#METABOLOMICS WORKBENCH tlange_20230801_045110 DATATRACK_ID:4196 STUDY_ID:ST002810 ANALYSIS_ID:AN004570 PROJECT_ID:PR001757 VERSION 1 CREATED_ON August 8, 2023, 4:43 pm #PROJECT PR:PROJECT_TITLE Functional GAS2 studies PR:PROJECT_TYPE GC-MS qualitative analysis PR:PROJECT_SUMMARY Liu et al. recently reported the characterization of Arabidopsis thaliana GAS2 PR:PROJECT_SUMMARY (Gain of Function in ABA-modulated Seed Germination 2), which was described as PR:PROJECT_SUMMARY an enzyme that catalyzes the stereospecific hydration of GA12 to produce GA12 PR:PROJECT_SUMMARY 16, 17-dihydro-16α-ol (DHGA12). A second paper describes the conversion of GA12 PR:PROJECT_SUMMARY to an unidentified product by GAS2 and also reports that this enzyme does not PR:PROJECT_SUMMARY convert ABA. However, as previously reported, we did not find any conversion of PR:PROJECT_SUMMARY [17-14C]-labeled or [1-,7-,12-,18-14C4]-labeled GA12 by GAS2. Instead, we PR:PROJECT_SUMMARY present here data showing that the recombinant GAS2 enzyme is capable of PR:PROJECT_SUMMARY catabolising abscisic acid (ABA) to phaseic acid (PA) and further to a second PR:PROJECT_SUMMARY product, putative 8’-carboxy-ABA (compound A; Fig. 1a). PR:INSTITUTE Technische Universität Braunschweig PR:DEPARTMENT Biochemie und Physiologie der Pflanzen PR:LABORATORY AG Lange PR:LAST_NAME Lange PR:FIRST_NAME Theo PR:ADDRESS Mendelssohnstr. 4 PR:EMAIL theo.lange@tu-bs.de PR:PHONE 00495313915880 PR:CONTRIBUTORS Nadiem Atiq, Maria João Pimenta Lange #STUDY ST:STUDY_TITLE GAS2 encodes a 2-oxoglutarate dependent dioxygenase involved in ABA catabolism ST:STUDY_SUMMARY Liu et al. [1] recently reported the characterization of Arabidopsis thaliana ST:STUDY_SUMMARY GAS2 (Gain of Function in ABA-modulated Seed Germination 2), which was described ST:STUDY_SUMMARY as an enzyme that catalyzes the stereospecific hydration of GA12 to produce ST:STUDY_SUMMARY GA12 16, 17-dihydro-16α-ol (DHGA12). A second paper describes the conversion of ST:STUDY_SUMMARY GA12 to an unidentified product by GAS2 and also reports that this enzyme does ST:STUDY_SUMMARY not convert ABA [2]. However, as previously reported [3], we did not find any ST:STUDY_SUMMARY conversion of [17-14C]-labeled or [1-,7-,12-,18-14C4]-labeled GA12 by GAS2. ST:STUDY_SUMMARY Instead, we present here data showing that the recombinant GAS2 enzyme is ST:STUDY_SUMMARY capable of catabolising abscisic acid (ABA) to phaseic acid (PA) and further to ST:STUDY_SUMMARY a second product, putative 8’-carboxy-ABA (compound A; Fig. 1a) [4]. ST:STUDY_SUMMARY References: [1] Liu, H. et al. Biosynthesis of DHGA12 and its roles in ST:STUDY_SUMMARY Arabidopsis seedling establishment. Nat. Commun. 10, 1768 (2019). [2] Xiong, W. ST:STUDY_SUMMARY et al. The dioxygenase GIM2 functions in seed germination by altering ST:STUDY_SUMMARY gibberellin production in Arabidopsis. J. Integr. Plant Biol. 60, 276-291 ST:STUDY_SUMMARY (2018). [3] Lange, T. & Pimenta Lange, M. J. The Multifunctional Dioxygenases of ST:STUDY_SUMMARY Gibberellin Synthesis. Plant Cell Physiol. 