#METABOLOMICS WORKBENCH shawn_christensen_20200803_201125 DATATRACK_ID:2110 STUDY_ID:ST001489 ANALYSIS_ID:AN002467 PROJECT_ID:PR001006 VERSION 1 CREATED_ON September 22, 2020, 10:04 am #PROJECT PR:PROJECT_TITLE Metabolomics by UHPLC-HRMS reveals the impact of heat stress on PR:PROJECT_TITLE pathogen-elicited immunity in maize PR:PROJECT_SUMMARY Studies investigating crop resistance to biotic and abiotic stress have largely PR:PROJECT_SUMMARY focused on plant responses to singular forms of stress and individual PR:PROJECT_SUMMARY biochemical pathways that only partially represent stress responses. Thus, PR:PROJECT_SUMMARY combined biotic and abiotic stress treatments and the global assessment of their PR:PROJECT_SUMMARY elicited metabolic expression remains largely unexplored. In this study, we PR:PROJECT_SUMMARY employed targeted and untargeted metabolomics to investigate the metabolic PR:PROJECT_SUMMARY responses of maize (Zea mays) to both individual and combinatorial stress PR:PROJECT_SUMMARY treatments using heat (abiotic) and Cochliobolus heterostrophus infection PR:PROJECT_SUMMARY (biotic) experiments. Ultra-high-performance liquid PR:PROJECT_SUMMARY chromatography-high-resolution mass spectrometry revealed significant metabolic PR:PROJECT_SUMMARY responses to C. heterostrophus infection and heat stress, and comparative PR:PROJECT_SUMMARY analyses between these individual forms of stress demonstrated differential PR:PROJECT_SUMMARY elicitation between the two global metabolomes. In combinatorial experiments, PR:PROJECT_SUMMARY treatment with heat stress prior to fungal inoculation negatively impacted maize PR:PROJECT_SUMMARY disease resistance against C. heterostrophus, and distinct metabolome separation PR:PROJECT_SUMMARY between combinatorial stressed plants and the non-heat stressed infected PR:PROJECT_SUMMARY controls was observed. Targeted analysis revealed inducible primary and PR:PROJECT_SUMMARY secondary metabolite responses to biotic/abiotic stress, and combinatorial PR:PROJECT_SUMMARY experiments indicated that deficiency in the hydroxycinnamic acid, p-coumaric PR:PROJECT_SUMMARY acid, may lead to the heat-induced susceptibility of maize to C. heterostrophus. PR:PROJECT_SUMMARY Collectively, these findings demonstrate that abiotic stress can predispose PR:PROJECT_SUMMARY crops to more severe disease symptoms, underlining the increasing need to PR:PROJECT_SUMMARY investigate defense chemistry in plants under combinatorial stress. PR:INSTITUTE Agricultural Research Service, United States Department of Agriculture PR:LAST_NAME Christensen PR:FIRST_NAME Shawn PR:ADDRESS 1600 SW 23rd Drive Gainesville, FL 32608, Gainesville, Florida, 32607, USA PR:EMAIL shawn.christensen@usda.gov PR:PHONE 3523745739 #STUDY ST:STUDY_TITLE Metabolomics by UHPLC-HRMS reveals the impact of heat stress on ST:STUDY_TITLE pathogen-elicited immunity in maize ST:STUDY_SUMMARY Studies investigating crop resistance to biotic and abiotic stress have largely ST:STUDY_SUMMARY focused on plant responses to singular forms of stress and individual ST:STUDY_SUMMARY biochemical pathways that only partially represent stress responses. Thus, ST:STUDY_SUMMARY combined biotic and abiotic stress