#METABOLOMICS WORKBENCH Tatiana_20240129_130504 DATATRACK_ID:4619 STUDY_ID:ST003064 ANALYSIS_ID:AN005019 PROJECT_ID:PR001910 VERSION 1 CREATED_ON January 31, 2024, 1:10 pm #PROJECT PR:PROJECT_TITLE Metabolic responses of Amaranthus caudatus roots and leaves to zinc stress PR:PROJECT_TYPE GCMS-based untargeted and targeted analysis PR:PROJECT_SUMMARY During the last decades pollution with heavy metals became an important stress PR:PROJECT_SUMMARY factor. Plants are characterized by significant biochemical plasticity and can PR:PROJECT_SUMMARY adjust their metabolism to ensure survival under changing environmental PR:PROJECT_SUMMARY conditions. In the most straightforward way these metabolic shifts can be PR:PROJECT_SUMMARY addressed by the untargeted mass spectrometry-based metabolomics approach. PR:PROJECT_SUMMARY However, so far this methodology was only minimally employed in studies of PR:PROJECT_SUMMARY Zn-induced metabolic shifts in plants. Moreover, the genus Amaranthus is still PR:PROJECT_SUMMARY not addressed in this respect. Therefore, here we propose, to the best of our PR:PROJECT_SUMMARY knowledge, the first gas chromatography-mass spectrometry (GC-MS)-based PR:PROJECT_SUMMARY metabolomics study of Zn2+-induced stress responses in Amaranthus caudatus PR:PROJECT_SUMMARY plants. The GC-MS-based study was performed with root and leaf aqueous PR:PROJECT_SUMMARY methanolic extracts after their lyophylization and sequential derivatization PR:PROJECT_SUMMARY with methoxylamine hydrochloride and N-trimethylsilyl-N-methyl PR:PROJECT_SUMMARY trifluoroacetamide. Thereby, 419 derivatives were detected, of which 144 could PR:PROJECT_SUMMARY be putatively annotated. The metabolic shifts in seven-week old A.caudatus PR:PROJECT_SUMMARY plants in response to a seven-day treatment with 300 µmol/L ZnSO4·7H2O in PR:PROJECT_SUMMARY nutrient solution were organ-specific and more pronounced in roots. The most of PR:PROJECT_SUMMARY the responsive metabolites were up-regulated and dominated with sugars and sugar PR:PROJECT_SUMMARY acids. These effects could be attributed to the involvement of these metabolites PR:PROJECT_SUMMARY in osmoregulation, ROS scavenging and complexation of Zn2+ ions. Galactose was PR:PROJECT_SUMMARY the most Zn2+-responsive root sugar that indicated its possible role in the PR:PROJECT_SUMMARY binding of Zn2+ ions to the root cell walls. A 59-fold up-regulation of gluconic PR:PROJECT_SUMMARY acid in roots clearly indicated its involvement in chelation of Zn2+. A high PR:PROJECT_SUMMARY Zn2+–induced up-regulation of salicylic acid in roots and shoots suggested a PR:PROJECT_SUMMARY key role of this hormone in the activation of Zn2+ stress tolerance mechanisms. PR:PROJECT_SUMMARY Thus, our study provides the first insight in the general trends in Zn-induced PR:PROJECT_SUMMARY biochemical rearrangements and main adaptive metabolic shifts in A. caudatus PR:PROJECT_SUMMARY plants. PR:INSTITUTE K.A. Timiryazev Institute of Plant Physiology RAS PR:LABORATORY