#METABOLOMICS WORKBENCH Cristina_20220616_023641 DATATRACK_ID:3307 STUDY_ID:ST002198 ANALYSIS_ID:AN003598 PROJECT_ID:PR001401 VERSION 1 CREATED_ON June 16, 2022, 6:45 am #PROJECT PR:PROJECT_TITLE Untargeted metabolomics revealed essential biochemical rearrangements towards PR:PROJECT_TITLE heat x drought stress acclimatization in Pinus pinaster PR:PROJECT_TYPE LC-MS analysis PR:PROJECT_SUMMARY Current projections for global climate change predict an increase in the PR:PROJECT_SUMMARY intensity and frequency of heat waves and droughts. The improvement in our PR:PROJECT_SUMMARY understanding of the mechanisms of how trees precisely can predict environmental PR:PROJECT_SUMMARY threats and cope with these stresses benefits our natural selection or genetic PR:PROJECT_SUMMARY improvement to the maintenance of forest sustainability. In this work, we PR:PROJECT_SUMMARY investigate the metabolic changes in heat and drought combined stress in Pinus PR:PROJECT_SUMMARY pinaster plantlets. Maritime pine is a coniferous tree with native populations PR:PROJECT_SUMMARY distributed across the European Atlantic and Mediterranean basins and the north PR:PROJECT_SUMMARY of Africa ranging from cool moist to warm dry climates. This species shows high PR:PROJECT_SUMMARY plasticity and a contrasting adaptive capacity and resilience. This plasticity PR:PROJECT_SUMMARY in the response to stress exposure may be associated with a differential ability PR:PROJECT_SUMMARY to modulate their secondary metabolism. For this reason, the current study aims PR:PROJECT_SUMMARY to investigate the gradual and synergetic metabolomic response using liquid PR:PROJECT_SUMMARY chromatography coupled to mass spectrometry (LC-MS) based on untargeted PR:PROJECT_SUMMARY metabolomic profiling of four stress levels. These metabolic profiles were PR:PROJECT_SUMMARY supported by physiological and biochemical determinations. Our results showed PR:PROJECT_SUMMARY that the metabolic profiles induced by low-stress exposition represent an PR:PROJECT_SUMMARY adaptive conditioning mode with metabolome changes that help seedlings to cope PR:PROJECT_SUMMARY with upcoming stress. The metabolism pathways involved in this response were PR:PROJECT_SUMMARY mainly included in amino acid metabolism and carbohydrate metabolism leading to PR:PROJECT_SUMMARY an enhanced accumulation of phenolics, flavonoids, and terpenoids. However, when PR:PROJECT_SUMMARY the plantlets were exposed to higher-stress exposition, the secondary PR:PROJECT_SUMMARY metabolites that starred the response are more complex and decorated, such as PR:PROJECT_SUMMARY alkaloids, lignans, and glycosyloxyflavones. Those changes could help to PR:PROJECT_SUMMARY maintain homeostasis and control the response magnitude on establishing and PR:PROJECT_SUMMARY facilitating the plantlets’ survival. Overall, our findings provide new PR:PROJECT_SUMMARY insights into the responsive mechanisms of the maritime pine under heat and PR:PROJECT_SUMMARY drought stress in terms of metabolic profiles. PR:INSTITUTE Universidad de Oviedo PR:DEPARTMENT Department of Organisms and Systems Biology PR:LABORATORY Plant Physiology PR:LAST_NAME López Hidalgo PR:FIRST_NAME Cristina PR:ADDRESS C/ Catedrático Rodrigo Uría s/n Oviedo 33071 PR:EMAIL lopezhcristina@uniovi.es PR:PHONE 985104774 PR:FUNDING_SOURCE This work is an output of the projects financed by the Spanish Ministry of PR:FUNDING_SOURCE Economy, Industry, and Competitiveness (AGL2017-83988-R) #STUDY ST:STUDY_TITLE