treatments and the global assessment of their ST:STUDY_SUMMARY elicited metabolic expression remains largely unexplored. In this study, we ST:STUDY_SUMMARY employed targeted and untargeted metabolomics to investigate the metabolic ST:STUDY_SUMMARY responses of maize (Zea mays) to both individual and combinatorial stress ST:STUDY_SUMMARY treatments using heat (abiotic) and Cochliobolus heterostrophus infection ST:STUDY_SUMMARY (biotic) experiments. Ultra-high-performance liquid ST:STUDY_SUMMARY chromatography-high-resolution mass spectrometry revealed significant metabolic ST:STUDY_SUMMARY responses to C. heterostrophus infection and heat stress, and comparative ST:STUDY_SUMMARY analyses between these individual forms of stress demonstrated differential ST:STUDY_SUMMARY elicitation between the two global metabolomes. In combinatorial experiments, ST:STUDY_SUMMARY treatment with heat stress prior to fungal inoculation negatively impacted maize ST:STUDY_SUMMARY disease resistance against C. heterostrophus, and distinct metabolome separation ST:STUDY_SUMMARY between combinatorial stressed plants and the non-heat stressed infected ST:STUDY_SUMMARY controls was observed. Targeted analysis revealed inducible primary and ST:STUDY_SUMMARY secondary metabolite responses to biotic/abiotic stress, and combinatorial ST:STUDY_SUMMARY experiments indicated that deficiency in the hydroxycinnamic acid, p-coumaric ST:STUDY_SUMMARY acid, may lead to the heat-induced susceptibility of maize to C. heterostrophus. ST:STUDY_SUMMARY Collectively, these findings demonstrate that abiotic stress can predispose ST:STUDY_SUMMARY crops to more severe disease symptoms, underlining the increasing need to ST:STUDY_SUMMARY investigate defense chemistry in plants under combinatorial stress. ST:INSTITUTE Agricultural Research Service, United States Department of Agriculture ST:DEPARTMENT Center of Medical, Agricultural, and Veterinary Entomology ST:LABORATORY Chemistry Research Unit ST:LAST_NAME Christensen ST:FIRST_NAME Shawn ST:ADDRESS 1600 SW 23rd Drive Gainesville, FL 32608 ST:EMAIL shawn.christensen@usda.gov ST:PHONE 3523745739 #SUBJECT SU:SUBJECT_TYPE Plant SU:SUBJECT_SPECIES Zea mays SU:TAXONOMY_ID 4577 #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 - 1_ Sample Type:SLB inoculation | Age:14 day | Growth Temperature:28 Description=3d post SLB (C. heterostrophus) inoculation on 3rd leaf, 14d old plant grown at 28°C under watered conditions; RAW_FILE_NAME=01_SLB_28_3d_H2O SUBJECT_SAMPLE_FACTORS - 2_ Sample Type:SLB inoculation | Age:14 day | Growth Temperature:28 Description=3d post SLB (C. heterostrophus) inoculation on 3rd leaf, 14d old plant grown at 28°C under watered conditions; RAW_FILE_NAME=02_SLB_28_3d_H2O SUBJECT_SAMPLE_FACTORS - 3_ Sample Type:SLB inoculation | Age:14 day | Growth Temperature:28 Description=3d post SLB (C. heterostrophus) inoculation on 3rd leaf, 14d old plant grown at 28°C under watered conditions; RAW_FILE_NAME=03_SLB_28_3d_H2O SUBJECT_SAMPLE_FACTORS - 4_ Sample Type:SLB inoculation | Age:14 day | Growth Temperature:28 Description=3d post SLB (C. heterostrophus) inoculation on 3rd leaf, 14d old plant grown at 28°C under watered conditions; RAW_FILE_NAME=04_SLB_28_3d_H2O