#METABOLOMICS WORKBENCH ManoelSouza_20220928_104649 DATATRACK_ID:3480 STUDY_ID:ST002430 ANALYSIS_ID:AN003955 PROJECT_ID:PR001563 VERSION 1 CREATED_ON January 5, 2023, 5:06 pm #PROJECT PR:PROJECT_TITLE Insights from a Multi-Omics Integration (MOI) Study in Oil Palm (Elaeis PR:PROJECT_TITLE guineensis Jacq.) Response to Abiotic Stresses: Part Two—Drought PR:PROJECT_TYPE Multi-Omics Integration (MOI) Study PR:PROJECT_SUMMARY Drought and salinity are two of the most severe abiotic stresses affecting PR:PROJECT_SUMMARY agriculture Worldwide and bear some similarities in the response of plants to PR:PROJECT_SUMMARY them. The first is also known as osmotic stress and shows similarities mainly PR:PROJECT_SUMMARY with the osmotic effect, the first phase of salinity stress. Multi-Omics PR:PROJECT_SUMMARY Integration (MOI) offers a new opportunity for the non-trivial challenge of PR:PROJECT_SUMMARY unraveling the mechanisms behind multigenic traits, such as drought and salinity PR:PROJECT_SUMMARY resistance. The current study carried out a comprehensive, large-scale, PR:PROJECT_SUMMARY single-omics analysis (SOA) and MOI studies on the leaves of young oil palm PR:PROJECT_SUMMARY plants submitted to water deprivation. After performing SOA, 1,955 DE enzymes PR:PROJECT_SUMMARY from transcriptomics analysis, 131 DE enzymes from proteomics analysis, and 269 PR:PROJECT_SUMMARY DE metabolites underwent MOI analysis, revealing several pathways affected by PR:PROJECT_SUMMARY this stress, with at least one DE molecule in all three omics platforms used. PR:PROJECT_SUMMARY Besides, the similarities and dissimilarities in the molecular response of those PR:PROJECT_SUMMARY plants to those two abiotic stresses underwent mapping. Cysteine and methionine PR:PROJECT_SUMMARY metabolism (map00270) was the most affected pathway in all scenarios evaluated. PR:PROJECT_SUMMARY The correlation analysis revealed that 91.55% of those enzymes expressed under PR:PROJECT_SUMMARY both stresses had similar qualitative profiles, corroborating the already known PR:PROJECT_SUMMARY fact that plant responses to drought and salinity show several similarities. At PR:PROJECT_SUMMARY last, the results shed light on some candidate genes for engineering crop PR:PROJECT_SUMMARY species resilient to both abiotic stresses. PR:INSTITUTE The Brazilian Agricultural Research Corporation (Embrapa) PR:DEPARTMENT Embrapa Agroenergy PR:LABORATORY Genetics and Plant Biotechnology PR:LAST_NAME Souza Jr PR:FIRST_NAME Manoel Teixeira PR:ADDRESS Parque Estacao Biologica, Final Avenida W3 Norte - Asa Norte, Brasilia, Distrito PR:ADDRESS Federal, 70770901, Brazil PR:EMAIL manoel.souza@embrapa.br PR:PHONE +55.61.3448.3210 PR:FUNDING_SOURCE FINEP (01.13.0315.00) PR:PROJECT_COMMENTS DendêPalm Project PR:PUBLICATIONS https://doi.org/10.1038/s41598-021-97835-x #STUDY ST:STUDY_TITLE Insights from a Multi-Omics Integration (MOI) Study in Oil Palm (Elaeis ST:STUDY_TITLE guineensis Jacq.) Response to Abiotic Stresses: Part Two—Drought ST:STUDY_TYPE Multi-Omics Integration (MOI) Study ST:STUDY_SUMMARY Drought and salinity are two of the most severe abiotic stresses affecting ST:STUDY_SUMMARY agriculture Worldwide and bear some similarities in the response of plants to ST:STUDY_SUMMARY them. The first is also known as