Untargeted metabolomics of Pinus pinaster needles under heat and drought stress ST:STUDY_TYPE Untargeted MS-based metabolomics ST:STUDY_SUMMARY Current projections for global climate change predict an increase in the ST:STUDY_SUMMARY intensity and frequency of heat waves and droughts. The improvement in our ST:STUDY_SUMMARY understanding of the mechanisms of how trees precisely can predict environmental ST:STUDY_SUMMARY threats and cope with these stresses benefits our natural selection or genetic ST:STUDY_SUMMARY improvement to the maintenance of forest sustainability. In this work, we ST:STUDY_SUMMARY investigate the metabolic changes in heat and drought combined stress in Pinus ST:STUDY_SUMMARY pinaster plantlets. Maritime pine is a coniferous tree with native populations ST:STUDY_SUMMARY distributed across the European Atlantic and Mediterranean basins and the north ST:STUDY_SUMMARY of Africa ranging from cool moist to warm dry climates. This species shows high ST:STUDY_SUMMARY plasticity and a contrasting adaptive capacity and resilience. This plasticity ST:STUDY_SUMMARY in the response to stress exposure may be associated with a differential ability ST:STUDY_SUMMARY to modulate their secondary metabolism. For this reason, the current study aims ST:STUDY_SUMMARY to investigate the gradual and synergetic metabolomic response using liquid ST:STUDY_SUMMARY chromatography coupled to mass spectrometry (LC-MS) based on untargeted ST:STUDY_SUMMARY metabolomic profiling of four stress levels. These metabolic profiles were ST:STUDY_SUMMARY supported by physiological and biochemical determinations. Our results showed ST:STUDY_SUMMARY that the metabolic profiles induced by low-stress exposition represent an ST:STUDY_SUMMARY adaptive conditioning mode with metabolome changes that help seedlings to cope ST:STUDY_SUMMARY with upcoming stress. The metabolism pathways involved in this response were ST:STUDY_SUMMARY mainly included in amino acid metabolism and carbohydrate metabolism leading to ST:STUDY_SUMMARY an enhanced accumulation of phenolics, flavonoids, and terpenoids. However, when ST:STUDY_SUMMARY the plantlets were exposed to higher-stress exposition, the secondary ST:STUDY_SUMMARY metabolites that starred the response are more complex and decorated, such as ST:STUDY_SUMMARY alkaloids, lignans, and glycosyloxyflavones. Those changes could help to ST:STUDY_SUMMARY maintain homeostasis and control the response magnitude on establishing and ST:STUDY_SUMMARY facilitating the plantlets’ survival. Overall, our findings provide new ST:STUDY_SUMMARY insights into the responsive mechanisms of the maritime pine under heat and ST:STUDY_SUMMARY drought stress in terms of metabolic profiles. ST:INSTITUTE Universidad de Oviedo ST:DEPARTMENT Department of Organisms and Systems Biology ST:LABORATORY Plant Physiology ST:LAST_NAME López Hidalgo ST:FIRST_NAME Cristina ST:ADDRESS C/ Catedrático Rodrigo Uría s/n Oviedo 33071 ST:EMAIL lopezhcristina@uniovi.es ST:PHONE 985104774 #SUBJECT SU:SUBJECT_TYPE Plant SU:SUBJECT_SPECIES Pinus pinaster SU:TAXONOMY_ID 71647 SU:AGE_OR_AGE_RANGE one-two years #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 - T0.30.WWC1_pos Factor:T0.30.WWC RAW_FILE_NAME=T0.30.WWC1_pos.mzXML SUBJECT_SAMPLE_FACTORS - T0.30.WWC2_pos Factor:T0.30.WWC RAW_FILE_NAME=T0.30.WWC2_pos.mzXML SUBJECT_SAMPLE_FACTORS - T0.30.WWC3_pos Factor:T0.30.WWC RAW_FILE_NAME=T0.30.WWC3_pos.mzXML SUBJECT_SAMPLE_FACTORS - T0.30.WWC4_pos Factor:T0.30.WWC