SUBJECT_SAMPLE_FACTORS - 5_ Sample Type:SLB inoculation | Age:14 day | Growth Temperature:28 Description=3d post SLB (C. heterostrophus) inoculation on 3rd leaf, 14d old plant grown at 28°C under watered conditions; RAW_FILE_NAME=05_SLB_28_3d_H2O SUBJECT_SAMPLE_FACTORS - 6_ Sample Type:SLB inoculation | Age:14 day | Growth Temperature:28 Description=3d post SLB (C. heterostrophus) inoculation on 3rd leaf, 14d old plant grown at 28°C under watered conditions; RAW_FILE_NAME=06_SLB_28_3d_H2O SUBJECT_SAMPLE_FACTORS - 7_ Sample Type:Control | Age:14 day | Growth Temperature:28 Description=Tween 20 control treated 3rd leaf, 14d old plant grown at 28°C under watered conditions; RAW_FILE_NAME=07_Tween_28_3d_H2O SUBJECT_SAMPLE_FACTORS - 8_ Sample Type:Control | Age:14 day | Growth Temperature:28 Description=Tween 20 control treated 3rd leaf, 14d old plant grown at 28°C under watered conditions; RAW_FILE_NAME=08_Tween_28_3d_H2O SUBJECT_SAMPLE_FACTORS - 9_ Sample Type:Control | Age:14 day | Growth Temperature:28 Description=Tween 20 control treated 3rd leaf, 14d old plant grown at 28°C under watered conditions; RAW_FILE_NAME=09_Tween_28_3d_H2O SUBJECT_SAMPLE_FACTORS - 10_ Sample Type:Control | Age:14 day | Growth Temperature:28 Description=Tween 20 control treated 3rd leaf, 14d old plant grown at 28°C under watered conditions; RAW_FILE_NAME=10_Tween_28_3d_H2O SUBJECT_SAMPLE_FACTORS - 11_ Sample Type:Control | Age:14 day | Growth Temperature:28 Description=Tween 20 control treated 3rd leaf, 14d old plant grown at 28°C under watered conditions; RAW_FILE_NAME=11_Tween_28_3d_H2O SUBJECT_SAMPLE_FACTORS - 12_ Sample Type:Control | Age:14 day | Growth Temperature:28 Description=Tween 20 control treated 3rd leaf, 14d old plant grown at 28°C under watered conditions; RAW_FILE_NAME=12_Tween_28_3d_H2O SUBJECT_SAMPLE_FACTORS - 25_ Sample Type:SLB inoculation | Age:14 day | Growth Temperature:38 returned to 28 Description=3d post SLB (C. heterostrophus) inoculation on 3rd leaf, 14d old plant grown at 38°C under watered conditions, then returned to 28°C; RAW_FILE_NAME=25_SLB_38_3d_H2O SUBJECT_SAMPLE_FACTORS - 26_ Sample Type:SLB inoculation | Age:14 day | Growth Temperature:38 returned to 28 Description=3d post SLB (C. heterostrophus) inoculation on 3rd leaf, 14d old plant grown at 38°C under watered conditions, then returned to 28°C; RAW_FILE_NAME=26_SLB_38_3d_H2O SUBJECT_SAMPLE_FACTORS - 27_ Sample Type:SLB inoculation | Age:14 day | Growth Temperature:38 returned to 28 Description=3d post SLB (C. heterostrophus) inoculation on 3rd leaf, 14d old plant grown at 38°C under watered conditions, then returned to 28°C; RAW_FILE_NAME=27_SLB_38_3d_H2O SUBJECT_SAMPLE_FACTORS - 28_ Sample Type:SLB inoculation | Age:14 day | Growth Temperature:38 returned to 28 Description=3d post SLB (C. heterostrophus) inoculation on 3rd leaf, 14d old plant grown at 38°C under watered conditions, then returned to 28°C; RAW_FILE_NAME=28_SLB_38_3d_H2O SUBJECT_SAMPLE_FACTORS - 29_ Sample Type:SLB inoculation | Age:14 day | Growth Temperature:38 returned to 28 Description=3d post SLB (C. heterostrophus) inoculation on 3rd leaf, 14d old plant grown at 38°C under watered conditions, then returned to 28°C; RAW_FILE_NAME=29_SLB_38_3d_H2O