osmotic stress and shows similarities mainly ST:STUDY_SUMMARY with the osmotic effect, the first phase of salinity stress. Multi-Omics ST:STUDY_SUMMARY Integration (MOI) offers a new opportunity for the non-trivial challenge of ST:STUDY_SUMMARY unraveling the mechanisms behind multigenic traits, such as drought and salinity ST:STUDY_SUMMARY resistance. The current study carried out a comprehensive, large-scale, ST:STUDY_SUMMARY single-omics analysis (SOA) and MOI studies on the leaves of young oil palm ST:STUDY_SUMMARY plants submitted to water deprivation. After performing SOA, 1,955 DE enzymes ST:STUDY_SUMMARY from transcriptomics analysis, 131 DE enzymes from proteomics analysis, and 269 ST:STUDY_SUMMARY DE metabolites underwent MOI analysis, revealing several pathways affected by ST:STUDY_SUMMARY this stress, with at least one DE molecule in all three omics platforms used. ST:STUDY_SUMMARY Besides, the similarities and dissimilarities in the molecular response of those ST:STUDY_SUMMARY plants to those two abiotic stresses underwent mapping. Cysteine and methionine ST:STUDY_SUMMARY metabolism (map00270) was the most affected pathway in all scenarios evaluated. ST:STUDY_SUMMARY The correlation analysis revealed that 91.55% of those enzymes expressed under ST:STUDY_SUMMARY both stresses had similar qualitative profiles, corroborating the already known ST:STUDY_SUMMARY fact that plant responses to drought and salinity show several similarities. At ST:STUDY_SUMMARY last, the results shed light on some candidate genes for engineering crop ST:STUDY_SUMMARY species resilient to both abiotic stresses. ST:INSTITUTE The Brazilian Agricultural Research Corporation (Embrapa) ST:DEPARTMENT Embrapa Agroenergy ST:LABORATORY Genetics and Plant Biotechnology ST:LAST_NAME Souza Jr ST:FIRST_NAME Manoel Teixeira ST:ADDRESS Parque Estacao Biologica, Final Avenida W3 Norte - Asa Norte, Brasilia, Distrito ST:ADDRESS Federal, 70770901, Brazil ST:EMAIL manoel.souza@embrapa.br ST:PHONE +55.61.3448.3210 ST:PUBLICATIONS https://doi.org/10.1038/s41598-021-97835-x #SUBJECT SU:SUBJECT_TYPE Plant SU:SUBJECT_SPECIES Elaeis guineensis Jacq. SU:TAXONOMY_ID NCBI:txid51953 #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 - OilPalm_Drought_Control_R1_POS Group:Control RAW_FILE_NAME=OilPalm_Drought_Control_R1_POS.mzXML SUBJECT_SAMPLE_FACTORS - OilPalm_Drought_Control_R2_POS Group:Control RAW_FILE_NAME=OilPalm_Drought_Control_R2_POS.mzXML SUBJECT_SAMPLE_FACTORS - OilPalm_Drought_Control_R3_POS Group:Control RAW_FILE_NAME=OilPalm_Drought_Control_R3_POS.mzXML SUBJECT_SAMPLE_FACTORS - OilPalm_Drought_Control_R4_POS Group:Control RAW_FILE_NAME=OilPalm_Drought_Control_R4_POS.mzXML SUBJECT_SAMPLE_FACTORS - OilPalm_Drought_Stressed_R1_POS Group:Stressed RAW_FILE_NAME=OilPalm_Drought_Stressed_R1_POS.mzXML SUBJECT_SAMPLE_FACTORS - OilPalm_Drought_Stressed_R2_POS Group:Stressed RAW_FILE_NAME=OilPalm_Drought_Stressed_R2_POS.mzXML SUBJECT_SAMPLE_FACTORS - OilPalm_Drought_Stressed_R3_POS Group:Stressed RAW_FILE_NAME=OilPalm_Drought_Stressed_R3_POS.mzXML SUBJECT_SAMPLE_FACTORS - OilPalm_Drought_Stressed_R4_POS Group:Stressed RAW_FILE_NAME=OilPalm_Drought_Stressed_R4_POS.mzXML SUBJECT_SAMPLE_FACTORS - OilPalm_Drought_Control_R1_NEG Group:Control RAW_FILE_NAME=OilPalm_Drought_Control_R1_NEG.mzXML