#METABOLOMICS WORKBENCH jrenaud86_20170312_095122 DATATRACK_ID:863 STUDY_ID:ST000591 ANALYSIS_ID:AN000906 PROJECT_ID:PR000432 VERSION 1 CREATED_ON April 16, 2017, 6:00 pm #PROJECT PR:PROJECT_TITLE Understanding the effects of acetyl-CoA carboxylase (ACC1) upon the metabolite PR:PROJECT_TITLE profiles of Arabidopsis PR:PROJECT_TYPE Single gene knockout impact on Arabidopsis metabolites PR:PROJECT_SUMMARY The arabidopsis gene, acetyl-CoA carboxylase1 (ACC1) catalyzes the carboxylation PR:PROJECT_SUMMARY of acetyl-CoA to malonyl-CoA. When this gene malfunctions, there are elevated PR:PROJECT_SUMMARY levels of acetyl-CoA which have been shown to increase levels of histone PR:PROJECT_SUMMARY acetylation. This project aims to understand how a malfunctioning ACC1 effects PR:PROJECT_SUMMARY the levels of primary metabolites by comparing to metabolite profiles of wild PR:PROJECT_SUMMARY type Arabidopsis grown under identical conditions. PR:INSTITUTE Agriculture and Agri-Food Canada PR:DEPARTMENT Chemistry PR:LABORATORY Renaud PR:LAST_NAME Renaud PR:FIRST_NAME Justin PR:ADDRESS 1391 Sandford Street PR:EMAIL justin.renaud@agr.gc.ca PR:PHONE 519-953-6698 PR:FUNDING_SOURCE Natural Science and Engineering Research Council of Canada (R4019A01) and PR:FUNDING_SOURCE Agriculture and Agri-Food Canada A-base #STUDY ST:STUDY_TITLE Metablomic profiling in acc1-5 mutant and wild type arabidiopsis ST:STUDY_SUMMARY This experiment tests the metabolic consequence of a mutation at the ACC1 gene ST:STUDY_SUMMARY (At1g36160). The allele of acc1-5 bearing an EMS mutation, which cause a single ST:STUDY_SUMMARY amino acid substitution from aspartic acid to asparagine. Seedlings from both ST:STUDY_SUMMARY the acc1-5 mutant and the wild type were harvested and analyzed via HILIC LC-MS. ST:STUDY_SUMMARY Of particular interest are metabolites which would be affected by depletion of ST:STUDY_SUMMARY malonyl-CoA pools (flavenoids) and primary metabolites. ST:INSTITUTE Agriculture and Agri-Food Canada ST:DEPARTMENT London Research and Developmental Centre ST:LABORATORY Renaud ST:LAST_NAME Renaud ST:FIRST_NAME Justin ST:ADDRESS 1391 Sandford street, London, Ontario, Canada ST:EMAIL justin.renaud@agr.gc.ca ST:PHONE 519-953-6698 #SUBJECT SU:SUBJECT_TYPE Plant SU:SUBJECT_SPECIES Arabidopsis thaliana SU:TAXONOMY_ID 3702 SU:GENOTYPE_STRAIN Col-0 SU:AGE_OR_AGE_RANGE 12 Days after germination SU:WEIGHT_OR_WEIGHT_RANGE 50 mg #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - WT-1 Genotype:Wild-type SUBJECT_SAMPLE_FACTORS - WT-2 Genotype:Wild-type SUBJECT_SAMPLE_FACTORS - WT-3 Genotype:Wild-type SUBJECT_SAMPLE_FACTORS - WT-4 Genotype:Wild-type SUBJECT_SAMPLE_FACTORS - WT-5 Genotype:Wild-type SUBJECT_SAMPLE_FACTORS - WT-6 Genotype:Wild-type SUBJECT_SAMPLE_FACTORS - WT-7 Genotype:Wild-type SUBJECT_SAMPLE_FACTORS - ACC1-1 Genotype:acc1-knockout mutant SUBJECT_SAMPLE_FACTORS - ACC1-2 Genotype:acc1-knockout mutant SUBJECT_SAMPLE_FACTORS - ACC1-3 Genotype:acc1-knockout mutant SUBJECT_SAMPLE_FACTORS - ACC1-4 Genotype:acc1-knockout mutant SUBJECT_SAMPLE_FACTORS - ACC1-5 Genotype:acc1-knockout mutant SUBJECT_SAMPLE_FACTORS - ACC1-6 Genotype:acc1-knockout mutant SUBJECT_SAMPLE_FACTORS - ACC1-7 Genotype:acc1-knockout mutant #COLLECTION CO:COLLECTION_SUMMARY Arabidopsis seeds obtained from the Arabidopsis Biological Resource Center CO:COLLECTION_SUMMARY (ABRC) at the Ohio State University were grown in half strength of Murashige and CO:COLLECTION_SUMMARY Skoog (½ MS) medium (0.5XMS salts, 1.5% [w/v] sucrose, and 0.8% agar [pH 5.8]) CO:COLLECTION_SUMMARY or soil under 16h/8h light/dark cycle at 23°C. Plants were harvested 12 days CO:COLLECTION_SUMMARY after germination. 