SUBJECT_SAMPLE_FACTORS - 30_ Sample Type:SLB inoculation | Age:14 day | Growth Temperature:38 returned to 28 Description=3d post SLB (C. heterostrophus) inoculation on 3rd leaf, 14d old plant grown at 38°C under watered conditions, then returned to 28°C; RAW_FILE_NAME=30_SLB_38_3d_H2O SUBJECT_SAMPLE_FACTORS - 31_ Sample Type:Control | Age:14 day | Growth Temperature:38 returned to 28 Description=Tween 20 control treated 3rd leaf, 14d old plant grown at 38°C under watered conditions, then returned to 28°C; RAW_FILE_NAME=31_Tween_38_3d_H2O SUBJECT_SAMPLE_FACTORS - 32_ Sample Type:Control | Age:14 day | Growth Temperature:38 returned to 28 Description=Tween 20 control treated 3rd leaf, 14d old plant grown at 38°C under watered conditions, then returned to 28°C; RAW_FILE_NAME=32_Tween_38_3d_H2O SUBJECT_SAMPLE_FACTORS - 33_ Sample Type:Control | Age:14 day | Growth Temperature:38 returned to 28 Description=Tween 20 control treated 3rd leaf, 14d old plant grown at 38°C under watered conditions, then returned to 28°C; RAW_FILE_NAME=33_Tween_38_3d_H2O SUBJECT_SAMPLE_FACTORS - 34_ Sample Type:Control | Age:14 day | Growth Temperature:38 returned to 28 Description=Tween 20 control treated 3rd leaf, 14d old plant grown at 38°C under watered conditions, then returned to 28°C; RAW_FILE_NAME=34_Tween_38_3d_H2O SUBJECT_SAMPLE_FACTORS - 49_ Sample Type:SLB inoculation | Age:14 day | Growth Temperature:38 returned to 38 Description=3d post SLB (C. heterostrophus) inoculation on 3rd leaf, 14d old plant grown at 38°C under watered conditions, then returned to 38°C; RAW_FILE_NAME=49_SLB_38_6d_H2O SUBJECT_SAMPLE_FACTORS - 50_ Sample Type:SLB inoculation | Age:14 day | Growth Temperature:38 returned to 38 Description=3d post SLB (C. heterostrophus) inoculation on 3rd leaf, 14d old plant grown at 38°C under watered conditions, then returned to 38°C; RAW_FILE_NAME=50_SLB_38_6d_H2O SUBJECT_SAMPLE_FACTORS - 51_ Sample Type:SLB inoculation | Age:14 day | Growth Temperature:38 returned to 38 Description=3d post SLB (C. heterostrophus) inoculation on 3rd leaf, 14d old plant grown at 38°C under watered conditions, then returned to 38°C; RAW_FILE_NAME=51_SLB_38_6d_H2O SUBJECT_SAMPLE_FACTORS - 52_ Sample Type:SLB inoculation | Age:14 day | Growth Temperature:38 returned to 38 Description=3d post SLB (C. heterostrophus) inoculation on 3rd leaf, 14d old plant grown at 38°C under watered conditions, then returned to 38°C; RAW_FILE_NAME=52_SLB_38_6d_H2O SUBJECT_SAMPLE_FACTORS - 53_ Sample Type:SLB inoculation | Age:14 day | Growth Temperature:38 returned to 38 Description=3d post SLB (C. heterostrophus) inoculation on 3rd leaf, 14d old plant grown at 38°C under watered conditions, then returned to 38°C; RAW_FILE_NAME=53_SLB_38_6d_H2O SUBJECT_SAMPLE_FACTORS - 54_ Sample Type:SLB inoculation | Age:14 day | Growth Temperature:38 returned to 38 Description=3d post SLB (C. heterostrophus) inoculation on 3rd leaf, 14d old plant grown at 38°C under watered conditions, then returned to 38°C; RAW_FILE_NAME=54_SLB_38_6d_H2O SUBJECT_SAMPLE_FACTORS - 55_ Sample Type:Control | Age:14 day | Growth Temperature:38 returned to 38 Description=Tween 20 control treated 3rd leaf, 14d old plant grown at 38°C under