Summary of Study ST002198

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001401. The data can be accessed directly via it's Project DOI: 10.21228/M8MT48 This work is supported by NIH grant, U2C- DK119886.

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This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST002198
Study TitleUntargeted metabolomics of Pinus pinaster needles under heat and drought stress
Study TypeUntargeted MS-based metabolomics
Study SummaryCurrent projections for global climate change predict an increase in the intensity and frequency of heat waves and droughts. The improvement in our understanding of the mechanisms of how trees precisely can predict environmental threats and cope with these stresses benefits our natural selection or genetic improvement to the maintenance of forest sustainability. In this work, we investigate the metabolic changes in heat and drought combined stress in Pinus pinaster plantlets. Maritime pine is a coniferous tree with native populations distributed across the European Atlantic and Mediterranean basins and the north of Africa ranging from cool moist to warm dry climates. This species shows high plasticity and a contrasting adaptive capacity and resilience. This plasticity in the response to stress exposure may be associated with a differential ability to modulate their secondary metabolism. For this reason, the current study aims to investigate the gradual and synergetic metabolomic response using liquid chromatography coupled to mass spectrometry (LC-MS) based on untargeted metabolomic profiling of four stress levels. These metabolic profiles were supported by physiological and biochemical determinations. Our results showed that the metabolic profiles induced by low-stress exposition represent an adaptive conditioning mode with metabolome changes that help seedlings to cope with upcoming stress. The metabolism pathways involved in this response were mainly included in amino acid metabolism and carbohydrate metabolism leading to an enhanced accumulation of phenolics, flavonoids, and terpenoids. However, when the plantlets were exposed to higher-stress exposition, the secondary metabolites that starred the response are more complex and decorated, such as alkaloids, lignans, and glycosyloxyflavones. Those changes could help to maintain homeostasis and control the response magnitude on establishing and facilitating the plantlets’ survival. Overall, our findings provide new insights into the responsive mechanisms of the maritime pine under heat and drought stress in terms of metabolic profiles.
Institute
Universidad de Oviedo
DepartmentDepartment of Organisms and Systems Biology
LaboratoryPlant Physiology
Last NameLópez Hidalgo
First NameCristina
AddressC/ Catedrático Rodrigo Uría s/n Oviedo 33071
Emaillopezhcristina@uniovi.es
Phone985104774
Submit Date2022-06-16
Raw Data AvailableYes
Raw Data File Type(s)mzXML
Analysis Type DetailLC-MS
Release Date2022-07-14
Release Version1
Cristina López Hidalgo Cristina López Hidalgo
https://dx.doi.org/10.21228/M8MT48
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:

Project:

Project ID:PR001401
Project DOI:doi: 10.21228/M8MT48
Project Title:Untargeted metabolomics revealed essential biochemical rearrangements towards heat x drought stress acclimatization in Pinus pinaster
Project Type:LC-MS analysis
Project Summary:Current projections for global climate change predict an increase in the intensity and frequency of heat waves and droughts. The improvement in our understanding of the mechanisms of how trees precisely can predict environmental threats and cope with these stresses benefits our natural selection or genetic improvement to the maintenance of forest sustainability. In this work, we investigate the metabolic changes in heat and drought combined stress in Pinus pinaster plantlets. Maritime pine is a coniferous tree with native populations distributed across the European Atlantic and Mediterranean basins and the north of Africa ranging from cool moist to warm dry climates. This species shows high plasticity and a contrasting adaptive capacity and resilience. This plasticity in the response to stress exposure may be associated with a differential ability to modulate their secondary metabolism. For this reason, the current study aims to investigate the gradual and synergetic metabolomic response using liquid chromatography coupled to mass spectrometry (LC-MS) based on untargeted metabolomic profiling of four stress levels. These metabolic profiles were supported by physiological and biochemical determinations. Our results showed that the metabolic profiles induced by low-stress exposition represent an adaptive conditioning mode with metabolome changes that help seedlings to cope with upcoming stress. The metabolism pathways involved in this response were mainly included in amino acid metabolism and carbohydrate metabolism leading to an enhanced accumulation of phenolics, flavonoids, and terpenoids. However, when the plantlets were exposed to higher-stress exposition, the secondary metabolites that starred the response are more complex and decorated, such as alkaloids, lignans, and glycosyloxyflavones. Those changes could help to maintain homeostasis and control the response magnitude on establishing and facilitating the plantlets’ survival. Overall, our findings provide new insights into the responsive mechanisms of the maritime pine under heat and drought stress in terms of metabolic profiles.
Institute:Universidad de Oviedo
Department:Department of Organisms and Systems Biology
Laboratory:Plant Physiology
Last Name:López Hidalgo
First Name:Cristina
Address:C/ Catedrático Rodrigo Uría s/n Oviedo 33071
Email:lopezhcristina@uniovi.es
Phone:985104774
Funding Source:This work is an output of the projects financed by the Spanish Ministry of Economy, Industry, and Competitiveness (AGL2017-83988-R)