50 mg of each plant (7 mutants, 7 wild type) were ground in CO:COLLECTION_SUMMARY liquid N2 using ball mill and immediately extracted following collection CO:COLLECTION_SUMMARY protocol. CO:COLLECTION_PROTOCOL_FILENAME collection_protocol.pdf CO:SAMPLE_TYPE Seedlings CO:COLLECTION_LOCATION London, Ontario CO:COLLECTION_FREQUENCY Single #TREATMENT TR:TREATMENT_SUMMARY Effects of single gene knockout on arabidopsis metabolites compared to wild-type TR:TREATMENT_SUMMARY control. 7 replicates of wild-type, 7-replicates of acc1-5 knockout. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY 50 mg of 12 DAG WT and acc1-5 seedlings were collected and grinded in liquid N2 SP:SAMPLEPREP_SUMMARY using a ball-mill. The fine powders were suspended in 1 mL ice cooled methanol: SP:SAMPLEPREP_SUMMARY water (4:1) by vortex. The mixtures were sonicated in water bath sonicator for SP:SAMPLEPREP_SUMMARY 15 mins and followed by centrifugation at 11,000g for 10 mins at 4 °C. 700 µL SP:SAMPLEPREP_SUMMARY of the supernatant was transferred into fresh tubes and evaporated to dryness SP:SAMPLEPREP_SUMMARY using a vacufuge at ambient temperature. The residue was re-dissolved in 1:1 SP:SAMPLEPREP_SUMMARY mixture of methanol: water and vortexed vigorously. All samples were filtered SP:SAMPLEPREP_SUMMARY using 0.2 μm PTFE syringe filter (Whatman) and 5 µL of 1 µg/mL 13C6 SP:SAMPLEPREP_SUMMARY phenylalanine internal standard (Cambridge Isotopes, Tewksbury, USA) were added SP:SAMPLEPREP_SUMMARY to all samples #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Samples were resolved using HILIC column CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Agilent 1290 CH:COLUMN_NAME SeQuant ZIC- pHILIC (100 x 2.1mm, 3.5um) CH:FLOW_GRADIENT 87% B for 5 minutes, decreased to 55% over 8 minutes and held for 4 minutes CH:FLOW_GRADIENT before returning to 87% over 3 minute CH:FLOW_RATE 0.3 ml min-1 CH:COLUMN_TEMPERATURE 30C CH:SOLVENT_A 5 mM ammonium acetate, pH = 4.00 CH:SOLVENT_B B = 90% acetonitrile, 0.1% formic acid CH:INTERNAL_STANDARD 13C6 phenylalanine internal standard #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME Renaud AN:OPERATOR_NAME Justin Renaud AN:DETECTOR_TYPE Orbitrap #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS High resolution 140k for sample comparison. The automatic gain control (AGC) and MS:MS_COMMENTS maximum injection time (IT) were 3×106 and 524 ms respectively. MS:CAPILLARY_TEMPERATURE 250°C MS:COLLISION_ENERGY - MS:DRY_GAS_FLOW 30 units MS:FRAGMENTATION_METHOD HCD MS:ION_SOURCE_TEMPERATURE 450 MS:ION_SPRAY_VOLTAGE 3.9 kV MS:IONIZATION ESI MS:MASS_ACCURACY <5 ppm MS:DATAFORMAT .raw MS:SCANNING_RANGE 93-1400 #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Peak area MS_METABOLITE_DATA_START Samples ACC1-1 ACC1-2 ACC1-3 ACC1-4 ACC1-5 ACC1-6 ACC1-7 WT-1 WT-2 WT-3 WT-4 WT-5 WT-6 WT-7 Factors Genotype:acc1-knockout mutant Genotype:acc1-knockout mutant Genotype:acc1-knockout mutant Genotype:acc1-knockout mutant Genotype:acc1-knockout mutant Genotype:acc1-knockout mutant Genotype:acc1-knockout mutant Genotype:Wild-type Genotype:Wild-type Genotype:Wild-type Genotype:Wild-type Genotype:Wild-type Genotype:Wild-type Genotype:Wild-type Valine 1251191058 1397100604 1627876569 1292553008 1209247387 1336199370 1181425154 431696029.5 445446298.7 455151809.2 350724867.5 378317578.8 540314322.1 