watered conditions, then returned to 38°C; RAW_FILE_NAME=55_Tween_38_6d_H2O SUBJECT_SAMPLE_FACTORS - 56_ Sample Type:Control | Age:14 day | Growth Temperature:38 returned to 38 Description=Tween 20 control treated 3rd leaf, 14d old plant grown at 38°C under watered conditions, then returned to 38°C; RAW_FILE_NAME=56_Tween_38_6d_H2O SUBJECT_SAMPLE_FACTORS - 57_ Sample Type:Control | Age:14 day | Growth Temperature:38 returned to 38 Description=Tween 20 control treated 3rd leaf, 14d old plant grown at 38°C under watered conditions, then returned to 38°C; RAW_FILE_NAME=57_Tween_38_6d_H2O SUBJECT_SAMPLE_FACTORS - 58_ Sample Type:Control | Age:14 day | Growth Temperature:38 returned to 38 Description=Tween 20 control treated 3rd leaf, 14d old plant grown at 38°C under watered conditions, then returned to 38°C; RAW_FILE_NAME=58_Tween_38_6d_H2O SUBJECT_SAMPLE_FACTORS - 59_ Sample Type:Control | Age:14 day | Growth Temperature:38 returned to 38 Description=Tween 20 control treated 3rd leaf, 14d old plant grown at 38°C under watered conditions, then returned to 38°C; RAW_FILE_NAME=59_Tween_38_6d_H2O #COLLECTION CO:COLLECTION_SUMMARY Samples were collected and flash frozen in liquid N2 CO:SAMPLE_TYPE Plant #TREATMENT TR:TREATMENT_SUMMARY For inoculations, single isolates of Cochliobolus heterostrophus were cultured TR:TREATMENT_SUMMARY on V8 agar for 3-4 weeks as described by (Christensen et al., 2018). Spot TR:TREATMENT_SUMMARY inoculations were performed on 14-d-old plants in which four 10 μl droplets of TR:TREATMENT_SUMMARY either C. heterostrophus suspension (1x106 spores mL-1) or control solution TR:TREATMENT_SUMMARY (sterile 0.1% Tween 20) were applied in a staggered fashion (Fig. 2a and Fig. TR:TREATMENT_SUMMARY 4a) onto the middle portion of the third leaf about 10cm from the distal tip. TR:TREATMENT_SUMMARY Following inoculation, both heat-stressed and non-heat-stressed plants were TR:TREATMENT_SUMMARY placed in a plastic humidity chamber and placed in the 28°C Percival. At 24 TR:TREATMENT_SUMMARY hours post-inoculation, infected and non-infected controls were removed from the TR:TREATMENT_SUMMARY humidity chambers and maintained at 28°C for the remainder of the experiment. TR:TREATMENT_SUMMARY At 72 hours post-inoculation, leaves (n=6) were photographed and immediately TR:TREATMENT_SUMMARY harvested into liquid N2. Lesions were digitally measured using ImageJ software TR:TREATMENT_SUMMARY (ImageJ 1.36b; Wayne Rasband, NIH, Bethesda, MD, USA). #SAMPLEPREP SP:SAMPLEPREP_SUMMARY LCMS grade reagents (Thermo Fisher Scientific) were used throughout the SP:SAMPLEPREP_SUMMARY extraction. Frozen 1.5-mL fast prep tubes with Zirmil® beads (1.1mm; SEPR SP:SAMPLEPREP_SUMMARY Ceramic Beads and Powders, Mountainside, NJ, USA) containing 50 mg of ground and SP:SAMPLEPREP_SUMMARY frozen tissue were carefully thawed to 4°C then transferred to ice. An internal SP:SAMPLEPREP_SUMMARY standard mix of 12 μL containing caffeine, D6-Abscisic acid, D5-Jasmonic acid, SP:SAMPLEPREP_SUMMARY D5-Cinnamic acid, D5-Indole-3-acetic acid, 13C-alpha linolenic acid, and SP:SAMPLEPREP_SUMMARY nicotine at 8.33 μg/mL was added to each sample along with 750 μL of 10 mM of SP:SAMPLEPREP_SUMMARY ammonium acetate