Subject:

Subject ID:SU002284
Subject Type:Plant
Subject Species:Pinus pinaster
Taxonomy ID:71647
Age Or Age Range:one-two years
Species Group:Plants

Factors:

Subject type: Plant; Subject species: Pinus pinaster (Factor headings shown in green)

mb_sample_id local_sample_id Factor
SA210588T0.30.WWC3_negT0.30.WWC
SA210589T0.30.WWC2_negT0.30.WWC
SA210590T0.30.WWC4_negT0.30.WWC
SA210591T0.30.WWC5_negT0.30.WWC
SA210592T0.30.WWC6_negT0.30.WWC
SA210593T0.30.WWC1_posT0.30.WWC
SA210594T0.30.WWC1_negT0.30.WWC
SA210595T0.30.WWC5_posT0.30.WWC
SA210596T0.30.WWC3_posT0.30.WWC
SA210597T0.30.WWC2_posT0.30.WWC
SA210598T0.30.WWC6_posT0.30.WWC
SA210599T0.30.WWC4_posT0.30.WWC
SA210600T0.40.WWC1_posT0.40.WWC
SA210601T0.40.WWC2_posT0.40.WWC
SA210602T0.40.WWC3_posT0.40.WWC
SA210603T0.40.WWC4_posT0.40.WWC
SA210604T0.40.WWC5_posT0.40.WWC
SA210605T0.40.WWC1_negT0.40.WWC
SA210606T0.40.WWC2_negT0.40.WWC
SA210607T0.40.WWC6_negT0.40.WWC
SA210608T0.40.WWC5_negT0.40.WWC
SA210609T0.40.WWC4_negT0.40.WWC
SA210610T0.40.WWC3_negT0.40.WWC
SA210611T0.40.WWC6_posT0.40.WWC
SA210612T1.30.HWS2_negT1.30.HWS
SA210613T1.30.HWS3_negT1.30.HWS
SA210614T1.30.HWS3_posT1.30.HWS
SA210615T1.30.HWS2_posT1.30.HWS
SA210616T1.30.HWS1_posT1.30.HWS
SA210617T1.30.HWS1_negT1.30.HWS
SA210618T1.30.LWS3_posT1.30.LWS
SA210619T1.30.LWS1_posT1.30.LWS
SA210620T1.30.LWS1_negT1.30.LWS
SA210621T1.30.LWS2_negT1.30.LWS
SA210622T1.30.LWS2_posT1.30.LWS
SA210623T1.30.LWS3_negT1.30.LWS
SA210624T1.40.HWS1_negT1.40.HWS
SA210625T1.40.HWS2_negT1.40.HWS
SA210626T1.40.HWS3_posT1.40.HWS
SA210627T1.40.HWS2_posT1.40.HWS
SA210628T1.40.HWS1_posT1.40.HWS
SA210629T1.40.HWS3_negT1.40.HWS
SA210630T1.40.LWS3_negT1.40.LWS
SA210631T1.40.LWS2_negT1.40.LWS
SA210632T1.40.LWS1_posT1.40.LWS
SA210633T1.40.LWS3_posT1.40.LWS
SA210634T1.40.LWS2_posT1.40.LWS