61, 1869-1879 (2020). [4] Lange, T., ST:STUDY_SUMMARY Atiq, N., Pimenta Lange. GAS2 encodes a 2-oxoglutarate dependent dioxygenase ST:STUDY_SUMMARY involved in ABA catabolism. bioRxiv, doi: 10.1101/2022.11.16.516706 (2022). ST:INSTITUTE Technische Universität Braunschweig ST:DEPARTMENT Biochemie und Physiology der Pflanzen ST:LABORATORY AG Lange ST:LAST_NAME Lange ST:FIRST_NAME Theo ST:ADDRESS Mendelssohnstr. 4 ST:EMAIL theo.lange@tu-bs.de ST:PHONE 00495313915880 #SUBJECT SU:SUBJECT_TYPE Plant SU:SUBJECT_SPECIES Arabidopsis thaliana #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 - 266_01 Treatment:DHGA12 Genotype=Standard; RAW_FILE_NAME=266_01.raw SUBJECT_SAMPLE_FACTORS - 403_02 Treatment:GAS2 Genotype=Wild-type; RAW_FILE_NAME=403_02.raw SUBJECT_SAMPLE_FACTORS - 479_01 Treatment:GAS2 Genotype=Wild-type; RAW_FILE_NAME=479_01.raw SUBJECT_SAMPLE_FACTORS - 480_02 Treatment:GAS2 Genotype=Wild-type; RAW_FILE_NAME=480_02.raw SUBJECT_SAMPLE_FACTORS - 481_03 Treatment:GAS2 Genotype=Wild-type; RAW_FILE_NAME=481_03.raw SUBJECT_SAMPLE_FACTORS - 482_04 Treatment:GAS2 Genotype=Wild-type; RAW_FILE_NAME=482_04.raw SUBJECT_SAMPLE_FACTORS - 485_05 Treatment:GAS2 Genotype=Wild-type; RAW_FILE_NAME=485_05.raw SUBJECT_SAMPLE_FACTORS - 486_06 Treatment:GAS2 Genotype=Wild-type; RAW_FILE_NAME=486_06.raw SUBJECT_SAMPLE_FACTORS - 538_01 Treatment:PA Genotype=Standard; RAW_FILE_NAME=538_01.raw SUBJECT_SAMPLE_FACTORS - 539_06 Treatment:d3-PA Genotype=Standard; RAW_FILE_NAME=539_06.raw SUBJECT_SAMPLE_FACTORS - 542_07 Treatment:GAS2 Genotype=Wild-type; RAW_FILE_NAME=542_07.raw SUBJECT_SAMPLE_FACTORS - 543_08 Treatment:GAS2 Genotype=Wild-type; RAW_FILE_NAME=543_08.raw SUBJECT_SAMPLE_FACTORS - 550_09 Treatment:20ox1 Genotype=Wild-type; RAW_FILE_NAME=550_09.raw SUBJECT_SAMPLE_FACTORS - 551_10 Treatment:20ox1 Genotype=Wild-type; RAW_FILE_NAME=551_10.raw SUBJECT_SAMPLE_FACTORS - 552_11 Treatment:20ox1 Genotype=Wild-type; RAW_FILE_NAME=552_11.raw SUBJECT_SAMPLE_FACTORS - 556_02 Treatment:GAS2 Genotype=Wild-type; RAW_FILE_NAME=556_02.raw SUBJECT_SAMPLE_FACTORS - 558_07 Treatment:GAS2 Genotype=Wild-type; RAW_FILE_NAME=558_07.raw SUBJECT_SAMPLE_FACTORS - 559_03 Treatment:ABA Genotype=Standard; RAW_FILE_NAME=559_03.raw SUBJECT_SAMPLE_FACTORS - 583_04 Treatment:GAS2 Genotype=Wild-type; RAW_FILE_NAME=583_04.raw SUBJECT_SAMPLE_FACTORS - 585_05 Treatment:GAS2 Genotype=Wild-type; RAW_FILE_NAME=585_05.raw SUBJECT_SAMPLE_FACTORS - 691_02 Treatment:GAS2 Genotype=Wild-type; RAW_FILE_NAME=691_02.raw SUBJECT_SAMPLE_FACTORS - 746_03 Treatment:GAS2 Genotype=Wild-type; RAW_FILE_NAME=746_03.raw SUBJECT_SAMPLE_FACTORS - 747_04 Treatment:GAS2 Genotype=Wild-type; RAW_FILE_NAME=747_04.raw SUBJECT_SAMPLE_FACTORS - 748_05 Treatment:GAS2 Genotype=Wild-type; RAW_FILE_NAME=748_05.raw SUBJECT_SAMPLE_FACTORS - 868_08 Treatment:d6-ABA Genotype=Standard; RAW_FILE_NAME=868_08.raw SUBJECT_SAMPLE_FACTORS - 907_09 Treatment:GAS2 Genotype=Wild-type; RAW_FILE_NAME=907_09.raw SUBJECT_SAMPLE_FACTORS - 920_10 