and 750 μL of methanol (MeOH). Samples were mixed by vortex SP:SAMPLEPREP_SUMMARY then homogenized at 6000 RPM for 30 seconds in a FastPrep® FP 120 tissue SP:SAMPLEPREP_SUMMARY homogenizer (Qbiogene) and then sonicated in an ambient water bath for 15 min. SP:SAMPLEPREP_SUMMARY Samples were centrifuged for 10 minutes at 20,000 RCF at ambient temperature, SP:SAMPLEPREP_SUMMARY then 1 mL of supernatant was transferred to individual 4 mL glass vials. The SP:SAMPLEPREP_SUMMARY supernatant was dried under a nitrogen stream at 30°C, reconstituted in 200 μL SP:SAMPLEPREP_SUMMARY of 0.1% formic acid in water, and vortexed for 30 seconds. Vial contents were SP:SAMPLEPREP_SUMMARY transferred to a 1.5-mL snap-cap tube, placed on ice for 10 minutes, then SP:SAMPLEPREP_SUMMARY centrifuged at 20,000 RCF for 10 minutes at ambient temperature. 150 μL of SP:SAMPLEPREP_SUMMARY supernatant was transferred to glass LC vials for UHPLC-HRMS analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME ACE Excel 2 C18-PFP (100 x 2.1mm, 2um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS The raw acquisition data were processed using a similar workflow described in MS:MS_COMMENTS previous work (Chamberlain et al., 2019a, b), which we detail here. Raw data MS:MS_COMMENTS files were converted from .raw to .mzxml format using RawConverter (He et al., MS:MS_COMMENTS 2015). MZmine 2 was used for processing the raw data including detecting masses, MS:MS_COMMENTS building chromatograms, grouping isotopic peaks, removing duplicate peaks, and MS:MS_COMMENTS aligning features (Pluskal et al., 2010). Identification was assigned to MS:MS_COMMENTS features by m/z (≤5 ppm) and retention time (0.2 min) (level 1 – MS:MS_COMMENTS identified compounds according to Metabolomics Standards Initiative standards MS:MS_COMMENTS (Sumner et al., 2007)) matching tousing our method-specific metabolite library MS:MS_COMMENTS produced from pure standards previously analyzed using this the above-mentioned MS:MS_COMMENTS chromatographic gradient. Processed data were exported from MZmine as a feature MS:MS_COMMENTS list containing the signal intensity for each feature in each sample. A small MS:MS_COMMENTS value (half the minimum value in the dataset) was used to replace zeros (no MS:MS_COMMENTS detection). The data were filtered to remove sample features with 10% signal MS:MS_COMMENTS contribution from their corresponding features in the extraction blanks. From MS:MS_COMMENTS this point, the data were further processed, normalized, and filtered using MS:MS_COMMENTS MetaboAnalyst 4.0 (Chong et al., 2018). For whole-metabolome comparative MS:MS_COMMENTS analyses, the data were normalized to total ion signal and feature intensities MS:MS_COMMENTS were auto-scaled to facilitate statistical comparisons (van den Berg et al., MS:MS_COMMENTS 2006). Statistical significance, defined as p 0.05, was determined using the MS:MS_COMMENTS two-tailed student’s t-test, and values for significance were adjusted for the MS:MS_COMMENTS false discovery rate with the Bonferroni-Holm method (HOLM, 1979). #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS peak intensity MS_METABOLITE_DATA_START Samples 1_ 2_ 3_ 4_ 5_ 6_ 7_ 8_ 9_ 10_ 11_ 