#METABOLOMICS WORKBENCH ManoelSouza_20220928_093934 DATATRACK_ID:3479 STUDY_ID:ST002429 ANALYSIS_ID:AN003953 PROJECT_ID:PR001563 VERSION 1 CREATED_ON January 5, 2023, 5:02 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 One—Salinity PR:PROJECT_TYPE Multi-Omics Integration (MOI) Study PR:PROJECT_SUMMARY Oil palm (Elaeis guineensis Jacq.) is the number one source of consumed PR:PROJECT_SUMMARY vegetable oil nowadays. It is cultivated in areas of tropical rainforest, where PR:PROJECT_SUMMARY it meets its natural condition of high rainfall throughout the year. The palm PR:PROJECT_SUMMARY oil industry faces criticism due to a series of practices that was considered PR:PROJECT_SUMMARY not environmentally sustainable, and it finds itself under pressure to adopt new PR:PROJECT_SUMMARY and innovative procedures to reverse this negative public perception. PR:PROJECT_SUMMARY Cultivating this oilseed crop outside the rainforest zone is only possible using PR:PROJECT_SUMMARY artificial irrigation. Close to 30% of the world’s irrigated agricultural PR:PROJECT_SUMMARY lands also face problems due to salinity stress. Consequently, the research PR:PROJECT_SUMMARY community must consider drought and salinity together when studying to empower PR:PROJECT_SUMMARY breeding programs in order to develop superior genotypes adapted to those PR:PROJECT_SUMMARY potential new areas for oil palm cultivation. Multi-Omics Integration (MOI) PR:PROJECT_SUMMARY offers a new window of opportunity for the non-trivial challenge of unraveling PR:PROJECT_SUMMARY the mechanisms behind multigenic traits, such as drought and salinity tolerance. PR:PROJECT_SUMMARY The current study carried out a comprehensive, large-scale, single-omics PR:PROJECT_SUMMARY analysis (SOA), and MOI study on the leaves of young oil palm plants submitted PR:PROJECT_SUMMARY to very high salinity stress. Taken together, a total of 1239 proteins were PR:PROJECT_SUMMARY positively regulated, and 1660 were negatively regulated in transcriptomics and PR:PROJECT_SUMMARY proteomics analyses. Meanwhile, the metabolomics analysis revealed 37 PR:PROJECT_SUMMARY metabolites that were upreg- ulated and 92 that were downregulated. After PR:PROJECT_SUMMARY performing SOA, 436 differentially expressed (DE) full-length transcripts, 74 DE PR:PROJECT_SUMMARY proteins, and 19 DE metabolites underwent MOI analysis, revealing sev- eral PR:PROJECT_SUMMARY pathways affected by this stress, with at least one DE molecule in all three PR:PROJECT_SUMMARY omics platforms used. The Cysteine and methionine metabolism (map00270) and PR:PROJECT_SUMMARY Glycolysis/Gluconeogenesis (map00010) pathways were the most affected ones, each PR:PROJECT_SUMMARY one with 20 DE molecules. 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/10.3390/plants11131755 #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 One—Salinity ST:STUDY_TYPE Multi-Omics Integration (MOI) Study ST:STUDY_SUMMARY Oil palm (Elaeis guineensis Jacq.) is the number one source of consumed ST:STUDY_SUMMARY vegetable oil nowadays. It is cultivated in areas of tropical rainforest, where ST:STUDY_SUMMARY it meets its natural condition of high rainfall throughout the year. The palm ST:STUDY_SUMMARY oil