Laboratory of Analytical Biochemistry and Biotechnology PR:LAST_NAME Frolov PR:FIRST_NAME Andrej PR:ADDRESS Botanicheskaya st. 35., Moskow, 127276, Russian Federation PR:EMAIL frolov@ifr.moscow PR:PHONE +79046097095 PR:FUNDING_SOURCE Russian Scientific Foundation (grant # 21-74-30003), Ministry of Science and PR:FUNDING_SOURCE Higher Education of the Russian Federation (theme # 122042700043-9) #STUDY ST:STUDY_TITLE Metabolic responses of Amaranthus caudatus roots and leaves to zinc stress ST:STUDY_TYPE GCMS-based untargeted and targeted analysis ST:STUDY_SUMMARY During the last decades pollution with heavy metals became an important stress ST:STUDY_SUMMARY factor. Plants are characterized by significant biochemical plasticity and can ST:STUDY_SUMMARY adjust their metabolism to ensure survival under changing environmental ST:STUDY_SUMMARY conditions. In the most straightforward way these metabolic shifts can be ST:STUDY_SUMMARY addressed by the untargeted mass spectrometry-based metabolomics approach. ST:STUDY_SUMMARY However, so far this methodology was only minimally employed in studies of ST:STUDY_SUMMARY Zn-induced metabolic shifts in plants. Moreover, the genus Amaranthus is still ST:STUDY_SUMMARY not addressed in this respect. Therefore, here we propose, to the best of our ST:STUDY_SUMMARY knowledge, the first gas chromatography-mass spectrometry (GC-MS)-based ST:STUDY_SUMMARY metabolomics study of Zn2+-induced stress responses in Amaranthus caudatus ST:STUDY_SUMMARY plants. The GC-MS-based study was performed with root and leaf aqueous ST:STUDY_SUMMARY methanolic extracts after their lyophylization and sequential derivatization ST:STUDY_SUMMARY with methoxylamine hydrochloride and N-trimethylsilyl-N-methyl ST:STUDY_SUMMARY trifluoroacetamide. Thereby, 419 derivatives were detected, of which 144 could ST:STUDY_SUMMARY be putatively annotated. The metabolic shifts in seven-week old A.caudatus ST:STUDY_SUMMARY plants in response to a seven-day treatment with 300 µmol/L ZnSO4·7H2O in ST:STUDY_SUMMARY nutrient solution were organ-specific and more pronounced in roots. The most of ST:STUDY_SUMMARY the responsive metabolites were up-regulated and dominated with sugars and sugar ST:STUDY_SUMMARY acids. These effects could be attributed to the involvement of these metabolites ST:STUDY_SUMMARY in osmoregulation, ROS scavenging and complexation of Zn2+ ions. Galactose was ST:STUDY_SUMMARY the most Zn2+-responsive root sugar that indicated its possible role in the ST:STUDY_SUMMARY binding of Zn2+ ions to the root cell walls. A 59-fold up-regulation of gluconic ST:STUDY_SUMMARY acid in roots clearly indicated its involvement in chelation of Zn2+. A high ST:STUDY_SUMMARY Zn2+–induced up-regulation of salicylic acid in roots and shoots suggested a ST:STUDY_SUMMARY key role of this hormone in the activation of Zn2+ stress tolerance mechanisms. ST:STUDY_SUMMARY Thus, our study provides the first insight in the general trends in Zn-induced ST:STUDY_SUMMARY biochemical