12_ 25_ 26_ 27_ 28_ 29_ 30_ 31_ 32_ 33_ 34_ 49_ 50_ 51_ 52_ 53_ 54_ 55_ 56_ 57_ 58_ 59_ Factors Sample Type:SLB inoculation | Age:14 day | Growth Temperature:28 Sample Type:SLB inoculation | Age:14 day | Growth Temperature:28 Sample Type:SLB inoculation | Age:14 day | Growth Temperature:28 Sample Type:SLB inoculation | Age:14 day | Growth Temperature:28 Sample Type:SLB inoculation | Age:14 day | Growth Temperature:28 Sample Type:SLB inoculation | Age:14 day | Growth Temperature:28 Sample Type:Control | Age:14 day | Growth Temperature:28 Sample Type:Control | Age:14 day | Growth Temperature:28 Sample Type:Control | Age:14 day | Growth Temperature:28 Sample Type:Control | Age:14 day | Growth Temperature:28 Sample Type:Control | Age:14 day | Growth Temperature:28 Sample Type:Control | Age:14 day | Growth Temperature:28 Sample Type:SLB inoculation | Age:14 day | Growth Temperature:38 returned to 28 Sample Type:SLB inoculation | Age:14 day | Growth Temperature:38 returned to 28 Sample Type:SLB inoculation | Age:14 day | Growth Temperature:38 returned to 28 Sample Type:SLB inoculation | Age:14 day | Growth Temperature:38 returned to 28 Sample Type:SLB inoculation | Age:14 day | Growth Temperature:38 returned to 28 Sample Type:SLB inoculation | Age:14 day | Growth Temperature:38 returned to 28 Sample Type:Control | Age:14 day | Growth Temperature:38 returned to 28 Sample Type:Control | Age:14 day | Growth Temperature:38 returned to 28 Sample Type:Control | Age:14 day | Growth Temperature:38 returned to 28 Sample Type:Control | Age:14 day | Growth Temperature:38 returned to 28 Sample Type:SLB inoculation | Age:14 day | Growth Temperature:38 returned to 38 Sample Type:SLB inoculation | Age:14 day | Growth Temperature:38 returned to 38 Sample Type:SLB inoculation | Age:14 day | Growth Temperature:38 returned to 38 Sample Type:SLB inoculation | Age:14 day | Growth Temperature:38 returned to 38 Sample Type:SLB inoculation | Age:14 day | Growth Temperature:38 returned to 38 Sample Type:SLB inoculation | Age:14 day | Growth Temperature:38 returned to 38 Sample Type:Control | Age:14 day | Growth Temperature:38 returned to 38 Sample Type:Control | Age:14 day | Growth Temperature:38 returned to 38 Sample Type:Control | Age:14 day | Growth Temperature:38 returned to 38 Sample Type:Control | Age:14 day | Growth Temperature:38 returned to 38 Sample Type:Control | Age:14 day | Growth Temperature:38 returned to 38 Catechol 176603.832 270123.4046 471865.2675 362157.0999 704936.667 395007.815 125094.9699 969175.9919 632927.4595 224647.1865 174560.5441 734276.3736 662433.4218 374088.8393 289154.8436 96907.79457 439849.7913 315687.8122 519948.5096 462429.0279 464860.2949 246120.3329 1437008.298 355346.6 545137.0003 1649.30528 179964.9176 99605.70964 151756.5625 459808.5568 109442.9097 596311.4603 113758.7951 Citric acid 518567107.3 496978734 1458279794 687724620.7 375680201.3 881591848.2 849659070.2 1063513055 1558383463 891833925.9 918070700 1305206496 313373112.6 512882310.1 1368930365 1027908639 519569498.1 877271401.7 1327821126 803145969.3 787500375.7 641239579.8 365132807.3 970084301.8 981341976.1 185873.9379 1903978827 1168260185 1706593537 840781639.7 598763210.6 227124175.4 654595975.7 