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MB Sample ID: SA343189
| Local Sample ID: | Young Leaf_Pyrus communis_YLR2 |
| Subject ID: | SU003292 |
| Subject Type: | Plant |
| Subject Species: | Malus domestica/Pyrus communis/Apple-pear intergeneric hybrid |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU003292 |
| Subject Type: | Plant |
| Subject Species: | Malus domestica/Pyrus communis/Apple-pear intergeneric hybrid |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| Young Leaf_Pyrus communis_YLR2 | SA343189 | FL040563 | Young Leaf | Sample source |
| Young Leaf_Pyrus communis_YLR2 | SA343189 | FL040563 | Pyrus communis | Genotype |
| Young Leaf_Pyrus communis_YLR2 | SA343189 | FL040563 | Control | Treatment |
Collection:
| Collection ID: | CO003285 |
| Collection Summary: | For metabolite profiling, ripe fruit and young and old leaves of apple, pear and hybrid were collected from each individual maintained in the germplasm collection of Fondazione Edmund Mach. 100 mg of fresh tissue (FW) was extracted in 4 mL 80% v·v-1 methanol, sonicated for 20 min at 60 Hz in a water bath at 25ºC, agitated for further 20 min and kept in dark for 48 h, filtered through a 0.22 µm PTFE filter and stored at 4 ºC. For enzyme assays, E. coli strains harbouring pGEX-4T-1 with putative AS were grown in Terrific Broth (12 g·L-1 tryptone, 24 g·L-1 yeast extract, 9.4 g·L-1 K2HPO4, 2.2 g·L-1 KH2PO4, 4 mL·L-1 glycerol) at 37 °C and recombinant proteins were induced by supplementation of 0.5 mM IPTG at optical density OD600 of 0.5 – 0.6 and incubation at 20 ºC with agitation at 200 rpm for 16 h. Protein extraction was carried out by resuspending cells with B-PER™ Complete reagent supplemented with cOmplete™ protease inhibitor cocktail (Roche) followed by protein purification by Pierce™ GST spin purification kit, according to manufacter’s instructions. Quantitation of proteins was carried out by Pierce™ BCA protein assay kit and Bradford reagent (Sigma) after crude extraction and Glutathione S-Transferase (GST) - fusion protein purification, respectively. Enzyme activity was assayed in 200 µL reactions using 1 mM hydroquinone, 2 mM UDP-glucose, 5 µg purified protein in 200 mM Tris HCl, pH 7.5 buffer, incubated at 50 °C for 1 h and terminated by adding 300 µL methanol, as previously described35. |
| Sample Type: | Plant tissue/Enzyme assay |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR003301 |
| Treatment Summary: | All samples were wildtype genotypes from Malus domestica, Pyrus communis and apple-pear hybrid, each in three replicates grown in the germplasm collection at Fondazione Edmund Mach, Italy. For recombinant protein assay, each protein was supplemented with the putative hydroquinone substrate. |
Sample Preparation:
| Sampleprep ID: | SP003299 |
| Sampleprep Summary: | For phenolic targeted profiling, 100 mg of fresh tissue (FW) was extracted in 4 mL 80% v·v-1 methanol, sonicated for 20 min at 60 Hz in a water bath at 25ºC, agitated for further 20 min and kept in dark for 48 h, filtered through a 0.22 µm PTFE filter and stored at 4 ºC. For recombinant protein assays, each 200 µL reaction was extracted with 300 µL methanol for injection. |
Combined analysis:
| Analysis ID | AN005207 | AN005208 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | Reversed phase | Reversed phase |
| Chromatography system | Waters Acquity | Waters Acquity |
| Column | Waters ACQUITY UPLC HSS T3 (100 x 2.1mm,1.8um) | Waters ACQUITY UPLC HSS T3 (100 x 2.1mm,1.8um) |
| MS Type | ESI | ESI |