rearrangements and main adaptive metabolic shifts in A. caudatus ST:STUDY_SUMMARY plants. ST:INSTITUTE K.A. Timiryazev Institute of Plant Physiology RAS, Moscow, Russia ST:LABORATORY Laboratory of Analytical Biochemistry and Biotechnology ST:LAST_NAME Frolov ST:FIRST_NAME Andrej ST:ADDRESS Botanicheskaya st. 35., Moskow, 127276, Russian Federation ST:EMAIL frolov@ifr.moscow ST:PHONE +79046097095 #SUBJECT SU:SUBJECT_TYPE Plant SU:SUBJECT_SPECIES Amaranthus caudatus L. SU:TAXONOMY_ID 3567 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 - LY_cont_1 Plant_organs:Young_leaves | Treatment:Control RAW_FILE_NAME=LY_cont_1.CDF SUBJECT_SAMPLE_FACTORS - LY_cont_2 Plant_organs:Young_leaves | Treatment:Control RAW_FILE_NAME=LY_cont_2.CDF SUBJECT_SAMPLE_FACTORS - LY_cont_3 Plant_organs:Young_leaves | Treatment:Control RAW_FILE_NAME=LY_cont_3.CDF SUBJECT_SAMPLE_FACTORS - LY_Zn_1 Plant_organs:Young_leaves | Treatment:Zn RAW_FILE_NAME=LY_Zn_1.CDF SUBJECT_SAMPLE_FACTORS - LY_Zn_2 Plant_organs:Young_leaves | Treatment:Zn RAW_FILE_NAME=LY_Zn_2.CDF SUBJECT_SAMPLE_FACTORS - LY_Zn_3 Plant_organs:Young_leaves | Treatment:Zn RAW_FILE_NAME=LY_Zn_3.CDF SUBJECT_SAMPLE_FACTORS - LM_cont_1 Plant_organs:Mature_leaves | Treatment:Control RAW_FILE_NAME=LM_cont_1.CDF SUBJECT_SAMPLE_FACTORS - LM_cont_2 Plant_organs:Mature_leaves | Treatment:Control RAW_FILE_NAME=LM_cont_2.CDF SUBJECT_SAMPLE_FACTORS - LM_cont_3 Plant_organs:Mature_leaves | Treatment:Control RAW_FILE_NAME=LM_cont_3.CDF SUBJECT_SAMPLE_FACTORS - LM_Zn_1 Plant_organs:Mature_leaves | Treatment:Zn RAW_FILE_NAME=LM_Zn_1.CDF SUBJECT_SAMPLE_FACTORS - LM_Zn_2 Plant_organs:Mature_leaves | Treatment:Zn RAW_FILE_NAME=LM_Zn_2.CDF SUBJECT_SAMPLE_FACTORS - LM_Zn_3 Plant_organs:Mature_leaves | Treatment:Zn RAW_FILE_NAME=LM_Zn_3.CDF SUBJECT_SAMPLE_FACTORS - R_cont_1 Plant_organs:Roots | Treatment:Control RAW_FILE_NAME=R_cont_1.CDF SUBJECT_SAMPLE_FACTORS - R_cont_2 Plant_organs:Roots | Treatment:Control RAW_FILE_NAME=R_cont_2.CDF SUBJECT_SAMPLE_FACTORS - R_cont_3 Plant_organs:Roots | Treatment:Control RAW_FILE_NAME=R_cont_3.CDF SUBJECT_SAMPLE_FACTORS - R_Zn_1 Plant_organs:Roots | Treatment:Zn RAW_FILE_NAME=R_Zn_1.CDF SUBJECT_SAMPLE_FACTORS - R_Zn_2 Plant_organs:Roots | Treatment:Zn RAW_FILE_NAME=R_Zn_2.CDF SUBJECT_SAMPLE_FACTORS - R_Zn_3 Plant_organs:Roots | Treatment:Zn RAW_FILE_NAME=R_Zn_3.CDF #COLLECTION CO:COLLECTION_SUMMARY Amaranthus caudatus L., variety Karwa dauta plants were used in the study. After CO:COLLECTION_SUMMARY two weeks culturing in the hydroponic system (nutrient solution (in mmol/L) was CO:COLLECTION_SUMMARY as follows: Ca(NO3)2·4H2O - 3.81; KNO3 - 6.44; MgSO4·7H2O - 0.81; KH2PO4 - CO:COLLECTION_SUMMARY 1.83; NH4NO3 - 0.87; Fe-EDTA - 0.09; H3BO3 - 0.047; MnSO4·5H2O - 0.007; CO:COLLECTION_SUMMARY ZnSO4·7H2O - 0.0007; CuSO4·5H2O - 0.0008; (NH4)2MoO4 - 0.0005), the vessels CO:COLLECTION_SUMMARY with six-week-old plants (experimental group) were subjected to Zn2+ stress for CO:COLLECTION_SUMMARY one week which was