industry faces criticism due to a series of practices that was considered ST:STUDY_SUMMARY not environmentally sustainable, and it finds itself under pressure to adopt new ST:STUDY_SUMMARY and innovative procedures to reverse this negative public perception. ST:STUDY_SUMMARY Cultivating this oilseed crop outside the rainforest zone is only possible using ST:STUDY_SUMMARY artificial irrigation. Close to 30% of the world’s irrigated agricultural ST:STUDY_SUMMARY lands also face problems due to salinity stress. Consequently, the research ST:STUDY_SUMMARY community must consider drought and salinity together when studying to empower ST:STUDY_SUMMARY breeding programs in order to develop superior genotypes adapted to those ST:STUDY_SUMMARY potential new areas for oil palm cultivation. Multi-Omics Integration (MOI) ST:STUDY_SUMMARY offers a new window of opportunity for the non-trivial challenge of unraveling ST:STUDY_SUMMARY the mechanisms behind multigenic traits, such as drought and salinity tolerance. ST:STUDY_SUMMARY The current study carried out a comprehensive, large-scale, single-omics ST:STUDY_SUMMARY analysis (SOA), and MOI study on the leaves of young oil palm plants submitted ST:STUDY_SUMMARY to very high salinity stress. Taken together, a total of 1239 proteins were ST:STUDY_SUMMARY positively regulated, and 1660 were negatively regulated in transcriptomics and ST:STUDY_SUMMARY proteomics analyses. Meanwhile, the metabolomics analysis revealed 37 ST:STUDY_SUMMARY metabolites that were upreg- ulated and 92 that were downregulated. After ST:STUDY_SUMMARY performing SOA, 436 differentially expressed (DE) full-length transcripts, 74 DE ST:STUDY_SUMMARY proteins, and 19 DE metabolites underwent MOI analysis, revealing sev- eral ST:STUDY_SUMMARY pathways affected by this stress, with at least one DE molecule in all three ST:STUDY_SUMMARY omics platforms used. The Cysteine and methionine metabolism (map00270) and ST:STUDY_SUMMARY Glycolysis/Gluconeogenesis (map00010) pathways were the most affected ones, each ST:STUDY_SUMMARY one with 20 DE molecules. 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/10.3390/plants11131755 #SUBJECT SU:SUBJECT_TYPE Plant SU:SUBJECT_SPECIES Elaeis guineensis Jacq. 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RAW_FILE_NAME=OilPalm_Salt_15_14DAT_R4_NEG.mzXML SUBJECT_SAMPLE_FACTORS - OilPalm_Salt_05_14DAT_R3_NEG Group:14 days Treatment=0.5g NaCl; RAW_FILE_NAME=OilPalm_Salt_05_14DAT_R3_NEG.mzXML SUBJECT_SAMPLE_FACTORS - OilPalm_Salt_10_14DAT_R2_NEG Group:14 days Treatment=1.0g NaCl; RAW_FILE_NAME=OilPalm_Salt_10_14DAT_R2_NEG.mzXML SUBJECT_SAMPLE_FACTORS - OilPalm_Salt_10_14DAT_R3_NEG Group:14 days Treatment=1.0g NaCl; RAW_FILE_NAME=OilPalm_Salt_10_14DAT_R3_NEG.mzXML SUBJECT_SAMPLE_FACTORS - OilPalm_Salt_20_14DAT_R3_NEG Group:14 days Treatment=2.0g NaCl; RAW_FILE_NAME=OilPalm_Salt_20_14DAT_R3_NEG.mzXML SUBJECT_SAMPLE_FACTORS - OilPalm_Salt_20_14DAT_R4_NEG Group:14 days Treatment=2.0g NaCl; RAW_FILE_NAME=OilPalm_Salt_20_14DAT_R4_NEG.mzXML SUBJECT_SAMPLE_FACTORS - OilPalm_Salt_10_14DAT_R4_NEG Group:14 days Treatment=1.0g NaCl; RAW_FILE_NAME=OilPalm_Salt_10_14DAT_R4_NEG.mzXML SUBJECT_SAMPLE_FACTORS - OilPalm_Salt_05_14DAT_R4_NEG