:STORAGE_CONDITIONS -80℃ CO:COLLECTION_TUBE_TEMP -80C #TREATMENT TR:TREATMENT_SUMMARY After 12 d hydroponic growth in nitrogen-containing media, then transferred to TR:TREATMENT_SUMMARY the nitrogen-free hydroponic media supplemented with 70 μM urea and nano urea, TR:TREATMENT_SUMMARY and were grown further for 21 days. On the 21st day, seedlings were harvested. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY To 500 mg of fine powder of crushed seedlings, 5 mL of HPLC-grade methanol was SP:SAMPLEPREP_SUMMARY added, and the sample was shaken/vortexed for over 30 minutes to extract SP:SAMPLEPREP_SUMMARY analytes. The sample was centrifuged at 8,000 rpm for 10 minutes to separate the SP:SAMPLEPREP_SUMMARY liquid phase, which was then transferred to a fresh tube and stored at -20°C. SP:SAMPLEPREP_SUMMARY Note: Derivatization was not done, as it was not required for non-volatile SP:SAMPLEPREP_SUMMARY compounds SP:SAMPLE_DERIVATIZATION Derivatization was not done, as it was not required for non-volatile compounds #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Oven temperature was initially held at 45°C for 2 min then increased to 140°C CH:CHROMATOGRAPHY_SUMMARY at 5°C/min and finally increased to 280°C and held isothermally for 10 min. CH:CHROMATOGRAPHY_SUMMARY The sample injection was 2 μL and the carrier gas was helium at 1 mL/min. The CH:CHROMATOGRAPHY_SUMMARY ionization of the sample components was carried out 70 eV. The running time of CH:CHROMATOGRAPHY_SUMMARY the GC was from 9.10 min – 52.0 min. CH:CHROMATOGRAPHY_TYPE GC CH:INSTRUMENT_NAME Shimadzu GCMS-QP2010 ultra CH:COLUMN_NAME Shimadzu SH-I-5Sil MS Capillary (30 m x 0.25 mm, 0.25um) CH:SOLVENT_A helium CH:SOLVENT_B helium CH:FLOW_GRADIENT - CH:FLOW_RATE 1 mL/min CH:COLUMN_TEMPERATURE 45 °C for 2 min; then increased to 140 °C at 5°C/min; and finally increased CH:COLUMN_TEMPERATURE to 280°C and held isothermally for 10 min #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Shimadzu QP2010 Ultra MS:INSTRUMENT_TYPE Single quadrupole MS:MS_TYPE EI MS:ION_MODE POSITIVE MS:MS_COMMENTS NIST14.L library (2020) was then searched to compare the structures of the MS:MS_COMMENTS compounds with that of the NIST database. Compounds were then identified based MS:MS_COMMENTS on the retention times and mass spectra with already known compounds in the NIST MS:MS_COMMENTS library (NIST14.L). #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS intensity MS_METABOLITE_DATA_START Samples 15d-c1-1 15d-c2-2 15d-c3-3 15d-nu1-7 15d-nu2-8 15d-nu3-9 15d-u1-4 15d-u2-5 15d-u3-6 Factors Sample source:Wild-type seedlings | Treatment:Control Sample source:Wild-type seedlings | Treatment:Control Sample source:Wild-type seedlings | Treatment:Control Sample source:Wild-type seedlings | Treatment:nanourea Sample source:Wild-type seedlings | Treatment:nanourea Sample source:Wild-type seedlings | Treatment:nanourea Sample source:Wild-type seedlings | Treatment:urea Sample source:Wild-type seedlings | Treatment:urea Sample source:Wild-type seedlings | Treatment:urea Methyl methanesulfinothioate 117459.2403 110558.268 92908.47482 63115.31789 55354.82813 50794.40067 88714.29381 91041.41804 76929.54351 Fluroxypyr 117264.9685 107685.2112 109818.4567 80042.19878 68293.12439 66384.52292 94711.03147 123306.9831 106730.8936 Dimethylcarbamoyl chloride 35447.81068 76356.16907 73754.7682 54627.38657 52527.40846 14559.08027 66160.81199 86023.22017 73611.74239 Ethyl 2-mercaptopropionate 18793.32397 180305.81 168191.9532 94081.75375 98793.30892 118991.5508 235700.2317 94213.55184 123153.6567 Fudosteine 16999.03798 13744.05576 7447.034666 11752.8425 8408.57141 11528.68676 7297.877038 10886.2247 9744.053139 Diisopropyl sulfate 93151.41692 83668.77209 42613.56952 64575.71176 47416.77926 47104.16987 49461.2946 39852.77064 40083.22985 Metam-sodium 203687.561 137376.6954 128175.229 61208.87028 35233.70059 45743.27395 94965.32625 112273.815 154611.3548 Digitalose 263290.0612 111085.2669 60097.07218 78633.13508 139017.3307 102480.2101 149366.969 344550.2738 122361.052 1-Piperazinecarbodithioic acid 75077.75511 179417.5581 208074.3157 193613.3447 186385.7973 233938.6086 196664.2538 206966.1643 214961.3637 Cyanophos 53879.12074 3015.581023 93558.64186 0 0 0 116843.8734 155812.1832 141367.6408 4-Chlorophenoxyacetate 97842.65372 51181.26338 74331.91127 77202.93543 175703.7606 91684.46892 141935.2656 136251.9816 100822.4963 Glycyl-Threonine 26714.81287 21349.63007 5928.055006 21550.28727 4922.308267 11936.59278 26505.10141 6338.953593 0 Pterostilbene 101532.9792 17682.89192 14265.61212 6876.862149 3326.345862 15217.18744 16841.46197 4911.169911 8430.640107 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name retention index quantified m/z Methyl methanesulfinothioate 42.345 117 Fluroxypyr 42.3475 130 Dimethylcarbamoyl chloride 44.14275 130 Ethyl 2-mercaptopropionate 48.3075 135.05 Fudosteine 36.94766667 144.05 Diisopropyl sulfate 40.715 147 Metam-sodium 44.56983333 147 Digitalose 45.362 185.05 1-Piperazinecarbodithioic acid 42.245 207 Cyanophos 44.0475 208 4-Chlorophenoxyacetate 49.57 209 Glycyl-Threonine 33.93441667 221.05 Pterostilbene 36.985 210.1 METABOLITES_END #END