576476445.7 Tyrosine 134351962.8 218512163.4 180005380.5 170607922.5 155543060.2 163580810.1 109258358.3 82419015.71 44701117.82 29766634.96 30866655 42713359.05 73947924.8 69135186.08 Tyramine 1197147162 820881927.8 1132435811 546423814.7 1101351042 664667667.6 598729316.1 432254230.9 329140848.2 425298143.8 187062583.6 245259482 248820956.5 277676472.7 Tryptophan 297827077.5 398002279.2 324418392.9 465241534.6 306797712.9 348849194.4 257717839.4 195232564.3 77908080.77 46933311.92 44179071.86 53279175.44 94939950.26 73772610.92 S-methylmethionine 1610436246 2067102275 1818734739 1450192851 1339751139 1205509466 1442429654 519343923.8 505639700.3 267291033.5 236062521.7 271195444.7 309762367.1 416003224.5 Serine 1096946003 1229927517 1381168529 779247104.6 835979301.1 797366995.4 814862903.4 434161917.4 332087287.4 399884684 286645734.1 368907664.2 396138912.4 435450279.7 Quercitin-glucose-rhamnoside 10802071.77 4013830.27 3882156.498 5486708.253 5648782.039 3801972.256 8703673.34 1860727422 1529994494 1613345784 852178811.6 1472744410 1276993427 1619422120 Quercitin 28614059.97 20088148.12 16921937.35 19165332.01 17921507.98 16109488.14 15745403.93 77549440.71 72362244.03 66696488.21 57739736.48 75561247.03 54054468.23 62542075.97 Proline 15089604960 4018071618 6834287705 2567818491 4332857070 5301066662 3476091855 2684104776 5335666856 1945452806 1391219217 1656055130 1836712047 2350402315 Phenylalanine 254471276.1 273747351.6 117108044.7 264243279.1 262105028.2 273809296.1 232247246 136098451.3 105722830.9 95394843.48 64047041.6 92659958.89 99829802.56 111425287.4 p-Coumaroylagmatine 23149342121 11678055396 28323122651 10577177746 9448940671 22673019232 8122146910 1258566850 1074066267 586149512.6 338572168.6 749630095.6 755026327.4 837450828.8 Ornithine 537774112.5 445965812.3 366084514.7 556246229.2 362628943.7 306122648.7 361175010.4 321155655.8 383238476.9 208091710.3 139339900.6 183843038.5 185114633.6 243156412.1 N-Acetylornithine 66257214.61 98430645.78 122088307.6 117050852.9 103481874.1 115890307.7 140310124.2 15688635.2 15507421.12 6859281.175 8009688.345 8973527.293 11182614.69 15746942.64 Methionine 172151118.1 186495690.1 203239309.1 82212787.18 80170170.27 96676437.67 93670375.05 133180622.1 74604980.36 64103369.8 48748502.38 68102468.82 53491210.41 51509940.33 Lysine 295384528.8 427963414 293660049.5 360938649.9 305351427.7 273762520 252873719.8 158450556.5 119904093.6 120092823.9 85002776.84 119374639.9 132728161.3 158642183.6 Leucine 1011640002 1187639158 992720241.7 1121461070 962500356.5 1161666816 771834589.9 999731700.6 701254819 653640292 430073010.9 630399721.3 784689988.4 800938300.3 Kaempferol-rhamnose-rhamnoside 194926887.3 146647118.8 133313940.3 171449115.6 116216120.3 154799157.8 100877083.1 841256512.1 954183257.1 464221018 232239586.5 443433611.5 431591598.5 550691796 Kaempferol-rhamnoside 13673937.7 8209326.254 7304680.2 9243016.072 6974424.616 6837798.446 5732703.557 126561888.1 105867571.6 71891657.85 32139467.86 63790745.33 73578176.14 87320553.93 Kaempferol -glucose-rhamnose-rhamnoside 69813227.44 37036073.64 41653211.04 43557682.26 37128435.45 39495531 30352422.18 696930637.5 639442129.2 508973139.9 281388601.7 451760880.1 506395939.9 512797795.2 Kaempferol- rhamnose- rhamnoside 48771622.58 28546704.61 32827698.35 36634424.76 27214206.63 34732300.91 24461348.14 202945364.9 169031272.6 106765571.4 67707551.99 83315969.2 93625227.07 132540405.6 Kaempferol-glucose-rhamnoside 72923468.44 38299368.51 31188204.04 39391216.58 