Treatment:GAS2 Genotype=Wild-type; RAW_FILE_NAME=920_10.raw SUBJECT_SAMPLE_FACTORS - 967_01 Treatment:GAS2 Genotype=Wild-type; RAW_FILE_NAME=967_01.raw SUBJECT_SAMPLE_FACTORS - 969_02 Treatment:GAS2 Genotype=Wild-type; RAW_FILE_NAME=969_02.raw SUBJECT_SAMPLE_FACTORS - 970_03 Treatment:GAS2 Genotype=Wild-type; RAW_FILE_NAME=970_03.raw SUBJECT_SAMPLE_FACTORS - 972_04 Treatment:GAS2 Genotype=Wild-type; RAW_FILE_NAME=972_04.raw SUBJECT_SAMPLE_FACTORS - 974_05 Treatment:GAS2 Genotype=Wild-type; RAW_FILE_NAME=974_05.raw SUBJECT_SAMPLE_FACTORS - 975_06 Treatment:GAS2 Genotype=Wild-type; RAW_FILE_NAME=975_06.raw SUBJECT_SAMPLE_FACTORS - 976_07 Treatment:GAS2 Genotype=Wild-type; RAW_FILE_NAME=976_07.raw SUBJECT_SAMPLE_FACTORS - 990_02 Treatment:GAS2 Genotype=Wild-type; RAW_FILE_NAME=990_02.raw #COLLECTION CO:COLLECTION_SUMMARY Recombinant GAS2 and AtGA20ox1 were produced as previously described [5,6]. [5] CO:COLLECTION_SUMMARY Pimenta Lange, M. J. et al. Functional characterization of gibberellin oxidases CO:COLLECTION_SUMMARY from cucumber, Cucumis sativus L. Phytochemistry 90, 62-69 (2013). [6] Pimenta CO:COLLECTION_SUMMARY Lange, M. J. et al. Cucumber gibberellin 1-oxidase/desaturase initiates novel CO:COLLECTION_SUMMARY gibberellin catabolic pathways. J. Biol. Chem. 295, 8442-8448 (2020). CO:SAMPLE_TYPE Bacterial cells #TREATMENT TR:TREATMENT_SUMMARY Recombinant GAS2 and AtGA20ox1 were produced as previously described [5,6]. [5] TR:TREATMENT_SUMMARY Pimenta Lange, M. J. et al. Functional characterization of gibberellin oxidases TR:TREATMENT_SUMMARY from cucumber, Cucumis sativus L. Phytochemistry 90, 62-69 (2013). [6] Pimenta TR:TREATMENT_SUMMARY Lange, M. J. et al. Cucumber gibberellin 1-oxidase/desaturase initiates novel TR:TREATMENT_SUMMARY gibberellin catabolic pathways. J. Biol. Chem. 295, 8442-8448 (2020). #SAMPLEPREP SP:SAMPLEPREP_SUMMARY 3’,5’,5’,7’,7’,7’-d6-labelled ABA and 17,17-d2-labelled GA12 were SP:SAMPLEPREP_SUMMARY purchased from OlChemIm, Czech Republic. PA and 7’,7’,7’-d3-PA were gifts SP:SAMPLEPREP_SUMMARY from Professor Eiji Nambara (University of Toronto, Canada). Preparations of E. SP:SAMPLEPREP_SUMMARY coli cell lysates were incubated in a total volume of 100 µl containing 100 mM SP:SAMPLEPREP_SUMMARY Tris-HCl, pH 7.0 at 30°C for 16 h with 2-oxoglutarate and ascorbate (100mM SP:SAMPLEPREP_SUMMARY each, final concentrations), FeSO4 (0.5 mM), catalase (1mg/ml), and the SP:SAMPLEPREP_SUMMARY substrates (5 µl in methanol for ABA, 3’,5’,5’,7’,7’,7’-d6-labeled SP:SAMPLEPREP_SUMMARY ABA (500 ng), PA (500 ng), and 7’,7’,7’-d3-labeled PA (50 ng), and 2 µl SP:SAMPLEPREP_SUMMARY in methanol per 5 ng 17,17-d2-labelled GA12. Variations to the standard SP:SAMPLEPREP_SUMMARY incubation conditions are indicated in the individual experiments. Incubation SP:SAMPLEPREP_SUMMARY products were extracted and analyzed by reverse-phase HPLC as described SP:SAMPLEPREP_SUMMARY previously12 using gradients of increasing methanol in water, containing 1% SP:SAMPLEPREP_SUMMARY acetic