Group:14 days Treatment=0.5g NaCl; RAW_FILE_NAME=OilPalm_Salt_05_14DAT_R4_NEG.mzXML SUBJECT_SAMPLE_FACTORS - OilPalm_Salt_Control_14DAT_R2_NEG Group:14 days Treatment=0.0g NaCl; RAW_FILE_NAME=OilPalm_Salt_Control_14DAT_R2_NEG.mzXML SUBJECT_SAMPLE_FACTORS - OilPalm_Salt_Control_14DAT_R3_NEG Group:14 days Treatment=0.0g NaCl; RAW_FILE_NAME=OilPalm_Salt_Control_14DAT_R3_NEG.mzXML SUBJECT_SAMPLE_FACTORS - OilPalm_Salt_Control_14DAT_R4_NEG Group:14 days Treatment=0.0g NaCl; RAW_FILE_NAME=OilPalm_Salt_Control_14DAT_R4_NEG.mzXML #COLLECTION CO:COLLECTION_SUMMARY The oil palm plants used in this study were clones regenerated out of CO:COLLECTION_SUMMARY embryogenic calluses obtained from the leaves of an adult plant—genotype AM33, CO:COLLECTION_SUMMARY a Deli x Ghana from ASD Costa Rica, as previously reported by [6]. Before CO:COLLECTION_SUMMARY starting the experiments, plants were standardized accordingly to the CO:COLLECTION_SUMMARY developmental stage, size, and number of leaves. They were in the growth stage CO:COLLECTION_SUMMARY known as bifid saplings, and the experiment was performed in March 2018 in a CO:COLLECTION_SUMMARY greenhouse at Embrapa Agroenergy in Brasília, DF, Brazil (S-15.732°, CO:COLLECTION_SUMMARY W-47.900°). The main environmental variables (temperature, humidity, and CO:COLLECTION_SUMMARY radiation) fluctuated according to the weather conditions and underwent CO:COLLECTION_SUMMARY monitoring throughout the entire experimental period using the data collected at CO:COLLECTION_SUMMARY a nearby meteorological station (S-15.789°, W-47.925°). We collected the CO:COLLECTION_SUMMARY apical leaves from control and stressed plants (0.0 and 2.0 g of NaCl per 100 g CO:COLLECTION_SUMMARY of substrate) 12 days after imposition of the treatments (DAT). CO:SAMPLE_TYPE Plant #TREATMENT TR:TREATMENT_SUMMARY The experiment consisted of five salinity levels (0.0, 0.5, 1.0, 1.5, and 2.0 g TR:TREATMENT_SUMMARY of NaCl per 100 g of substrate (a mixture of vermiculite, soil, and the Bioplant TR:TREATMENT_SUMMARY commercial substrate (Bioplant Agrícola Ltd.a., Nova Ponte, MG, Brazil), in a TR:TREATMENT_SUMMARY 1:1:1 ratio, on a dry basis), with four replicates in a completely randomized TR:TREATMENT_SUMMARY design. The substrate mixture was fertilized using 2.5 g L−1 of the N-P2O5-K2O TR:TREATMENT_SUMMARY formula (20-20-20). #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Leaves harvested from control and stressed plants were immediately immersed in SP:SAMPLEPREP_SUMMARY liquid nitrogen and stored at −80 °C until metabolite extraction: four plants SP:SAMPLEPREP_SUMMARY for treatments. Before solvent extraction, all samples underwent grounding in SP:SAMPLEPREP_SUMMARY liquid nitrogen. The solvents used were methanol grade UHPLC, acetonitrile grade SP:SAMPLEPREP_SUMMARY LC-MS, formic acid grade LC-MS, sodium hydroxide ACS grade LC-MS, all from SP:SAMPLEPREP_SUMMARY Sigma-Aldrich, and water treated in a Milli-Q system from Millipore. We employed SP:SAMPLEPREP_SUMMARY a protocol to extract the metabolites in three phases (polar, non-polar, and SP:SAMPLEPREP_SUMMARY protein pellet). Aliquots of 50 mg of ground sample were transferred to 2 mL SP:SAMPLEPREP_SUMMARY microtubes, and