23589885.89 27610828.04 39518787.79 1831340325 1884577080 1473457131 819998878.9 1487201028 1568880327 1713135603 Isorharmnetin-glucose-rhamnoside 3876006.183 1326719.724 1634534.728 2693428.529 2497699.409 1590625.236 1911057.038 301052271.4 241966426.2 214307198.5 101629657 203934788.9 184681786.9 239772551.8 Isoleucine 598859009.3 580327275.9 599899083.9 567378351.8 561007409.1 656689515.1 451611860.3 610524351.6 240697223.1 239317476.5 168892658.9 266693115.7 328637674 341427921.9 Histidine 1361386300 2155358938 2230963905 1963501435 1536882470 1527899833 1774443496 561465863.7 494345525.8 385821575 403391543.8 534442371.8 607974607.5 786028623.4 sn-glycero-3-phosphocholine 552847488.9 279252279 435044524.6 413081597.3 493065991.8 284892241 251858601 1830164266 1125981761 1394698049 880298922.8 925330138.9 576802447.6 1650502045 Glutamine 19851713844 26544821660 27361888148 19817513281 19902919482 20951073035 24438183194 3604070517 3838237445 1754308673 1198833115 1666367937 1859437715 2635590195 Glutamic acid 1143299475 1688727626 1786075250 1155101521 1430770886 1503452841 1374445366 786524247 662069914.3 569886260.2 418613126.7 546921183.9 617719386.5 593862132.5 Gamma-Aminobutyric acid 1133606914 1224979671 1507862275 1007383951 1017790796 1476549135 795274005 196202688.8 636807759.9 209739262 194408655.7 251381782 299503358.2 422256977.1 Feruloylagmatine 4405036040 3526363008 6191350762 3874148187 4145550419 4400866057 2617171160 145966014.1 180783028.7 81433157.86 52315085.46 95424806.14 63123840.18 106404027.2 Docosenamide 4634809633 4695302421 2687711373 5163229661 5116384256 4334733164 5344525445 4107514389 3220921641 5097002822 3533445643 4648172717 4658424929 5165784423 Citrulline 637663862.5 703065163.4 713560383.1 684056561.1 420382995.3 379161930.7 553877595.9 78458552.68 86240291.59 34185706.21 15100682.12 22573059.12 21316455.49 50013996.81 Choline 31730854033 31763528950 30285880184 30348032405 29615307333 28864511311 28130898902 26511625652 27607414494 27055815756 21660731908 17665022738 24957080809 27303291232 Aspartic acid 367343290.6 512353315.1 528880803.5 475048349.2 497680818.5 497600681.3 238563654.7 254120846 258490744.4 333390547.5 274118713.1 309508810.5 307458541.9 313378737.9 Asparagine 1971505078 1455530297 3770870260 2051561973 1882571703 1684035073 2682225277 472780464.7 389384610.3 252990241.2 204762366.4 277785777.6 301784511.6 382666714.4 Arginine 13784768252 16445611225 12305273738 14284381406 11102024078 9459301187 12384722835 5957916681 4803257838 3960073124 2419781384 3915664357 3512512851 5305659377 Anthranillic acid 21425021.89 26191861.36 24487156.45 40408567.99 37968992.14 41110571.24 35343181.26 17250491.78 10766017.23 13437957.78 17768790.08 19053452.33 18637593.09 14355569.36 Aminoadipic acid 224639456.1 207235064.9 193862336 138045254.7 178526272.7 162470909.4 183881649.5 184456284.4 92104274.36 100340568.7 49259523.1 46081708.9 56761840.28 76734243.24 Acetylagmatine 4249107107 5941240292 8476256414 5478388200 4606540560 5663793431 7001161145 450640954.3 375870418.7 147648976.8 115343350.7 166205657 234490976.4 524238325.2 8-(methylsulfinyl)octylamine related III 2153253274 4656279630 3288533812 2460692971 2207049704 2409392901 1843791221 864040777.6 483256461.1 148776168.3 180336939 251764071.4 396823685.5 378556165.6 8-(methylsulfinyl)octylamine related II 4899113018 3948170271 3551556019 2045304673 1891172514 2510314641 1414605596 284143474.8 153838723 74569478.26 81004417.71 76219239.76 118852357.8 