SA210635T1.40.LWS1_negT1.40.LWS
SA210636T3.30.HWS1_negT3.30.HWS
SA210637T3.30.HWS1_posT3.30.HWS
SA210638T3.30.HWS2_posT3.30.HWS
SA210639T3.30.HWS3_posT3.30.HWS
SA210640T3.30.HWS3_negT3.30.HWS
SA210641T3.30.HWS2_negT3.30.HWS
SA210642T3.30.LWS2_posT3.30.LWS
SA210643T3.30.LWS1_posT3.30.LWS
SA210644T3.30.LWS3_negT3.30.LWS
SA210645T3.30.LWS1_negT3.30.LWS
SA210646T3.30.LWS3_posT3.30.LWS
SA210647T3.30.LWS2_negT3.30.LWS
SA210648T3.40.HWS1_posT3.40.HWS
SA210649T3.40.HWS2_posT3.40.HWS
SA210650T3.40.HWS3_posT3.40.HWS
SA210651T3.40.HWS2_negT3.40.HWS
SA210652T3.40.HWS1_negT3.40.HWS
SA210653T3.40.HWS3_negT3.40.HWS
SA210654T3.40.LWS1_posT3.40.LWS
SA210655T3.40.LWS3_negT3.40.LWS
SA210656T3.40.LWS2_negT3.40.LWS
SA210657T3.40.LWS1_negT3.40.LWS
SA210658T3.40.LWS3_posT3.40.LWS
SA210659T3.40.LWS2_posT3.40.LWS
SA210660T5.30.HWS3_negT5.30.HWS
SA210661T5.30.HWS1_negT5.30.HWS
SA210662T5.30.HWS2_negT5.30.HWS
SA210663T5.30.HWS2_posT5.30.HWS
SA210664T5.30.HWS3_posT5.30.HWS
SA210665T5.30.HWS1_posT5.30.HWS
SA210666T5.30.LWS3_negT5.30.LWS
SA210667T5.30.LWS2_negT5.30.LWS
SA210668T5.30.LWS1_posT5.30.LWS
SA210669T5.30.LWS3_posT5.30.LWS
SA210670T5.30.LWS1_negT5.30.LWS
SA210671T5.30.LWS2_posT5.30.LWS
SA210672T5.40.HWS3_posT5.40.HWS
SA210673T5.40.HWS1_negT5.40.HWS
SA210674T5.40.HWS2_posT5.40.HWS
SA210675T5.40.HWS2_negT5.40.HWS
SA210676T5.40.HWS1_posT5.40.HWS
SA210677T5.40.HWS3_negT5.40.HWS
SA210678T5.40.LWS1_negT5.40.LWS
SA210679T5.40.LWS1_posT5.40.LWS
SA210680T5.40.LWS3_negT5.40.LWS
SA210681T5.40.LWS3_posT5.40.LWS
SA210682T5.40.LWS2_posT5.40.LWS
SA210683T5.40.LWS2_negT5.40.LWS
SA210684T7.30.HWS1_negT7.30.HWS
SA210685T7.30.HWS3_negT7.30.HWS
SA210686T7.30.HWS2_posT7.30.HWS
SA210687T7.30.HWS3_posT7.30.HWS
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Collection:

Collection ID:CO002277
Collection Summary:Plantlets were sampled on the first-day assay (T0) under well-watered conditions before the temperature change in both chambers. The following day to T0, the exposure to combined stress began. HWS plantlets were watering with 25 % of the weight loss each day, while LWS plantlets with 50 %. Afterward, heat-stressed and water-stressed plantlets were sampled at the end of the 6-h heat exposure on day 1 (T1), day 3 (T3), day 5 (T5), and day 7 (T7). The water deficit more or less severe was imposed for seven days by progressively depleting soil water content. Immediately after sampling, cell membrane damage and leaf water status were measured in fresh needles by quantifying relative EL and RWC (see below). Other needles were frozen in liquid nitrogen, lyophilized, and stored in the dark and cold (-20 ºC) until use.
Sample Type:Plant
Storage Conditions:-20℃

Treatment:

Treatment ID:TR002296
Treatment Summary:The experimental layout was based on a factorial design with two factors: temperature and water availability. Before starting the experiment, plants were divided into two chambers for testing two temperatures (30 ºC and 40 ºC), which in turn were split again into two levels of water availability (“low-water-stress”, LWS, and “high-water-stress”, HWS). Consequently, four stress levels, 40 ºC-HWS, 40 ºC-LWS, 30 ºC-HWS, and 30 ºC-LWS were tested. In both chambers, twelve plants were divided into six pools of two plants (three for HWS and three for LWS). These pools of two plants were kept across the sampling and formed the three independent biological replicates analyzed for each stress level.
Treatment:Heat and Drought
Treatment Dose:High Water Stress and Low Water Stress in 30ºC and 40ºC.
Treatment Vehicle:Fitoclima 1200, Aralab Ltd, Sintra, Portugal
Plant Growth Support:Fitoclima 1200, Aralab Ltd, Sintra, Portugal
Plant Growth Location:Oviedo, Asturias
Plant Plot Design:Randomized design
Plant Light Period:During this month, the plants in the growth chamber (Fitoclima 1200, Aralab Ltd, Sintra, Portugal) were kept at a light intensity of 400 µmol m−2·s−1 under long-day conditions (16 h light/8 h dark for photoperiod).
Plant Humidity:Relative humidity (RH) were set to 25 ºC and 50 % RH during the day, and 15 ºC and 60 % RH during the night
Plant Temp:25ºC, 30ºC, and 40ºC
Plant Watering Regime:Plants were well-watered to field capacity until soil dropped every two days.
Plant Nutritional Regime:efore trial, seedlings had been acclimated over one month inside the chamber and were watered to field capacity with nutritive solution (N:P:K; 5:8:10).
Plant Growth Stage:Two-year-old seedlings
Plant Metab Quench Method:Liquid N2
Plant Harvest Method:Liquid N2
Plant Storage:Lyophilized

Sample Preparation:

Sampleprep ID:SP002290
Sampleprep Summary:Metabolites were extracted from 20 mg (lyophilized weight) of needles. Metabolite extraction was performed according to Valledor et al., (2014). Briefly, 600 µL of cold (4 ºC) metabolite extraction solution (methanol: chloroform: H2O (2.5:1:0.5) was added to each tube and strongly vortexed. Then, the tubes were incubated in a cold ultrasound bath for 10 min. Later, the tubes were centrifuged at 20.000 x g for 6 min at 4 °C. The supernatant containing metabolites from each tube was transferred to a new tube containing 300 µL of chloroform: water (1:1) to allow phase separation. Six hundred µL of cold (4 ºC) metabolite extraction solution were added to the remaining pellets, and vortexing, ultrasound bath, and centrifugation were repeated. The new supernatant was transferred to the previous tube that contained the phase separation solution and the old supernatant. These tubes were vortexed and then centrifuged at 15.000 x g for 5 min at 4 °C. After centrifugation, two layers are formed; the upper-aqueous layer (methanol: water) containing the polar metabolites was transferred to a separate microcentrifuge tube and then cleaned from non-polar metabolites adding 300 µL of cold (4 ºC) chloroform: water (1:1), vortexed, and centrifuged at 15.000 x g for 4 min at 4 °C. The new upper phase was transferred to a new tube. The polar extract was dried using a speedvac at 25 °C.
Processing Method:Methanol:Chloroform:Water
Processing Storage Conditions:On ice
Extraction Method:Methanol:Chloroform:Water
Extract Enrichment:Polar metabolites
Extract Cleanup:Centrifugation
Extract Storage:-80℃
Sample Resuspension:Methanol
Sample Derivatization:NO
Sample Spiking:NO

Combined analysis:

Analysis ID AN003597 AN003598
Analysis type MS MS
Chromatography type Reversed phase Reversed phase
Chromatography system Thermo Dionex Ultimate 3000 Thermo Dionex Ultimate 3000
Column Phenomenex Luna Omega Polar C18 (100 x 2.1 mm,1.7um) Phenomenex Luna Omega Polar C18 (100 x 2.1 mm,1.7um)
MS Type ESI ESI
MS instrument type QTOF QTOF
MS instrument name Bruker Impact II HD Bruker Impact II HD
Ion Mode POSITIVE NEGATIVE
Units peak area peak area

Chromatography:

Chromatography ID:CH002658
Chromatography Summary:A fifty-three-minute mobile phase gradient was employed. Gradient elution chromatography was performed starting with 100 % A to 98 % A in 1 min; hold for 9 min, gradient to 60 % A in 21 min, gradient to 45 % A in 5 min; hold for 2 min, gradient to 5 % A in 3 min; hold 5 min, return to initial conditions in 3 min and equilibrate for 5 min (total run time: 53 min). Solvent A was 100 % H2O containing 0.1 % formic acid, and solvent B was 100 % ACN containing 0.1 % formic acid. A flow rate of 0.1 mL/min was used.
Instrument Name:Thermo Dionex Ultimate 3000
Column Name:Phenomenex Luna Omega Polar C18 (100 x 2.1 mm,1.7um)
Column Temperature:30 ºC
Flow Gradient:Gradient elution chromatography was performed starting with 100 % A to 98 % A in 1 min; hold for 9 min, gradient to 60 % A in 21 min, gradient to 45 % A in 5 min; hold for 2 min, gradient to 5 % A in 3 min; hold 5 min, return to initial conditions in 3 min and equilibrate for 5 min (total run time: 53 min)
Flow Rate:0.1 mL/min
Retention Time:53 min
Sample Injection:5 uL
Solvent A:100% water; 0.1% formic acid
Solvent B:100% acetonitrile; 0.1% formic acid
Capillary Voltage:4.5 kV
Washing Buffer:IPA and Methanol
Randomization Order:True
Chromatography Type:Reversed phase

MS:

MS ID:MS003352
Analysis ID:AN003597
Instrument Name:Bruker Impact II HD
Instrument Type:QTOF
MS Type:ESI
MS Comments:The column eluent was analyzed using a Bruker Impact II HD (Bruker, Karlsruhe, Germany) quadrupole time-of-flight (Q-TOF) mass spectrometer equipped with an ESI source operating in positive polarity and negative polarity. Mass spectra were acquired with the following parameters of mass spectrometer: ion capillary voltage 4.5 kV (same for positive and negative mode), dry gas flow 6 L/min, dry gas temperature 250 ºC, nebulizer pressure 2 bar, collision RF 650 V, transfer time 80 µs and prepulse storage 5 µs. Spectra data, MS1 and MS2, were acquired in a data-dependent manner at 2 Hz, fragmenting the three most abundant precursor ions per MS1 scan, acquiring MS/MS data between 50 and 1300 m/z. Repetitive MS/MS sampling was limited by exclusion after 3 spectra at a particular mass within a window of 0.2 min. MS/MS fragmentation of the 3 most intense selected ions per spectrum was performed using ramped collision-induced dissociation energy of 7–17.5 eV. Hexakis (1H, 1H, 3H-tetrafluoropropoxy) phosphazene (Agilent Technologies, Santa Clara, CA, USA) was introduced as an internal calibrant after the run ends . Each sample was analyzed twice, first using the positive ion mode and then the negative.
Ion Mode:POSITIVE
Capillary Voltage:4.5 kV
Collision Energy:ramped collision-induced dissociation energy of 7–17.5 eV
Dry Gas Flow:6 L/min
Dry Gas Temp:250 ºC
Gas Pressure:2 bar
  
MS ID:MS003353
Analysis ID:AN003598
Instrument Name:Bruker Impact II HD
Instrument Type:QTOF
MS Type:ESI
MS Comments:The column eluent was analyzed using a Bruker Impact II HD (Bruker, Karlsruhe, Germany) quadrupole time-of-flight (Q-TOF) mass spectrometer equipped with an ESI source operating in positive polarity and negative polarity. Mass spectra were acquired with the following parameters of mass spectrometer: ion capillary voltage 4.5 kV (same for positive and negative mode), dry gas flow 6 L/min, dry gas temperature 250 ºC, nebulizer pressure 2 bar, collision RF 650 V, transfer time 80 µs and prepulse storage 5 µs. Spectra data, MS1 and MS2, were acquired in a data-dependent manner at 2 Hz, fragmenting the three most abundant precursor ions per MS1 scan, acquiring MS/MS data between 50 and 1300 m/z. Repetitive MS/MS sampling was limited by exclusion after 3 spectra at a particular mass within a window of 0.2 min. MS/MS fragmentation of the 3 most intense selected ions per spectrum was performed using ramped collision-induced dissociation energy of 7–17.5 eV. Hexakis (1H, 1H, 3H-tetrafluoropropoxy) phosphazene (Agilent Technologies, Santa Clara, CA, USA) was introduced as an internal calibrant after the run ends . Each sample was analyzed twice, first using the positive ion mode and then the negative.
Ion Mode:NEGATIVE
Capillary Voltage:4.5 kV
Collision Energy:ramped collision-induced dissociation energy of 7–17.5 eV
Dry Gas Flow:6 L/min
Dry Gas Temp:250 ºC
Gas Pressure:2 bar
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