SUBJECT_SAMPLE_FACTORS - OilPalm_Drought_Control_R2_NEG Group:Control RAW_FILE_NAME=OilPalm_Drought_Control_R2_NEG.mzXML SUBJECT_SAMPLE_FACTORS - OilPalm_Drought_Control_R3_NEG Group:Control RAW_FILE_NAME=OilPalm_Drought_Control_R3_NEG.mzXML SUBJECT_SAMPLE_FACTORS - OilPalm_Drought_Control_R4_NEG Group:Control RAW_FILE_NAME=OilPalm_Drought_Control_R4_NEG.mzXML SUBJECT_SAMPLE_FACTORS - OilPalm_Drought_Stressed_R1_NEG Group:Stressed RAW_FILE_NAME=OilPalm_Drought_Stressed_R1_NEG.mzXML SUBJECT_SAMPLE_FACTORS - OilPalm_Drought_Stressed_R2_NEG Group:Stressed RAW_FILE_NAME=OilPalm_Drought_Stressed_R2_NEG.mzXML SUBJECT_SAMPLE_FACTORS - OilPalm_Drought_Stressed_R3_NEG Group:Stressed RAW_FILE_NAME=OilPalm_Drought_Stressed_R3_NEG.mzXML SUBJECT_SAMPLE_FACTORS - OilPalm_Drought_Stressed_R4_NEG Group:Stressed RAW_FILE_NAME=OilPalm_Drought_Stressed_R4_NEG.mzXML #COLLECTION CO:COLLECTION_SUMMARY The oil palm plants used in this study are clones of the ones used in the CO:COLLECTION_SUMMARY Bittencourt et al. (2022) study. All plants—from both studies—came from the CO:COLLECTION_SUMMARY same embryogenic calluses. The young oil palm plants used in both studies were CO:COLLECTION_SUMMARY clones regenerated out of embryogenic calluses obtained from the leaves of an CO:COLLECTION_SUMMARY adult plant—genotype AM33, a Deli x Ghana from ASD Costa Rica; and were CO:COLLECTION_SUMMARY subjected to treatments when they were in the growth stage known as “bifid” CO:COLLECTION_SUMMARY saplings. Before starting the experiments, plants were standardized according to CO:COLLECTION_SUMMARY their developmental stage, size, and the number of leaves. The experiment CO:COLLECTION_SUMMARY consisted of two water availability levels (field capacity—control and water CO:COLLECTION_SUMMARY deprivation—stressed), with four replicates in a completely randomized design. CO:COLLECTION_SUMMARY For the metabolomics analysis, we collected the apical leaves from control and CO:COLLECTION_SUMMARY stressed plants 14 days after imposing the treatments (DAT). CO:SAMPLE_TYPE Plant #TREATMENT TR:TREATMENT_SUMMARY The experiment consisted of treatments—control and drought-stressed TR:TREATMENT_SUMMARY plants—with four plants kept in a substrate in the field capacity (control), TR:TREATMENT_SUMMARY and four plants submitted to drought stress. The young oil palm plants were TR:TREATMENT_SUMMARY subjected to treatments when they were in the growth stage known as “bifid” TR:TREATMENT_SUMMARY saplings. Drought stress consisted of total suppression of irrigation for 14 TR:TREATMENT_SUMMARY consecutive days. At the end of this period, the substrate water potential, as TR:TREATMENT_SUMMARY measured by the water potential meter Decagon mod. WP4C (Decagon Devices, TR:TREATMENT_SUMMARY Pullman, WA, USA), was 0.19 ± 0.03 MPa (control) and − 13.61 ± 1.79 MPa TR:TREATMENT_SUMMARY (drought stress), while the relative water content of leaves was 90.50 ± 0.95% TR:TREATMENT_SUMMARY (control) and 49.18 ± 9.76% (stressed plants). Before the onset of drought TR:TREATMENT_SUMMARY stress, oil palm leaves had the highest gas exchange rates, as measured by an TR:TREATMENT_SUMMARY infrared gas analyzer Li-Cor model 6400XT (Li-Cor, Lincoln, NE, USA). Under TR:TREATMENT_SUMMARY drought, leaf gas exchange rates in