144634865.5 8-(methylsulfinyl)octylamine related I 18617152127 16557023775 14399797630 11467948690 10055313114 11224773302 4934164657 1728314562 1488630227 625346583.6 663834660.5 756325651.2 1196468043 1365658440 4-Guanidinobutanoic acid 1599545819 1401724816 1997249210 1187638397 1250702675 1244539274 1072817358 791685898 541303208.9 629708847.5 335814684.4 427398724.5 442559492 501020026.2 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name retention index quantified m/z PubChem ID KEGG ID Mass P( benjamini-hochberg) fold change (log 2) Valine 8.09 118.0866177 117.0791956 0.00000051 1.54836999 Tyrosine 8.35 182.0813646 181.0739425 0.00007609 1.59932223 Tyramine 5.02 138.0914242 137.0840021 0.00095041 1.498384945 Tryptophan 5.69 205.097232 204.0898099 0.0000301 2.032770332 S-methylmethionine 15.27 164.0740134 163.0665913 0.0000043 2.11431624 Serine 10.73 106.0503484 105.0429263 0.00014666 1.386224535 Quercitin-glucose-rhamnoside 2.63 611.1604015 610.1529794 0.0000013 -7.915948658 Quercitin 2.45 303.1049516 302.0975295 0.00000092 -1.793582085 Proline 9.06 116.0709983 115.0635762 0.11199113 1.274893859 Phenylalanine 9.95 164.0294464 163.0220243 0.00022976 1.250452054 p-Coumaroylagmatine 4.48 277.1658992 276.1584771 0.00108681 4.347244921 Ornithine 14.9 133.0972962 132.0898741 0.00802675 0.819247364 N-Acetylornithine 8.31 161.0922022 160.0847801 0.00000229 3.219511135 Methionine 6.55 150.0584053 149.0509832 0.05121991 0.889411254 Lysine 14.63 147.1128268 146.1054047 0.00003776 1.305341722 Leucine 5.87 132.1020479 131.0946258 0.008592 0.527753563 Kaempferol-rhamnose-rhamnoside 1.48 579.1706483 578.1632262 0.00310371 -1.943913842 Kaempferol-rhamnoside 2.87 433.1128671 432.105445 0.00020234 -3.274861734 Kaempferol -glucose-rhamnose-rhamnoside 2.88 741.2231475 740.2157254 0.00000739 -3.588676634 Kaempferol- rhamnose- rhamnoside 1.01 579.170808 578.1633859 0.0016024 -1.87599967 Kaempferol-glucose-rhamnoside 1.81 595.1654926 594.1580705 0.00000207 -5.305653278 Isorharmnetin-glucose-rhamnoside 1.66 625.1759965 624.1685744 0.00000882 -6.581530798 Isoleucine 4.87 132.1020526 131.0946305 0.00270406 0.870674393 Histidine 14.06 156.0768522 155.0694301 0.00000789 1.733773775 sn-glycero-3-phosphocholine 12.43 258.1101886 257.1027665 0.00191476 -1.629284856 Glutamine 10.61 147.0764844 146.0690623 0.00000022 3.262329839 Glutamic acid 10.27 148.060432 147.0530099 0.00001928 1.264815924 Gamma-Aminobutyric acid 9.14 104.0711498 103.0637277 0.00005278 1.884935898 Feruloylagmatine 4.1 307.1763625 306.1689404 0.00000447 5.328994833 Docosenamide 0.89 338.3417063 337.3342842 0.72691866 0.071466497 Citrulline 11.23 176.1030724 175.0956503 0.0000037 3.732243491 Choline 7.86 104.1074628 103.1000407 0.00938163 0.286679991 Aspartic acid 11 134.0449278 133.0375057 0.00861903 0.604423615 Asparagine 10.74 133.0609544 132.0535323 0.00017835 2.763641457 Arginine 14.33 175.1190865 174.1116644 0.00001076 1.587238216 Anthranillic acid 9.18 138.0551005 137.0476784 0.0011023 1.028218362 Aminoadipic acid 6.55 162.0760809 161.0686588 0.00191476 1.08910585 Acetylagmatine 7.31 173.1397776 172.1323555 0.00000351 4.361755412 8-(methylsulfinyl)octylamine related III 6.39 192.1416566 191.1342345 0.00021105 2.81451168 8-(methylsulfinyl)octylamine related II 6.12 192.1416712 191.1342491 0.00037701 4.440224206 8-(methylsulfinyl)octylamine related I 5.82 192.1416855 191.1342634 0.00012696 3.479172323 4-Guanidinobutanoic acid 7.33 146.0924487 145.0850266 0.00012669 1.410446237 METABOLITES_END #END