RAW_FILE_NAME=T0.30.WWC4_pos.mzXML SUBJECT_SAMPLE_FACTORS - T0.30.WWC5_pos Factor:T0.30.WWC RAW_FILE_NAME=T0.30.WWC5_pos.mzXML SUBJECT_SAMPLE_FACTORS - 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T3.40.HWS2_neg Factor:T3.40.HWS RAW_FILE_NAME=T3.40.HWS2_neg.mzXML SUBJECT_SAMPLE_FACTORS - T3.40.LWS2_neg Factor:T3.40.LWS RAW_FILE_NAME=T3.40.LWS2_neg.mzXML SUBJECT_SAMPLE_FACTORS - T3.40.HWS3_neg Factor:T3.40.HWS RAW_FILE_NAME=T3.40.HWS3_neg.mzXML SUBJECT_SAMPLE_FACTORS - T3.40.LWS3_neg Factor:T3.40.LWS RAW_FILE_NAME=T3.40.LWS3_neg.mzXML SUBJECT_SAMPLE_FACTORS - T5.40.HWS1_neg Factor:T5.40.HWS RAW_FILE_NAME=T5.40.HWS1_neg.mzXML SUBJECT_SAMPLE_FACTORS - T5.40.LWS1_neg Factor:T5.40.LWS RAW_FILE_NAME=T5.40.LWS1_neg.mzXML SUBJECT_SAMPLE_FACTORS - T5.40.HWS2_neg Factor:T5.40.HWS RAW_FILE_NAME=T5.40.HWS2_neg.mzXML SUBJECT_SAMPLE_FACTORS - T5.40.LWS2_neg Factor:T5.40.LWS RAW_FILE_NAME=T5.40.LWS2_neg.mzXML SUBJECT_SAMPLE_FACTORS - T5.40.HWS3_neg Factor:T5.40.HWS RAW_FILE_NAME=T5.40.HWS3_neg.mzXML SUBJECT_SAMPLE_FACTORS - T5.40.LWS3_neg Factor:T5.40.LWS RAW_FILE_NAME=T5.40.LWS3_neg.mzXML SUBJECT_SAMPLE_FACTORS - T7.40.HWS1_neg Factor:T7.40.HWS RAW_FILE_NAME=T7.40.HWS1_neg.mzXML SUBJECT_SAMPLE_FACTORS - T7.40.LWS1_neg Factor:T7.40.LWS RAW_FILE_NAME=T7.40.LWS1_neg.mzXML SUBJECT_SAMPLE_FACTORS - T7.40.HWS2_neg Factor:T7.40.HWS RAW_FILE_NAME=T7.40.HWS2_neg.mzXML SUBJECT_SAMPLE_FACTORS - T7.40.LWS2_neg Factor:T7.40.LWS RAW_FILE_NAME=T7.40.LWS2_neg.mzXML SUBJECT_SAMPLE_FACTORS - T7.40.HWS3_neg Factor:T7.40.HWS RAW_FILE_NAME=T7.40.HWS3_neg.mzXML SUBJECT_SAMPLE_FACTORS - T7.40.LWS3_neg Factor:T7.40.LWS RAW_FILE_NAME=T7.40.LWS3_neg.mzXML #COLLECTION CO:COLLECTION_SUMMARY Plantlets were sampled on the first-day assay (T0) under well-watered conditions CO:COLLECTION_SUMMARY before the temperature change in both chambers. The following day to T0, the CO:COLLECTION_SUMMARY exposure to combined stress began. HWS plantlets were watering with 25 % of the CO:COLLECTION_SUMMARY weight loss each day, while LWS plantlets with 50 %. Afterward, heat-stressed CO:COLLECTION_SUMMARY and water-stressed plantlets were sampled at the end of the 6-h heat exposure on CO:COLLECTION_SUMMARY day 1 (T1), day 3 (T3), day 5 (T5), and day 7 (T7). The water deficit more or CO:COLLECTION_SUMMARY less severe was imposed for seven days by progressively depleting soil water CO:COLLECTION_SUMMARY content. Immediately after sampling, cell membrane damage and leaf water status CO:COLLECTION_SUMMARY were measured in fresh needles by quantifying relative EL and RWC (see below). CO:COLLECTION_SUMMARY Other needles were frozen in liquid nitrogen, lyophilized, and stored in the CO:COLLECTION_SUMMARY dark and cold (-20 ºC) until use. CO:SAMPLE_TYPE Plant CO:STORAGE_CONDITIONS -20℃ #TREATMENT TR:TREATMENT_SUMMARY The experimental layout was based on a factorial design with two factors: TR:TREATMENT_SUMMARY temperature and water availability. Before starting the experiment, plants were TR:TREATMENT_SUMMARY divided into two chambers for testing two temperatures (30 ºC and 40 ºC), TR:TREATMENT_SUMMARY which in turn were split again into two levels of water availability TR:TREATMENT_SUMMARY (“low-water-stress”, LWS, and “high-water-stress”, HWS). Consequently, TR:TREATMENT_SUMMARY four stress levels, 40 ºC-HWS, 40 ºC-LWS, 30 ºC-HWS, and 30 ºC-LWS were TR:TREATMENT_SUMMARY tested. In both chambers, twelve plants were divided into six pools of two TR:TREATMENT_SUMMARY plants (three for HWS and three for LWS). These