| MS instrument type | Triple quadrupole | Triple quadrupole |
| MS instrument name | Waters Xevo TQ-XS | Waters Xevo TQ-XS |
| Ion Mode | POSITIVE | NEGATIVE |
| Units | mg/L | mg/L |
Chromatography:
| Chromatography ID: | CH003940 |
| Chromatography Summary: | Ultraperformance liquid chromatography was performed on a Waters Acquity UPLC system (Milford, MA) consisting of a binary pump, an online vacuum degasser, an autosampler, and a column compartment. Separation of the phenolic compounds was achieved on a Waters Acquity HSS T3 column 1.8 μm, 100 mm × 2.1 mm (Milford, MA, USA), kept at 40 °C. Mobile phase A was water containing 0.1% formic acid; mobile phase B was acetonitrile containing 0.1% formic acid. The flow was 0.4 mL/min, and the gradient profile was 0-0.1 min, 5% B; from 0 to 3 min, linear gradient to 20% B; from 3 to 4.3 min, isocratic 20% B; from 4.3 to 9 min, linear gradient to 45% B; from 9 to 11 min, linear gradient to 100% B; from 11 to 13 min, wash at 100% B; from 13.01 to 15 min, back to the initial conditions of 5% B. The injection volume of both the standard solutions and the samples was 2 μL. After each injection, the needle was rinsed with 600 μL of weak wash solution (water/methanol, 90:10) and 200 μL of strong wash solution (methanol/water, 90:10). Samples were kept at 6 °C during the analysis. |
| Instrument Name: | Waters Acquity |
| Column Name: | Waters ACQUITY UPLC HSS T3 (100 x 2.1mm,1.8um) |
| Column Temperature: | 40 |
| Flow Gradient: | 0-0.1 min: 5% B, 0.1-3.0 min: linear 20%B, 3.0-4.3 min: isocratic 20% B, 4.3-9.0 min: linear 45% B, 9.0-11.0 min: linear 100% B, 11.0-13.0 min: wash 100% B, 13.01 – 15.0 min: back to initial 5% B |
| Flow Rate: | 0.4 ml/min |
| Solvent A: | 99.9% water/0.1% formic acid |
| Solvent B: | 99.9% acetonitrile/0.1% formic acid |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS004940 |
| Analysis ID: | AN005207 |
| Instrument Name: | Waters Xevo TQ-XS |
| Instrument Type: | Triple quadrupole |
| MS Type: | ESI |
| MS Comments: | Capillary voltage was 3.5 kV in positive mode and −2.5 kV in negative mode; the source was kept at 150 °C; desolvation temperature was 500 °C; cone gas flow, 50 L/h; and desolvation gas flow, 800 L/h. Unit resolution was applied to each quadrupole. Flow injections of each individual metabolite were used to optimize the MRM conditions. For the majority of the metabolites, this was done automatically by the Waters Intellistart software, whereas for some compounds the optimal cone voltages and collision energies were identified during collision-induced dissociation (CID) experiments and manually set. A dwell time of at least 25 ms was applied to each MRM transition. |
| Ion Mode: | POSITIVE |
| MS ID: | MS004941 |
| Analysis ID: | AN005208 |
| Instrument Name: | Waters Xevo TQ-XS |
| Instrument Type: | Triple quadrupole |
| MS Type: | ESI |
| MS Comments: | Capillary voltage was 3.5 kV in positive mode and −2.5 kV in negative mode; the source was kept at 150 °C; desolvation temperature was 500 °C; cone gas flow, 50 L/h; and desolvation gas flow, 800 L/h. Unit resolution was applied to each quadrupole. Flow injections of each individual metabolite were used to optimize the MRM conditions. For the majority of the metabolites, this was done automatically by the Waters Intellistart software, whereas for some compounds the optimal cone voltages and collision energies were identified during collision-induced dissociation (CID) experiments and manually set. A dwell time of at least 25 ms was applied to each MRM transition. |
| Ion Mode: | NEGATIVE |