accomplished by supplementation of 300 µmol/L ZnSO4·7H2O in CO:COLLECTION_SUMMARY the nutrient solution. Control plants remained untreated. Roots, young and CO:COLLECTION_SUMMARY mature leaves of seven-week-old Zn-treated and control plants were collected CO:COLLECTION_SUMMARY separately. Approximately 10 and 20 mg of ground dry leaf and root material, CO:COLLECTION_SUMMARY respectively, were extracted with 1 mL methanol. After vortexing (3000 g, 30 s) CO:COLLECTION_SUMMARY and centrifugation (12000 g, 4 °C, 10 min) of the suspensions, the resulted CO:COLLECTION_SUMMARY supernatants were collected. The plant material residues were additionally CO:COLLECTION_SUMMARY supplemented with 0.1 mL of deionized water. After a following vortex and CO:COLLECTION_SUMMARY centrifugation cycle, the obtained supernatants were combined with the first CO:COLLECTION_SUMMARY portions. The total extract volume was 1090 μL. Aliquots (30 μL) of the CO:COLLECTION_SUMMARY resulted aq. methanolic extracts were freeze-dried under reduced pressure with CO:COLLECTION_SUMMARY Labconco CentriVap centrifugal concentrator. The residues were sequentially CO:COLLECTION_SUMMARY derivatized with methoxyamine hydrochloride in pyridine, and CO:COLLECTION_SUMMARY N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) according to the CO:COLLECTION_SUMMARY established procedure (Leonova et al., 2020, CO:COLLECTION_SUMMARY http://dx.doi.org/10.3390/ijms21020567) CO:SAMPLE_TYPE Plant leaves and roots #TREATMENT TR:TREATMENT_SUMMARY Amaranthus caudatus L., variety Karwa dauta plants were used in the study. After TR:TREATMENT_SUMMARY two weeks culturing in the hydroponic system (nutrient solution (in mmol/L) was TR:TREATMENT_SUMMARY as follows: Ca(NO3)2·4H2O - 3.81; KNO3 - 6.44; MgSO4·7H2O - 0.81; KH2PO4 - TR:TREATMENT_SUMMARY 1.83; NH4NO3 - 0.87; Fe-EDTA - 0.09; H3BO3 - 0.047; MnSO4·5H2O - 0.007; TR:TREATMENT_SUMMARY ZnSO4·7H2O - 0.0007; CuSO4·5H2O - 0.0008; (NH4)2MoO4 - 0.0005), the vessels TR:TREATMENT_SUMMARY with six-week-old plants (experimental group) were subjected to Zn2+ stress for TR:TREATMENT_SUMMARY one week which was accomplished by supplementation of 300 µmol/L ZnSO4·7H2O in TR:TREATMENT_SUMMARY the nutrient solution. Control plants remained untreated. Roots, young and TR:TREATMENT_SUMMARY mature leaves of seven-week-old Zn-treated and control plants were collected TR:TREATMENT_SUMMARY separately. TR:TREATMENT_PROTOCOL_COMMENTS 6 sample groups: LY_cont - young leaves of control plants; LY_Zn - young leaves TR:TREATMENT_PROTOCOL_COMMENTS of Zn-treated plants; LM_cont - mature leaves of control plants; LM_Zn - mature TR:TREATMENT_PROTOCOL_COMMENTS leaves of Zn-treated plants; R_cont - roots of control plants; R_Zn - roots of TR:TREATMENT_PROTOCOL_COMMENTS Zn-treated plants. TR:TREATMENT Heavy metal stress TR:TREATMENT_COMPOUND ZnSO4·7H2O TR:TREATMENT_ROUTE supplementation in the nutrient solution TR:TREATMENT_DOSE 300 µmol/L TR:TREATMENT_DOSEDURATION 1 week TR:PLANT_PLOT_DESIGN total 27 plants