pools of two plants were kept TR:TREATMENT_SUMMARY across the sampling and formed the three independent biological replicates TR:TREATMENT_SUMMARY analyzed for each stress level. TR:TREATMENT Heat and Drought TR:TREATMENT_DOSE High Water Stress and Low Water Stress in 30ºC and 40ºC. TR:TREATMENT_VEHICLE Fitoclima 1200, Aralab Ltd, Sintra, Portugal TR:PLANT_GROWTH_SUPPORT Fitoclima 1200, Aralab Ltd, Sintra, Portugal TR:PLANT_GROWTH_LOCATION Oviedo, Asturias TR:PLANT_PLOT_DESIGN Randomized design TR:PLANT_LIGHT_PERIOD During this month, the plants in the growth chamber (Fitoclima 1200, Aralab Ltd, TR:PLANT_LIGHT_PERIOD Sintra, Portugal) were kept at a light intensity of 400 µmol m−2·s−1 under TR:PLANT_LIGHT_PERIOD long-day conditions (16 h light/8 h dark for photoperiod). TR:PLANT_HUMIDITY Relative humidity (RH) were set to 25 ºC and 50 % RH during the day, and 15 ºC TR:PLANT_HUMIDITY and 60 % RH during the night TR:PLANT_TEMP 25ºC, 30ºC, and 40ºC TR:PLANT_WATERING_REGIME Plants were well-watered to field capacity until soil dropped every two days. TR:PLANT_NUTRITIONAL_REGIME efore trial, seedlings had been acclimated over one month inside the chamber and TR:PLANT_NUTRITIONAL_REGIME were watered to field capacity with nutritive solution (N:P:K; 5:8:10). TR:PLANT_GROWTH_STAGE Two-year-old seedlings TR:PLANT_METAB_QUENCH_METHOD Liquid N2 TR:PLANT_HARVEST_METHOD Liquid N2 TR:PLANT_STORAGE Lyophilized #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Metabolites were extracted from 20 mg (lyophilized weight) of needles. SP:SAMPLEPREP_SUMMARY Metabolite extraction was performed according to Valledor et al., (2014). SP:SAMPLEPREP_SUMMARY Briefly, 600 µL of cold (4 ºC) metabolite extraction solution (methanol: SP:SAMPLEPREP_SUMMARY chloroform: H2O (2.5:1:0.5) was added to each tube and strongly vortexed. Then, SP:SAMPLEPREP_SUMMARY the tubes were incubated in a cold ultrasound bath for 10 min. Later, the tubes SP:SAMPLEPREP_SUMMARY were centrifuged at 20.000 x g for 6 min at 4 °C. The supernatant containing SP:SAMPLEPREP_SUMMARY metabolites from each tube was transferred to a new tube containing 300 µL of SP:SAMPLEPREP_SUMMARY chloroform: water (1:1) to allow phase separation. Six hundred µL of cold (4 SP:SAMPLEPREP_SUMMARY ºC) metabolite extraction solution were added to the remaining pellets, and SP:SAMPLEPREP_SUMMARY vortexing, ultrasound bath, and centrifugation were repeated. The new SP:SAMPLEPREP_SUMMARY supernatant was transferred to the previous tube that contained the phase SP:SAMPLEPREP_SUMMARY separation solution and the old supernatant. These tubes were vortexed and then SP:SAMPLEPREP_SUMMARY centrifuged at 15.000 x g for 5 min at 4 °C. After centrifugation, two layers SP:SAMPLEPREP_SUMMARY are formed; the upper-aqueous layer (methanol: water) containing the polar SP:SAMPLEPREP_SUMMARY metabolites was transferred to a separate microcentrifuge tube and then cleaned SP:SAMPLEPREP_SUMMARY from non-polar metabolites adding 300 µL of cold (4 ºC) chloroform: water SP:SAMPLEPREP_SUMMARY (1:1), vortexed, and centrifuged at 15.000 x g for 4 min at 4 °C. The new upper SP:SAMPLEPREP_SUMMARY phase was transferred to a new tube. The polar extract was dried using a SP:SAMPLEPREP_SUMMARY speedvac at 25 °C. SP:PROCESSING_METHOD Methanol:Chloroform:Water SP:PROCESSING_STORAGE_CONDITIONS On ice SP:EXTRACTION_METHOD Methanol:Chloroform:Water SP:EXTRACT_ENRICHMENT Polar metabolites SP:EXTRACT_CLEANUP Centrifugation SP:EXTRACT_STORAGE -80℃ SP:SAMPLE_RESUSPENSION