Summary of Study ST003173
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 PR001973. The data can be accessed directly via it's Project DOI: 10.21228/M8PQ8C This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
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.
| Study ID | ST003173 |
| Study Title | Assessment and partial characterization of candidate genes in dihydrochalcone and arbutin biosynthesis in an apple-pear hybrid by de novo transcriptome assembly |
| Study Summary | The goal of the study was to determine the phenolic profile of young and old leaves, as well as fruit of apple (Malus x domestica), pear (Pyrus communis) and an intergeneric apple-pear hybrid. Three independent replicates were obtained for each genotype from the germplasm collection at Fondazione Edmund Mach (Italy) and analyzed by a targeted phenolic LC/MS-MS method. In addition, candidate genes from apple, pear and apple-pear hybrid retrieved from a de novo transcriptome assembly were expressed in E. coli and recombinant proteins were tested (in triplicate) to determine the conversion of hydroquinone to arbutin. Combining RNA-Seq, in silico functional annotation prediction, targeted gene expression analysis and expression – metabolite correlations with the data submitted to Metabolomics Workbench, we identified candidate genes for functional characterisation, resulting in the identification of active arbutin synthases in the hybrid and parental genotypes. We found that the putative arbutin synthases of pear (PcAS) and apple-pear hybrid (HybAS) were able to convert hydroquinone into arbutin. Interestingly, also one out of two putative arbutin synthases isolated from apple (MdAS1) could produce arbutin in vitro. However, the metabolomic profiling of phenolic compounds showed that apple lacks of arbutin and was found to accumulate the precursor hydroquinone in traces in young and old leaves of apple. Although quercetin was accumulated in similar amounts in the same tissues, a luminiscence-based assay showed that quercetin was converted only 25% compared to activity towards hydroquinone in the tested conditions. In summary, the metabolomic profiling submitted to Metabolomics workbench also shows that: 1) arbutin is accumulated mainly in young leaves of pear, followed by the apple-pear hybrid and was found in traces in apple fruit; 2) rutin was found mainly in pear and apple-pear hybrid tissues; 3) phenolic profile of apple is dominated by phloridzin and undetectable in all pear tissues analyzed, with young leaves being the tissue showing highest accumulation. |
| Institute | Fondazione Edmund Mach |
| Last Name | Miranda Chavez |
| First Name | Simon David |
| Address | Via Mach, 1, San Michele all'Adige, Trento, 38098, Italy |
| simondavid.mirandachavez@fmach.it | |
| Phone | +390461615231 |
| Submit Date | 2024-04-12 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzML |
| Analysis Type Detail | LC-MS |
| Release Date | 2024-05-03 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR001973 |
| Project DOI: | doi: 10.21228/M8PQ8C |
| Project Title: | Assessment and partial characterization of candidate genes in dihydrochalcone and arbutin biosynthesis in an apple-pear hybrid by de novo transcriptome assembly |
| Project Summary: | The goal of the study was to determine the phenolic profile of young and old leaves, as well as fruit of apple (Malus x domestica), pear (Pyrus communis) and an intergeneric apple-pear hybrid. Three independent replicates were obtained for each genotype from the germplasm collection at Fondazione Edmund Mach (Italy) and analyzed by a phenolic targeted LC/MS-MS method. In addition, candidate genes retrieved from a de novo transcriptome assembly were tested in recombinant proteins (n = 3) to determine the conversion of hydroquinone to arbutin. Combining RNA-Seq, in silico functional annotation prediction, targeted gene expression analysis and expression – metabolite correlations with the data submitted to Metabolomics Workbench, we identified candidate genes for functional characterisation, resulting in the identification of active arbutin synthases in the hybrid and parental genotypes. We found that the putative arbutin synthases of pear (PcAS) and apple-pear hybrid (HybAS) were able to convert hydroquinone into arbutin. Interestingly, also one out of two putative arbutin synthases isolated from apple (MdAS1) could produce arbutin in vitro. However, the metabolomic profiling of phenolic compounds showed that apple lacks of arbutin and was found to accumulate the precursor hydroquinone in traces in young and old leaves of apple. Although quercetin was accumulated in similar amounts in the same tissues, a luminiscence-based assay showed that quercetin was converted only 25% compared to activity towards hydroquinone in the tested conditions. In summary, the metabolomic profiling submitted to Metabolomics workbench also shows that: 1) arbutin is accumulated mainly in young leaves of pear, followed by the apple-pear hybrid and was found in traces in apple fruit; 2) rutin was found mainly in pear and apple-pear hybrid tissues; 3) phenolic profile of apple is dominated by phloridzin and undetectable in all pear tissues analyzed, with young leaves being the tissue showing highest accumulation. |