in nine vessels TR:PLANT_LIGHT_PERIOD 16 : 8 day/night regimen TR:PLANT_HUMIDITY 70-75% relative humidity TR:PLANT_TEMP day/night temperatures of 24/18° C TR:PLANT_WATERING_REGIME plant were culturing in the hydroponic system TR:PLANT_NUTRITIONAL_REGIME nutrient solution in mmol/L as follows: Ca(NO3)2·4H2O - 3.81; KNO3 - 6.44; TR:PLANT_NUTRITIONAL_REGIME MgSO4·7H2O - 0.81; KH2PO4 - 1.83; NH4NO3 - 0.87; Fe-EDTA - 0.09; H3BO3 - 0.047; TR:PLANT_NUTRITIONAL_REGIME MnSO4·5H2O - 0.007; ZnSO4·7H2O - 0.0007; CuSO4·5H2O - 0.0008; (NH4)2MoO4 - TR:PLANT_NUTRITIONAL_REGIME 0.0005 TR:PLANT_GROWTH_STAGE vegetative stage #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Approximately 10 and 20 mg of ground dry leaf and root material, respectively, SP:SAMPLEPREP_SUMMARY were extracted with 1 mL methanol. After vortexing (3000 g, 30 s) and SP:SAMPLEPREP_SUMMARY centrifugation (12000 g, 4 °C, 10 min) of the suspensions, the resulted SP:SAMPLEPREP_SUMMARY supernatants were collected. The plant material residues were additionally SP:SAMPLEPREP_SUMMARY supplemented with 0.1 mL of deionized water. After a following vortex and SP:SAMPLEPREP_SUMMARY centrifugation cycle, the obtained supernatants were combined with the first SP:SAMPLEPREP_SUMMARY portions. The total extract volume was 1090 μL. Aliquots (30 μL) of the SP:SAMPLEPREP_SUMMARY resulted aq. methanolic extracts were freeze-dried under reduced pressure with SP:SAMPLEPREP_SUMMARY Labconco CentriVap centrifugal concentrator. The residues were sequentially SP:SAMPLEPREP_SUMMARY derivatized with methoxyamine hydrochloride in pyridine, and SP:SAMPLEPREP_SUMMARY N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) according to the SP:SAMPLEPREP_SUMMARY established procedure (Leonova et al., 2020, SP:SAMPLEPREP_SUMMARY http://dx.doi.org/10.3390/ijms21020567). SP:PROCESSING_STORAGE_CONDITIONS 4℃ SP:EXTRACT_STORAGE -20℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY The samples (1μL) were injected with CTC GC PAL Liquid Injector (Shimadzu CH:CHROMATOGRAPHY_SUMMARY Deutschland GmbH, Duisburg, Germany) into GC2010 gas chromatograph coupled CH:CHROMATOGRAPHY_SUMMARY online to a quadrupole mass selective detector Shimadzu GCMS QP201 operating CH:CHROMATOGRAPHY_SUMMARY under the instrumental settings summarized in PR2.pdf CH:CHROMATOGRAPHY_TYPE GC CH:INSTRUMENT_NAME Shimadzu GC-2010 CH:COLUMN_NAME Phenomenex ZB-5MS (30 m × 0.25 mm, 0.25 μm) CH:SOLVENT_A - CH:SOLVENT_B - CH:SOLVENT_C - CH:FLOW_GRADIENT - CH:FLOW_RATE 1 mL/min CH:COLUMN_TEMPERATURE 1 min at 40°C, ramp 15°C/min to 70°C, 1 min at 70°C, ramp 6°C/min to CH:COLUMN_TEMPERATURE 320°C, 12 min at 320°C CH:INJECTION_TEMPERATURE 250 CH:SAMPLE_INJECTION 1μm CH:ANALYTICAL_TIME 5.5-55 min #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Shimadzu QP2010 Plus MS:INSTRUMENT_TYPE Single quadrupole MS:MS_TYPE EI MS:ION_MODE POSITIVE MS:MS_COMMENTS Targeted GC-MS analysis The samples (1μL) were injected with CTC GC PAL Liquid MS:MS_COMMENTS Injector (Shimadzu Deutschland