#METABOLOMICS WORKBENCH Favela_20250701_122835 DATATRACK_ID:6124 STUDY_ID:ST004211 ANALYSIS_ID:AN007005 PROJECT_ID:PR002654 VERSION 1 CREATED_ON September 22, 2025, 8:12 pm #PROJECT PR:PROJECT_TITLE Lost and Found: Rediscovering Microbiome-Associated Phenotypes that Reshape PR:PROJECT_TITLE Agricultural Sustainability PR:PROJECT_SUMMARY Modern agriculture faces an urgent need to improve nutrient use efficiency while PR:PROJECT_SUMMARY reducing environmental impacts. Here, we show that ancestral traits controlling PR:PROJECT_SUMMARY rhizosphere microbiome functions can be reintroduced into elite maize through PR:PROJECT_SUMMARY targeted teosinte introgressions. Using near-isogenic lines, we mapped PR:PROJECT_SUMMARY microbiome-associated phenotypes (MAPs) derived from teosinte that suppress PR:PROJECT_SUMMARY nitrification and denitrification—key microbial processes contributing to PR:PROJECT_SUMMARY nitrogen loss. These introgressions altered root exudate chemistry, resulting in PR:PROJECT_SUMMARY distinct microbial assemblies and enhanced nitrogen retention. We identified PR:PROJECT_SUMMARY candidate loci and metabolites responsible for suppressive activity and PR:PROJECT_SUMMARY demonstrated their functional effects in vitro. Our findings reveal a genetic PR:PROJECT_SUMMARY and biochemical basis for rewilding microbiome-mediated ecosystem services in PR:PROJECT_SUMMARY crops, offering a scalable path toward sustainable nutrient management in global PR:PROJECT_SUMMARY agriculture. ---- These maize root exduate metabolomics data are a subset of PR:PROJECT_SUMMARY this larger project and make up a phenotyping for candidate lines. PR:INSTITUTE University of Arizona PR:DEPARTMENT School of Plant Sciences PR:LABORATORY Favela Lab PR:LAST_NAME Favela PR:FIRST_NAME Alonso PR:ADDRESS Forbes Building PR:EMAIL alonsof@arizona.edu PR:PHONE 4802552527 #STUDY ST:STUDY_TITLE Lost and Found: Rediscovering Microbiome-Associated Phenotypes that Reshape ST:STUDY_TITLE Agricultural Sustainability ST:STUDY_SUMMARY Modern agriculture faces an urgent need to improve nutrient use efficiency while ST:STUDY_SUMMARY reducing environmental impacts. Here, we show that ancestral traits controlling ST:STUDY_SUMMARY rhizosphere microbiome functions can be reintroduced into elite maize through ST:STUDY_SUMMARY targeted teosinte introgressions. Using near-isogenic lines, we mapped ST:STUDY_SUMMARY microbiome-associated phenotypes (MAPs) derived from teosinte that suppress ST:STUDY_SUMMARY nitrification and denitrification—key microbial processes contributing to ST:STUDY_SUMMARY nitrogen loss. These introgressions altered root exudate chemistry, resulting in ST:STUDY_SUMMARY distinct microbial assemblies and enhanced nitrogen retention. We identified ST:STUDY_SUMMARY candidate loci and exudate metabolites responsible for suppressive activity and ST:STUDY_SUMMARY demonstrated their functional effects in vitro. These findings