drought-stressed plants dropped to TR:TREATMENT_SUMMARY negligible values (data not shown). #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Leaf samples with approximately 50 mg were collected for the metabolomics SP:SAMPLEPREP_SUMMARY analysis; four replicates per plant. After harvesting, samples were immediately SP:SAMPLEPREP_SUMMARY frozen in liquid nitrogen and stored at − 80 °C until metabolites extraction SP:SAMPLEPREP_SUMMARY and analysis. Each sample was ground in a ball mill (Biospec Products, USA) SP:SAMPLEPREP_SUMMARY before solvent extraction. Metabolites were extracted using an adapted protocol SP:SAMPLEPREP_SUMMARY from The Max Planck Institute, called "All-in-One", which provides a polar SP:SAMPLEPREP_SUMMARY fraction for secondary metabolite analysis, a nonpolar fraction for lipidomics, SP:SAMPLEPREP_SUMMARY and a protein pellet for proteomics; all obtained from the same plant sample. SP:SAMPLEPREP_SUMMARY Each ground sample was added to a microtube and mixed with 1 mL of a methanol SP:SAMPLEPREP_SUMMARY and methyl-tert-butyl-ether (1:3) solution at − 20°C. After homogenization, SP:SAMPLEPREP_SUMMARY they were incubated at 4 °C for 10 min. Each microtube was ultrasonicated in an SP:SAMPLEPREP_SUMMARY ice bath for another 10 min. Then, 500 μL of a methanol and water (1:3) SP:SAMPLEPREP_SUMMARY solution was added to the microtube before centrifugation (12,000 rpm at 4 °C SP:SAMPLEPREP_SUMMARY for 5 min). Three phases were separate: an upper non-polar (green), a lower SP:SAMPLEPREP_SUMMARY polar (brown), and a remaining protein pellet. Samples were transferred to fresh SP:SAMPLEPREP_SUMMARY microtubes and vacuum-dried in a speed vac (Centrivap, Labconco, Kansas City, SP:SAMPLEPREP_SUMMARY MO, USA) overnight at room temperature (~ 22 °C). #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Shimadzu Nexera X2 CH:COLUMN_NAME Waters Acquity BEH C18 (150 x 2mm, 1.7um) CH:SOLVENT_A - CH:SOLVENT_B - CH:FLOW_GRADIENT - CH:FLOW_RATE - CH:COLUMN_TEMPERATURE - #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Bruker maXis Impact qTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS High-resolution mass spectrometry (HRMS) was performed in a MaXis 4G Q-TOF MS MS:MS_COMMENTS system (Bruker Daltonics, Germany) using an electrospray source in the positive MS:MS_COMMENTS and negative ion modes (ESI(+)–MS and ESI(−)–MS). The MS instrument MS:MS_COMMENTS settings used were: endplate offset, 500 V; capillary voltage, 3800 V; nebulizer MS:MS_COMMENTS pressure, 4 bar; dry gas flow, 9 L/min, dry temperature, 200 °C; and column MS:MS_COMMENTS temperature, 40 °C. The acquisition spectra rate was 3.00 Hz, monitoring a mass MS:MS_COMMENTS range from 70 to 1200 m/z. Sodium formate solution (10 mM NaOH solution in 50/50 MS:MS_COMMENTS v/v isopropanol/water containing 0.2% formic acid) was directly injected through MS:MS_COMMENTS a 6-port valve at the beginning of each chromatographic run to external MS:MS_COMMENTS calibration. UHPLC–MS data was acquired by the HyStar Application version 3.2 MS:MS_COMMENTS (Bruker Daltonics, Germany), and data processing was done using Data Analysis MS:MS_COMMENTS 4.2 (Bruker Daltonics, Germany). MS:MS_RESULTS_FILE ST002430_AN003955_Results.txt UNITS:Peak intensity Has m/z:Yes Has RT:No RT units:No RT data #END