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MB Sample ID: SA210704
| Local Sample ID: | T7.40.LWS2_neg |
| Subject ID: | SU002284 |
| Subject Type: | Plant |
| Subject Species: | Pinus pinaster |
| Taxonomy ID: | 71647 |
| Age Or Age Range: | one-two years |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002284 |
| Subject Type: | Plant |
| Subject Species: | Pinus pinaster |
| Taxonomy ID: | 71647 |
| Age Or Age Range: | one-two years |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| T7.40.LWS2_neg | SA210704 | FL025737 | T7.40.LWS | Factor |
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 |