reveal a genetic ST:STUDY_SUMMARY and biochemical basis for rewilding microbiome-mediated ecosystem services in ST:STUDY_SUMMARY crops, offering a scalable path toward sustainable nutrient management in global ST:STUDY_SUMMARY agriculture. These maize root exduate metabolomics data are a subset of this ST:STUDY_SUMMARY larger project and make up a phenotyping for candidate lines. ST:INSTITUTE University of Arizona ST:LAST_NAME Favela ST:FIRST_NAME Alonso ST:ADDRESS Forbes Building ST:EMAIL alonsof@arizona.edu ST:PHONE 14802552527 #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 - B73 1 Sample source:Maize Exudates | Genotype:B73 Rep=1; RAW_FILE_NAME()=HILIC_Neg_B73_1.raw; RAW_FILE_NAME_RP=RP_Pos_B73_1.raw SUBJECT_SAMPLE_FACTORS - B73 2 Sample source:Maize Exudates | Genotype:B73 Rep=2; RAW_FILE_NAME()=HILIC_Neg_B73_2.raw; RAW_FILE_NAME_RP=RP_Pos_B73_2.raw SUBJECT_SAMPLE_FACTORS - B73 3 Sample source:Maize Exudates | Genotype:B73 Rep=3; RAW_FILE_NAME()=HILIC_Neg_B73_3.raw; RAW_FILE_NAME_RP=RP_Pos_B73_3.raw SUBJECT_SAMPLE_FACTORS - B73 4 Sample source:Maize Exudates | Genotype:B73 Rep=4; RAW_FILE_NAME()=HILIC_Neg_B73_4.raw; RAW_FILE_NAME_RP=RP_Pos_B73_4.raw SUBJECT_SAMPLE_FACTORS - B73 5 Sample source:Maize Exudates | Genotype:B73 Rep=5; RAW_FILE_NAME()=HILIC_Neg_B73_5.raw; RAW_FILE_NAME_RP=RP_Pos_B73_5.raw SUBJECT_SAMPLE_FACTORS - B73 6 Sample source:Maize Exudates | Genotype:B73 Rep=6; RAW_FILE_NAME()=HILIC_Neg_B73_6.raw; RAW_FILE_NAME_RP=RP_Pos_B73_6.raw SUBJECT_SAMPLE_FACTORS - Z47 1 Sample source:Maize Exudates | Genotype:NIL047 Rep=1; RAW_FILE_NAME()=HILIC_Neg_Z47_1.raw; RAW_FILE_NAME_RP=RP_Pos_Z47_1.raw SUBJECT_SAMPLE_FACTORS - Z47 2 Sample source:Maize Exudates | Genotype:NIL047 Rep=2; RAW_FILE_NAME()=HILIC_Neg_Z47_2.raw; RAW_FILE_NAME_RP=RP_Pos_Z47_2.raw SUBJECT_SAMPLE_FACTORS - Z47 3 Sample source:Maize Exudates | Genotype:NIL047 Rep=3; RAW_FILE_NAME()=HILIC_Neg_Z47_3.raw; RAW_FILE_NAME_RP=RP_Pos_Z47_3.raw SUBJECT_SAMPLE_FACTORS - Z47 4 Sample source:Maize Exudates | Genotype:NIL047 Rep=4; RAW_FILE_NAME()=HILIC_Neg_Z47_4.raw; RAW_FILE_NAME_RP=RP_Pos_Z47_4.raw SUBJECT_SAMPLE_FACTORS - Z47 5 Sample source:Maize Exudates | Genotype:NIL047 Rep=5; RAW_FILE_NAME()=HILIC_Neg_Z47_5.raw; RAW_FILE_NAME_RP=RP_Pos_Z47_5.raw SUBJECT_SAMPLE_FACTORS - Z47 6 Sample source:Maize Exudates | Genotype:NIL047 Rep=6; RAW_FILE_NAME()=HILIC_Neg_Z47_6.raw; RAW_FILE_NAME_RP=RP_Pos_Z47_6.raw SUBJECT_SAMPLE_FACTORS - Z21 1 Sample source:Maize Exudates | Genotype:NIL021 Rep=1; RAW_FILE_NAME()=HILIC_Neg_Z21_1.raw; RAW_FILE_NAME_RP=RP_Pos_Z21_1.raw SUBJECT_SAMPLE_FACTORS - Z21 2 Sample source:Maize Exudates | Genotype:NIL021 Rep=2; RAW_FILE_NAME()=HILIC_Neg_Z21_2.raw; RAW_FILE_NAME_RP=RP_Pos_Z21_2.raw SUBJECT_SAMPLE_FACTORS - Z21 3 Sample source:Maize Exudates | Genotype:NIL021 Rep=3; RAW_FILE_NAME()=HILIC_Neg_Z21_3.raw; RAW_FILE_NAME_RP=RP_Pos_Z21_3.raw SUBJECT_SAMPLE_FACTORS - Z21 4 Sample source:Maize Exudates | Genotype:NIL021 Rep=4; RAW_FILE_NAME()=HILIC_Neg_Z21_4.raw; RAW_FILE_NAME_RP=RP_Pos_Z21_4.raw SUBJECT_SAMPLE_FACTORS - Z21 5 Sample source:Maize Exudates | Genotype:NIL021 Rep=5; RAW_FILE_NAME()=HILIC_Neg_Z21_5.raw; RAW_FILE_NAME_RP=RP_Pos_Z21_5.raw SUBJECT_SAMPLE_FACTORS - Blank MS1 Sample source:Blank | Genotype:NA Rep=1; RAW_FILE_NAME()=HILIC_Neg_Blank_MS1.raw; RAW_FILE_NAME_RP=RP_Pos_Blank_MS1.raw SUBJECT_SAMPLE_FACTORS - Blank MS2 Sample source:Blank | Genotype:NA Rep=2; RAW_FILE_NAME()=HILIC_Neg_Blank_MS2.raw; RAW_FILE_NAME_RP=RP_Pos_Blank_MS2.raw SUBJECT_SAMPLE_FACTORS - Pooled MS2 Sample source:Pool | Genotype:NA Rep=2; RAW_FILE_NAME()=HILIC_Neg_Pooled_MS2.raw; RAW_FILE_NAME_RP=RP_Pos_Pooled_MS2.raw SUBJECT_SAMPLE_FACTORS - Pooled QC1 Sample source:Pool | Genotype:NA Rep=1; RAW_FILE_NAME()=HILIC_Neg_Pooled_QC1.raw; RAW_FILE_NAME_RP=RP_Pos_Pooled_QC1.raw SUBJECT_SAMPLE_FACTORS - Pooled QC2 Sample source:Pool | Genotype:NA Rep=2; RAW_FILE_NAME()=HILIC_Neg_Pooled_QC2.raw; RAW_FILE_NAME_RP=RP_Pos_Pooled_QC2.raw SUBJECT_SAMPLE_FACTORS - Pooled QC3 Sample source:Pool | Genotype:NA Rep=3; RAW_FILE_NAME()=HILIC_Neg_Pooled_QC3.raw; RAW_FILE_NAME_RP=RP_Pos_Pooled_QC3.raw #COLLECTION CO:COLLECTION_SUMMARY Root exudates were collected from candidate BNI NILs to assess whether these CO:COLLECTION_SUMMARY lines exhibit distinct exudation profiles, in addition to previously observed CO:COLLECTION_SUMMARY differences in root tissue metabolomes. Seeds were sterilized prior to CO:COLLECTION_SUMMARY introduction to the hydroponic environment with a dilute bleach and ethanol CO:COLLECTION_SUMMARY solution, as in (2). To collect exudates, seedlings of B73, NIL021, and NIL047 CO:COLLECTION_SUMMARY were grown for four weeks in sterilized Hoagland’s nutrient solution with CO:COLLECTION_SUMMARY inert glass beads as a support matrix. Plants were maintained in a Hettich CO:COLLECTION_SUMMARY growth chamber under a 12 h light / 12 h dark photoperiod. At the time of CO:COLLECTION_SUMMARY sampling, plants were hydroponically extracted in a series of washes. First, the CO:COLLECTION_SUMMARY existing growth solution was drained and replaced with fresh deionized water CO:COLLECTION_SUMMARY (“trap solution”) to induce an osmotic shock response and stimulate CO:COLLECTION_SUMMARY exudation. Following a short incubation, the trap solution was removed and CO:COLLECTION_SUMMARY collected. A total of 40 mL of hydroponic exudate was recovered per plant. CO:SAMPLE_TYPE Maize root exudates #TREATMENT TR:TREATMENT_SUMMARY No further treatment. This is a genotype study. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY To capture the chemical diversity of root exudates, we conducted untargeted SP:SAMPLEPREP_SUMMARY metabolomic profiling using both reverse phase liquid chromatography (RP-LC) and SP:SAMPLEPREP_SUMMARY hydrophilic interaction liquid chromatography (HILIC) coupled to high-resolution SP:SAMPLEPREP_SUMMARY mass spectrometry. RP-LC was used to analyze non-polar to moderately polar SP:SAMPLEPREP_SUMMARY metabolites, while HILIC enabled the detection of highly polar and ionic SP:SAMPLEPREP_SUMMARY compounds that are often poorly retained on reverse phase columns. This SP:SAMPLEPREP_SUMMARY complementary approach allowed for broader metabolite coverage of root exudation SP:SAMPLEPREP_SUMMARY chemistry. As with the root tissue analysis, metabolite identifications derived SP:SAMPLEPREP_SUMMARY from these LC-MS datasets are considered putative due to limitations in compound SP:SAMPLEPREP_SUMMARY libraries and structural resolution. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Waters ACQUITY Premier HSS T3 (150 x 2.1mm, 1.8um) CH:SOLVENT_A 100% Water; 0.1% formic acid CH:SOLVENT_B 100% Methanol; 0.1% formic acid CH:FLOW_GRADIENT 0–3 min held at 1% B; 3–19 min 1% B – 95% B; 19–20 min 95% B. CH:FLOW_RATE 300 μL/min CH:COLUMN_TEMPERATURE 45 C #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Orbitrap Exploris 480 MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Samples were analyzed in both positive and negative ionization modes using HCD MS:MS_COMMENTS (higher-energy collision dissociation). The HESI source parameters were set as MS:MS_COMMENTS follows: spray voltage 3.5 or 2.5 kV for positive and negative modes MS:MS_COMMENTS respectively; capillary temperature 350 °C; S lens RF level 50 arbitrary units, MS:MS_COMMENTS and aux gas heater temperature 350 °C. Full MS scan data were acquired at a MS:MS_COMMENTS resolving power of 120,000 FWHM at m/z 200 with the scanning range of m/z MS:MS_COMMENTS 65–975. The automatic gain control (AGC) target was set at 50%, with the MS:MS_COMMENTS maximum injection time of 100 ms. The data dependent acquisition (dd-MS2) MS:MS_COMMENTS parameters used to obtain product ion spectra were as follows: resolving power MS:MS_COMMENTS 30,000 FWHM at m/z 200, AGC target of 50% ions with maximum injection time is MS:MS_COMMENTS set to auto, isolation width 1.2 m/z, and HCD collision energies of: 20, 40, 80 MS:MS_COMMENTS %. For data generated, confident metabolite identifications were made using MS:MS_COMMENTS Thermo Compound Discoverer 3.3. For RP and HILIC positive and negative mode, MS:MS_COMMENTS spectra were aligned using an adaptive curve with a maximum of 0.5 min RT shift MS:MS_COMMENTS respectively and a 5-ppm mass tolerance. Peaks were selected based on a minimum MS:MS_COMMENTS intensity of 1 × 10⁶ and a chromatographic S/N of 3. Detected features were MS:MS_COMMENTS grouped based on a mass tolerance of 5 ppm and a RT tolerance of 0.5. Compounds MS:MS_COMMENTS were assigned based on Isotopic pattern, RT, MS1, and/or MS2. All MS:MS_COMMENTS identifications and integrated peaks were manually validated and exported for MS:MS_COMMENTS statistical analysis. MS:MS_RESULTS_FILE ST004211_AN007005_Results.txt UNITS:Area Has m/z:Yes Has RT:Yes RT units:Minutes #END