Methanol SP:SAMPLE_DERIVATIZATION NO SP:SAMPLE_SPIKING NO #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY A fifty-three-minute mobile phase gradient was employed. Gradient elution CH:CHROMATOGRAPHY_SUMMARY chromatography was performed starting with 100 % A to 98 % A in 1 min; hold for CH:CHROMATOGRAPHY_SUMMARY 9 min, gradient to 60 % A in 21 min, gradient to 45 % A in 5 min; hold for 2 CH:CHROMATOGRAPHY_SUMMARY min, gradient to 5 % A in 3 min; hold 5 min, return to initial conditions in 3 CH:CHROMATOGRAPHY_SUMMARY min and equilibrate for 5 min (total run time: 53 min). Solvent A was 100 % H2O CH:CHROMATOGRAPHY_SUMMARY containing 0.1 % formic acid, and solvent B was 100 % ACN containing 0.1 % CH:CHROMATOGRAPHY_SUMMARY formic acid. A flow rate of 0.1 mL/min was used. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 CH:COLUMN_NAME Phenomenex Luna Omega Polar C18 column (1.7 µm, 100 x 2.1 mm) CH:FLOW_GRADIENT Gradient elution chromatography was performed starting with 100 % A to 98 % A in CH:FLOW_GRADIENT 1 min; hold for 9 min, gradient to 60 % A in 21 min, gradient to 45 % A in 5 CH:FLOW_GRADIENT min; hold for 2 min, gradient to 5 % A in 3 min; hold 5 min, return to initial CH:FLOW_GRADIENT conditions in 3 min and equilibrate for 5 min (total run time: 53 min) CH:FLOW_RATE 0.1 mL/min CH:COLUMN_TEMPERATURE 30 ºC CH:SOLVENT_A 100 % H2O containing 0.1 % formic acid CH:SOLVENT_B 100 % ACN containing 0.1 % formic acid CH:RETENTION_TIME 53 min CH:SAMPLE_INJECTION 5 uL CH:CAPILLARY_VOLTAGE 4.5 kV CH:WASHING_BUFFER IPA and Methanol CH:RANDOMIZATION_ORDER True #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME Severo Ochoa AN:DATA_FORMAT .D #MS MS:INSTRUMENT_NAME Bruker Impact II HD MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS The column eluent was analyzed using a Bruker Impact II HD (Bruker, Karlsruhe, MS:MS_COMMENTS Germany) quadrupole time-of-flight (Q-TOF) mass spectrometer equipped with an MS:MS_COMMENTS ESI source operating in positive polarity and negative polarity. Mass spectra MS:MS_COMMENTS were acquired with the following parameters of mass spectrometer: ion capillary MS:MS_COMMENTS voltage 4.5 kV (same for positive and negative mode), dry gas flow 6 L/min, dry MS:MS_COMMENTS gas temperature 250 ºC, nebulizer pressure 2 bar, collision RF 650 V, transfer MS:MS_COMMENTS time 80 µs and prepulse storage 5 µs. Spectra data, MS1 and MS2, were acquired MS:MS_COMMENTS in a data-dependent manner at 2 Hz, fragmenting the three most abundant MS:MS_COMMENTS precursor ions per MS1 scan, acquiring MS/MS data between 50 and 1300 m/z. MS:MS_COMMENTS Repetitive MS/MS sampling was limited by exclusion after 3 spectra at a MS:MS_COMMENTS particular mass within a window of 0.2 min. MS/MS fragmentation of the 3 most MS:MS_COMMENTS intense selected ions per spectrum was performed using ramped collision-induced MS:MS_COMMENTS dissociation energy of 7–17.5 eV. Hexakis (1H, 1H, 3H-tetrafluoropropoxy) MS:MS_COMMENTS phosphazene (Agilent Technologies, Santa Clara, CA, USA) was introduced as an MS:MS_COMMENTS internal calibrant after the run ends . Each sample was analyzed twice, first MS:MS_COMMENTS using the positive ion mode and then the negative. MS:CAPILLARY_VOLTAGE 4.5 kV MS:COLLISION_ENERGY ramped collision-induced dissociation energy of 7–17.5 eV MS:DRY_GAS_FLOW 6 L/min MS:DRY_GAS_TEMP 250 ºC MS:GAS_PRESSURE 2 bar MS:MS_RESULTS_FILE ST002198_AN003598_Results.txt UNITS:peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END