| Institute: | Fondazione Edmund Mach |
| Last Name: | Miranda Chavez |
| First Name: | Simon David |
| Address: | Via Mach, 1, San Michele all'Adige, Trento, 38098, Italy |
| Email: | simondavid.mirandachavez@fmach.it |
| Phone: | +390461615231 |
Subject:
| Subject ID: | SU003292 |
| Subject Type: | Plant |
| Subject Species: | Malus domestica;Pyrus communis;Apple-pear intergeneric hybrid |
| Taxonomy ID: | 3750;23211 |
Factors:
Subject type: Plant; Subject species: Malus domestica;Pyrus communis;Apple-pear intergeneric hybrid (Factor headings shown in green)
| mb_sample_id | local_sample_id | Sample source | Genotype | Treatment |
|---|---|---|---|---|
| SA343152 | Enzyme assay_Apple pear hybrid_HybASR1 | Enzyme assay | Apple pear hybrid | 5ug |
| SA343153 | Enzyme assay_Apple pear hybrid_HybASR2 | Enzyme assay | Apple pear hybrid | 5ug |
| SA343154 | Enzyme assay_Apple pear hybrid_HybASR3 | Enzyme assay | Apple pear hybrid | 5ug |
| SA343155 | Enzyme assay_Malus domestica_MdAS1R1 | Enzyme assay | Malus domestica | 5ug |
| SA343156 | Enzyme assay_Malus domestica_MdAS2R1 | Enzyme assay | Malus domestica | 5ug |
| SA343157 | Enzyme assay_Malus domestica_MdAS2R2 | Enzyme assay | Malus domestica | 5ug |
| SA343158 | Enzyme assay_Malus domestica_MdAS2R3 | Enzyme assay | Malus domestica | 5ug |
| SA343159 | Enzyme assay_Malus domestica_MdAS1R3 | Enzyme assay | Malus domestica | 5ug |
| SA343160 | Enzyme assay_Malus domestica_MdAS1R2 | Enzyme assay | Malus domestica | 5ug |
| SA343161 | Enzyme assay_Pyrus communis_PcASR2 | Enzyme assay | Pyrus communis | 5ug |
| SA343162 | Enzyme assay_Pyrus communis_PcASR3 | Enzyme assay | Pyrus communis | 5ug |
| SA343163 | Enzyme assay_Pyrus communis_PcASR1 | Enzyme assay | Pyrus communis | 5ug |
| SA343164 | Fruit_Apple pear hybrid_FR1 | Fruit | Apple pear hybrid | Control |
| SA343165 | Fruit_Apple pear hybrid_FR2 | Fruit | Apple pear hybrid | Control |
| SA343166 | Fruit_Apple pear hybrid_FR3 | Fruit | Apple pear hybrid | Control |
| SA343167 | Fruit_Malus domestica_FR1 | Fruit | Malus domestica | Control |
| SA343168 | Fruit_Malus domestica_FR3 | Fruit | Malus domestica | Control |
| SA343169 | Fruit_Malus domestica_FR2 | Fruit | Malus domestica | Control |
| SA343170 | Fruit_Pyrus communis_FR1 | Fruit | Pyrus communis | Control |
| SA343171 | Fruit_Pyrus communis_FR3 | Fruit | Pyrus communis | Control |
| SA343172 | Fruit_Pyrus communis_FR2 | Fruit | Pyrus communis | Control |
| SA343173 | Old Leaf_Apple pear hybrid_OLR3 | Old Leaf | Apple pear hybrid | Control |
| SA343174 | Old Leaf_Apple pear hybrid_OLR2 | Old Leaf | Apple pear hybrid | Control |
| SA343175 | Old Leaf_Apple pear hybrid_OLR1 | Old Leaf | Apple pear hybrid | Control |
| SA343176 | Old Leaf_Malus domestica_OLR2 | Old Leaf | Malus domestica | Control |
| SA343177 | Old Leaf_Malus domestica_OLR1 | Old Leaf | Malus domestica | Control |
| SA343178 | Old Leaf_Malus domestica_OLR3 | Old Leaf | Malus domestica | Control |
| SA343179 | Old Leaf_Pyrus communis_OLR3 | Old Leaf | Pyrus communis | Control |
| SA343180 | Old Leaf_Pyrus communis_OLR2 | Old Leaf | Pyrus communis | Control |
| SA343181 | Old Leaf_Pyrus communis_OLR1 | Old Leaf | Pyrus communis | Control |
| SA343182 | Young Leaf_Apple pear hybrid_YLR2 | Young Leaf | Apple pear hybrid | Control |
| SA343183 | Young Leaf_Apple pear hybrid_YLR3 | Young Leaf | Apple pear hybrid | Control |
| SA343184 | Young Leaf_Apple pear hybrid_YLR1 | Young Leaf | Apple pear hybrid | Control |
| SA343185 | Young Leaf_Malus domestica_YLR2 | Young Leaf | Malus domestica | Control |
| SA343186 | Young Leaf_Malus domestica_YLR1 | Young Leaf | Malus domestica | Control |
| SA343187 | Young Leaf_Malus domestica_YLR3 | Young Leaf | Malus domestica | Control |
| SA343188 | Young Leaf_Pyrus communis_YLR3 | Young Leaf | Pyrus communis | Control |
| SA343189 | Young Leaf_Pyrus communis_YLR2 | Young Leaf | Pyrus communis | Control |
| SA343190 | Young Leaf_Pyrus communis_YLR1 | Young Leaf | Pyrus communis | Control |
| Showing results 1 to 39 of 39 |
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 |