acid, at 1 ml.min-1 as follows: 50% methanol, followed by five 0.5-min SP:SAMPLEPREP_SUMMARY steps to 57.4%, 60.6%, 61.8%, 62.3%, 62.5%, one 5-min step to 62.7%, one 1-min SP:SAMPLEPREP_SUMMARY step to 63.2% and seven 2-min steps to 64.3%, 67.2%, 70.5%, 74.9%, 81%, 89% and SP:SAMPLEPREP_SUMMARY 100% methanol. PA and compound A eluted between 3 and 6 min, and ABA between 6 SP:SAMPLEPREP_SUMMARY and 9 min. The dried HPLC-fractions were redissolved in 100 µl methanol and SP:SAMPLEPREP_SUMMARY methylated with 100 µl ethereal diazomethane. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE GC CH:INSTRUMENT_NAME Thermo Scientific Trace 1300 CH:COLUMN_NAME Agilent DB5-MS (30m x 0.25mm, 0.25um) CH:SOLVENT_A Helium CH:SOLVENT_B Helium CH:FLOW_GRADIENT 60-280 CH:FLOW_RATE 1.2 ml/min CH:COLUMN_TEMPERATURE 60-280 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME ThermoScientific ISQ7000 MS:INSTRUMENT_TYPE Single quadrupole MS:MS_TYPE EI MS:ION_MODE POSITIVE MS:MS_COMMENTS ThermoScientific ISQ7000 with Advanced Electron Ionization (AEI) source. Software used was Chromeleon 7.2.10 #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Peak area MS_METABOLITE_DATA_START Samples 266_01 403_02 479_01 480_02 481_03 482_04 485_05 486_06 538_01 539_06 542_07 543_08 550_09 551_10 552_11 556_02 558_07 559_03 583_04 585_05 691_02 746_03 747_04 748_05 868_08 907_09 920_10 967_01 969_02 970_03 972_04 974_05 975_06 976_07 990_02 Factors Treatment:DHGA12 Treatment:GAS2 Treatment:GAS2 Treatment:GAS2 Treatment:GAS2 Treatment:GAS2 Treatment:GAS2 Treatment:GAS2 Treatment:PA Treatment:d3-PA Treatment:GAS2 Treatment:GAS2 Treatment:20ox1 Treatment:20ox1 Treatment:20ox1 Treatment:GAS2 Treatment:GAS2 Treatment:ABA Treatment:GAS2 Treatment:GAS2 Treatment:GAS2 Treatment:GAS2 Treatment:GAS2 Treatment:GAS2 Treatment:d6-ABA Treatment:GAS2 Treatment:GAS2 Treatment:GAS2 Treatment:GAS2 Treatment:GAS2 Treatment:GAS2 Treatment:GAS2 Treatment:GAS2 Treatment:GAS2 Treatment:GAS2 Abscisic acid (ABA) 393009 8095803 3',5',5',7',7',7'-d6-ABA 3400698 50431663 34785395 53959422 3553267 86045238 Phaseic acid (PA) 38044587 4741346 4809508 7',7',7''-d3-PA 14376696 18385753 4191184 1877480 57568775 Compound A 24375974 877561 18610937 3207229 3',5',5',7',7',7'-d6-Compound A 1170504 9209255 53959908 1930882 16332480 4422398 7',7',7''-d3-compound A 449336 17,17-d2-GA12 16988102 283071 761845 767473 1377490 1664488 2985792 GA12 25549 46996 59468 17,17-d2-DHGA12 7875399 44566 109720 113535 17-14C-DHGA12 127886867 17-14C-GA9 10797510 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Kovats retention indices Retention time Base peak Abscisic acid (ABA) 2086 13.16 190 3',5',5',7',7',7'-d6-ABA 2083 13.15 194 Phaseic acid (PA) 2146 14.21 125 7',7',7''-d3-PA 2143 14.19 125 Compound A 2201 15.24 190 3',5',5',7',7',7'-d6-Compound A 2196 15.19 194 7',7',7''-d3-compound A 2199 15.22 193 17,17-d2-GA12 2373 18.81 302 GA12 2374 18.82 300 17,17-d2-DHGA12 2592 22.95 288 17-14C-DHGA12 2599 22.7 288 17-14C-GA9 2361 18.29 300 METABOLITES_END #END