GmbH, Duisburg, Germany) into GC2010 gas MS:MS_COMMENTS chromatograph coupled online to a quadrupole mass selective detector Shimadzu MS:MS_COMMENTS GCMS QP201. The GC-MS instrumental settings are summarized in PR2.pdf. The MS:MS_COMMENTS quality of the acquired chromatograms was assessed by verification of the MS:MS_COMMENTS baseline regularity, background MS noise, the symmetry, width and height of MS:MS_COMMENTS chromatographic peaks. To obtain qualitative information about the Zn-related MS:MS_COMMENTS dynamics of individual metabolites, the chromatograms were processed by AMDIS MS:MS_COMMENTS software (www.amdis.net/) to accomplish deconvolution of mass spectra, peak MS:MS_COMMENTS picking, calculation of Kovach retention indices (RI) and annotation of MS:MS_COMMENTS analytes. The analytes annotated in the experimental samples were quantified by MS:MS_COMMENTS integration of the corresponding extracted ion chromatograms (XIC, m/z ± 0.5 MS:MS_COMMENTS Da) for representative intense signals at specific retention times. This analyte MS:MS_COMMENTS quantification procedure was accomplished with XcaliburTM (version 2.0.7), MS:MS_COMMENTS LCquanTM (version 2.5.6, TermoFisher Scientific Inc., Bremen, Germany) and MS:MS_COMMENTS MSDial (http://prime.psc.riken.jp/compms/msdial/main.html) softwares which MS:MS_COMMENTS perform alignment of chromatograms by retention times of analytes and the MS:MS_COMMENTS integration of analyte peak areas. Metabolite identification and targeted MS:MS_COMMENTS absolute quantitative analysis relied on external standardization with 29 MS:MS_COMMENTS authentic standards (oxalic acid, malonic acid, succinic acid, tartaric acid, MS:MS_COMMENTS malic acid, aconitic acid, citric acid, fumaric acid, benzoic acid, ascorbic MS:MS_COMMENTS acid, erythronic acid, glycerol, arabinose, glucose, galactose, myo-inositol, MS:MS_COMMENTS sucrose, urea, Ala, Trp, Ile, Leu, Asn, Asp, Glu, Pro, Val, Ser, Thr) prepared MS:MS_COMMENTS as a total mix serially diluted in the range from 0.2 pmol/μL to 200 pmol/μL. MS:MS_COMMENTS Among these, only 21 compounds were confirmed in leaves and roots of control and MS:MS_COMMENTS Zn2+-treated A. caudatus plants (Result table). MS:ION_SOURCE_TEMPERATURE 240 MS:IONIZATION EI MS:IONIZATION_ENERGY 70eV #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS μmol/g DW MS_METABOLITE_DATA_START Samples LY_cont_1 LY_cont_2 LY_cont_3 LY_Zn_1 LY_Zn_2 LY_Zn_2 LM_cont_1 LM_cont_2 LM_cont_3 LM_Zn_1 LM_Zn_3 R_cont_1 R_cont_2 R_cont_3 R_Zn_1 R_Zn_2 R_Zn_3 Factors Plant_organs:Young_leaves | Treatment:Control Plant_organs:Young_leaves | Treatment:Control Plant_organs:Young_leaves | Treatment:Control Plant_organs:Young_leaves | Treatment:Zn Plant_organs:Young_leaves | Treatment:Zn Plant_organs:Young_leaves | Treatment:Zn Plant_organs:Mature_leaves | Treatment:Control Plant_organs:Mature_leaves | Treatment:Control Plant_organs:Mature_leaves | Treatment:Control Plant_organs:Mature_leaves | Treatment:Zn Plant_organs:Mature_leaves | Treatment:Zn Plant_organs:Roots | Treatment:Control Plant_organs:Roots | Treatment:Control Plant_organs:Roots | Treatment:Control Plant_organs:Roots | Treatment:Zn Plant_organs:Roots | Treatment:Zn Plant_organs:Roots | Treatment:Zn Oxalic acid 5.13 19.16 4.65 12.24 11.23 3.38 20.97 26.45 21.82 27.64 1.53 11.49 12.68 11.21 0.58 6.02 0.61 Malonic acid 0.48 0.66 8.17 0.98 0.74 0.30 0.86 1.01 0.66 0.78 1.01 0.53 0.41 0.50 0.83 1.08 1.01 Succinic acid 4.14 4.18 4.48 2.84 2.49 1.36 4.70 5.37 4.37 2.44 3.33 2.74 2.54 2.77 1.53 1.78 1.78 Fumaric acid 0.40 0.47 0.45 0.46 0.40 0.30 0.58 0.64 0.52 0.53 0.77 0.79 0.75 0.81 0.74 0.83 0.76 Malic acid 2.20 1.98 2.31 3.54 1.87 1.58 2.81 2.90 2.39 6.09 7.57 4.62 4.70 4.89 4.98 6.04 5.83 Pyroglutamic acid 16.40 9.86 4.27 33.01 18.86 23.42 6.37 2.87 3.10 3.96 10.71 17.48 19.51 21.35 21.83 15.41 15.13 Citric acid 0.25 0.32 6.13 0.58 0.20 0.23 0.49 0.15 1.95 0.24 0.56 0.13 0.12 0.11 0.27 0.38 0.36 Aconitic acid 0.82 1.37 1.90 1.47 1.12 0.77 1.94 3.41 3.28 2.02 2.62 0.07 0.03 0.04 0.25 0.03 0.03 Erythronic acid 2.39 2.51 2.73 3.66 2.99 2.24 2.69 3.20 2.82 4.50 6.23 1.05 0.97 1.12 1.86 2.02 2.03 Benzoic acid 7.78 7.83 7.59 4.02 5.51 4.18 9.80 13.58 11.68 8.43 9.19 6.83 6.01 12.19 9.50 10.02 9.64 Alanine 10.23 4.14 9.47 4.57 4.08 2.86 5.87 3.85 3.42 2.33 3.64 1.29 1.42 1.40 3.10 3.32 2.41 Valine 0.82 0.50 0.46 0.39 0.44 0.26 0.74 0.46 0.37 0.18 0.17 0.49 0.50 0.54 1.45 0.84 0.88 Isoleucine 0.32 0.14 0.26 0.12 0.11 0.09 0.26 0.06 0.08 0.06 0.11 0.27 0.35 0.38 1.07 0.44 0.44 Proline 1.32 0.32 1.46 0.65 1.10 0.76 1.51 1.07 2.17 2.18 2.13 Urea 10.34 6.42 1.53 0.54 3.01 1.04 6.32 2.66 2.62 Glycerol 1.01 0.90 1.07 0.44 0.66 2.44 1.08 0.56 1.06 1.62 1.44 2.82 2.79 3.09 4.10 4.32 4.25 Arabinose 0.23 0.20 0.19 0.23 0.15 0.16 0.16 0.22 0.16 0.27 0.37 0.06 0.05 0.06 0.11 0.14 0.12 Galactose 0.04 0.04 0.06 0.12 0.11 0.07 0.06 0.04 0.02 0.12 0.17 0.05 0.04 0.05 0.37 0.40 0.38 Glucose 0.24 0.21 0.24 1.04 0.70 0.55 0.28 0.30 0.22 0.64 0.84 0.96 0.88 1.04 3.01 3.52 3.49 Myo-inositol 0.35 0.24 0.34 0.69 0.67 0.41 0.13 0.18 0.15 0.18 0.32 0.36 0.34 0.39 0.66 0.82 0.74 Sucrose 2.04 1.83 3.24 29.73 12.72 11.93 3.97 4.75 4.00 8.27 11.39 7.74 8.05 8.42 7.74 9.39 9.01 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name KEGG ID PubChem ID RI quantitated m/z Oxalic acid C00209 971 1145 190 Malonic acid C04025 867 1211 233 Succinic acid C00042 1110 1316 247 Fumaric acid C00122 444972 1352 245 Malic acid C00149 222656 1487 233 Pyroglutamic acid C01879 7405 1516 156 Citric acid C00158 311 1814 273 Aconitic acid C00417 643757 1747 229 Erythronic acid C21593 2781043 1540 292 Benzoic acid C00180 243 1253 179 Alanine C00041 5950 1113 116 Valine C00183 6287 1217 144 Isoleucine C00407 6306 1292 158 Proline C00148 145742 1577 142 Urea C00086 1176 1249 189 Glycerol C00116 753 1276 205 Arabinose C00259 439195 1758 307 Galactose C00984 439357 1979 319 Glucose C00031 5793 1991 319 Myo-inositol C00137 892 2065 305 Sucrose C00089 5988 2457 361 METABOLITES_END #END