then 1 mL of a mixture of 1:3 (v:v) methanol/methyl tert-butyl SP:SAMPLEPREP_SUMMARY ether (MTBE) at −20 °C was added. Homogenization on an orbital shaker at 4.0 SP:SAMPLEPREP_SUMMARY °C and ultrasound treatment in an ice bath were each performed for 10 min. As SP:SAMPLEPREP_SUMMARY the next step, we added 500 μL of a mixture of 1:3 (v:v) methanol/water to each SP:SAMPLEPREP_SUMMARY microtube. After centrifugation (15,300× g at 4.0 °C for 5 min), an upper SP:SAMPLEPREP_SUMMARY non-polar (green) and a lower polar (brown) phase and a protein pellet remained SP:SAMPLEPREP_SUMMARY in each microtube. After transferring both fractions separately to 1.5 mL SP:SAMPLEPREP_SUMMARY microtubes, they were submitted to a Speed vac system (Centrivap, Labconco) to SP:SAMPLEPREP_SUMMARY be vacuum dried. Finally, the dry-fraction, resuspended in 500 μL of 1:3 (v:v) SP:SAMPLEPREP_SUMMARY methanol and water mixture and transferred to vials, were now ready for SP:SAMPLEPREP_SUMMARY analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Solvent A was 0.1% (v:v) formic acid in water and solvent B was 0.1% (v:v) CH:CHROMATOGRAPHY_SUMMARY formic acid in acetonitrile/methanol (70:30, v:v). The gradient elution used, CH:CHROMATOGRAPHY_SUMMARY with a flow rate of 0.4 mL min–1, was as follows: 0–1 min isocratic, 0% B; CH:CHROMATOGRAPHY_SUMMARY 1–3 min, 5% B; 3–10 min, 50% B; 10–13 min, 100% B; 13–15 min isocratic, CH:CHROMATOGRAPHY_SUMMARY 100% B; then, 5 min rebalancing was conducted to the initial conditions. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Shimadzu Nexera X2 CH:COLUMN_NAME Waters Acquity BEH HSS T3 (100 x 2.1mm, 1.8um) CH:SOLVENT_A 100% water; 0.1% formic acid CH:SOLVENT_B 70% acetonitrile/30% methanol; 0.1% formic acid CH:FLOW_GRADIENT 0–1 min isocratic, 0% B; 1–3 min, 5% B; 3–10 min, 50% B; 10–13 min, 100% CH:FLOW_GRADIENT B; 13–15 min isocratic, 100% B; then, 5 min rebalancing was conducted to the CH:FLOW_GRADIENT initial conditions. CH:FLOW_RATE 0.4 mL/min 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 The rate of acquisition spectra was 3.00 Hz, mass range m/z 70–1200 for the MS:MS_COMMENTS polar fraction analysis and m/z 300–1600 for the lipidic fraction. MS:MS_COMMENTS High-resolution mass spectrometry was used for detection (MaXis 4G Q-TOF MS, MS:MS_COMMENTS Bruker Daltonics) equipped with an electrospray source in positive (ESI-(+)-MS) MS:MS_COMMENTS and negative (ESI-(−)-MS) modes. The settings of the mass spectrometer were as MS:MS_COMMENTS follows: capillary voltage, 3800 V; dry gas flow, 9 L min−1; dry temperature, MS:MS_COMMENTS 200 °C; nebulizer pressure, 4 bar; final plate offset, 500 V. For the external MS:MS_COMMENTS calibration of the equipment, we used a sodium formate solution (10 mM HCOONa MS:MS_COMMENTS solution in 50:50 v:v isopropanol and water containing 0.2% formic acid) MS:MS_COMMENTS injected through a six-way valve at the beginning of each chromatographic run. MS:MS_COMMENTS Ampicillin ([M+H] + m/z 350.1186729 and [M-H]- m/z 348.1028826) was the internal MS:MS_COMMENTS standard for later peak normalization on data analysis. MS:MS_RESULTS_FILE ST002429_AN003953_Results.txt UNITS:Peak intensity Has m/z:Yes Has RT:No RT units:No RT data #END