Cryptochlorogenic acid 135826.5719 43277.15678 484296.3257 25467.11166 2611024.035 605650.3397 4575462.944 4179984.231 2702415.01 759617.2724 71235.64353 692179.1344 698896.1792 41898.06966 5097544.328 594305.5439 271890.2975 42304593.16 205919711 21936.25973 660355.5196 116029.1421 2228832.912 637001.2114 99574.89227 497.4088837 17994227.71 8041482.477 19623040.47 817333.6618 3791428.736 137325.1025 855522.5247 Mevalonic acid 606083.3017 757993.2822 2726708.659 938582.5096 966438.8224 2843702.08 2071516.349 2689738.755 3762972.941 1473680.727 1446876.533 2350952.632 1113202.622 1037348.406 2869223.256 2171554.161 1682130.257 1657342.551 5454204.333 1684655.702 2594017.998 2338767.111 1772007.701 1812789.47 1925029.209 8434.744316 4881163.247 2280997.512 3233592.093 2259811.919 1799492.245 1027053.375 1782278.669 Ribose-5-Phosphate 12723521.15 12711436.93 7242452.69 14666897.45 12034303.4 10421408.81 3482266.522 3618743.763 3632940.636 8578909.733 17270846.97 13778661.84 10473073.46 9382992.076 1352116.904 75265.43262 7321980.655 10074911.87 8044334.157 9906414.809 3761975.043 7719477.295 9611054.812 2217118.746 9811750.772 45908.14858 2053123.076 1469413.707 3175671.551 2353099.364 5774654.156 7682311.121 5065569.715 Salicylic acid 3068548.58 3190018.081 10761842.55 4831981.198 5829247.311 5897326.952 21517472.31 17795377.73 23824189.86 6494262.011 3542471.522 6099924.055 4261939.068 3398469.45 12249572.49 9316116.458 5179139.869 8894124.007 10847201.34 8157476.539 18512609.86 4819824.644 6597557.857 6585791.314 8669883.126 1128539.627 10917402.97 10126942.22 14958214.33 19291141.21 15715719.03 3807904.563 21258797.22 Shikimic acid 40899262.23 33448553.97 88290894.79 62638183.55 49852056.11 63853240.42 20511643.57 57536456.4 44667334.86 42269483.82 43131025.7 94866752.69 61703089.78 38246019.99 34239395.64 28780584.61 47332775.52 37978457.02 168768231.4 19278475.72 19411273.51 32944117.66 107410927.9 29169587.74 41661059.88 2313.130649 88734209.91 36808556.79 53440049.67 31441027.74 10916545.76 54473700.6 27557196.13 Valine 4295135.365 4154951.486 16225362.06 3964097.69 17710362.31 7033356.783 7349628.305 11286474.96 11503563.82 5207066.223 4230253.972 16458810.99 4633852.416 3439378.586 15096260.11 9133744.644 1717772.011 7571303.538 41009809.83 3301924.896 7553606.038 3321536.417 16428341.97 11304827.75 13864643.42 497.4088837 13992302.18 11134399.14 15577218.8 7339735.106 6866768.108 6470095.87 7536229.597 ZealexinA4 519802.7399 517495.4505 666611.7469 637203.4388 226360.1108 806126.695 166981.8129 331176.9912 324834.4659 499976.4144 589318.5147 523103.0448 403863.6177 364922.7875 235017.7881 219834.6106 396311.9706 351692.2062 205874.6803 772487.8828 100437.1942 652649.4609 228956.6508 297741.4903 229410.3167 497.4088837 517998.3117 262371.6478 454853.9149 127519.0998 339165.8511 206080.9945 78481.90429 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name quantitated m/z [M-H]- PubChem Catechol 109.0289 289 Citric acid 191.01918 311 Cryptochlorogenic acid 353.0872 9798666 Mevalonic acid 147.0657 439230 Ribose-5-Phosphate 229.0113 77982 Salicylic acid 137.0239 338 Shikimic acid 173.045524 8742 Valine 116.0711 6287 ZealexinA4 247.1334 135398014 METABOLITES_END #END