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.

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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 IDST003173
Study TitleAssessment and partial characterization of candidate genes in dihydrochalcone and arbutin biosynthesis in an apple-pear hybrid by de novo transcriptome assembly
Study SummaryThe 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 NameMiranda Chavez
First NameSimon David
AddressVia Mach, 1, San Michele all'Adige, Trento, 38098, Italy
Emailsimondavid.mirandachavez@fmach.it
Phone+390461615231
Submit Date2024-04-12
Raw Data AvailableYes
Raw Data File Type(s)mzML
Analysis Type DetailLC-MS
Release Date2024-05-03
Release Version1
Simon David Miranda Chavez Simon David Miranda Chavez
https://dx.doi.org/10.21228/M8PQ8C
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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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
SA343152Enzyme assay_Apple pear hybrid_HybASR1Enzyme assay Apple pear hybrid 5ug
SA343153Enzyme assay_Apple pear hybrid_HybASR2Enzyme assay Apple pear hybrid 5ug
SA343154Enzyme assay_Apple pear hybrid_HybASR3Enzyme assay Apple pear hybrid 5ug
SA343155Enzyme assay_Malus domestica_MdAS1R1Enzyme assay Malus domestica 5ug
SA343156Enzyme assay_Malus domestica_MdAS2R1Enzyme assay Malus domestica 5ug
SA343157Enzyme assay_Malus domestica_MdAS2R2Enzyme assay Malus domestica 5ug
SA343158Enzyme assay_Malus domestica_MdAS2R3Enzyme assay Malus domestica 5ug
SA343159Enzyme assay_Malus domestica_MdAS1R3Enzyme assay Malus domestica 5ug
SA343160Enzyme assay_Malus domestica_MdAS1R2Enzyme assay Malus domestica 5ug
SA343161Enzyme assay_Pyrus communis_PcASR2Enzyme assay Pyrus communis 5ug
SA343162Enzyme assay_Pyrus communis_PcASR3Enzyme assay Pyrus communis 5ug
SA343163Enzyme assay_Pyrus communis_PcASR1Enzyme assay Pyrus communis 5ug
SA343164Fruit_Apple pear hybrid_FR1Fruit Apple pear hybrid Control
SA343165Fruit_Apple pear hybrid_FR2Fruit Apple pear hybrid Control
SA343166Fruit_Apple pear hybrid_FR3Fruit Apple pear hybrid Control
SA343167Fruit_Malus domestica_FR1Fruit Malus domestica Control
SA343168Fruit_Malus domestica_FR3Fruit Malus domestica Control
SA343169Fruit_Malus domestica_FR2Fruit Malus domestica Control
SA343170Fruit_Pyrus communis_FR1Fruit Pyrus communis Control
SA343171Fruit_Pyrus communis_FR3Fruit Pyrus communis Control
SA343172Fruit_Pyrus communis_FR2Fruit Pyrus communis Control
SA343173Old Leaf_Apple pear hybrid_OLR3Old Leaf Apple pear hybrid Control
SA343174Old Leaf_Apple pear hybrid_OLR2Old Leaf Apple pear hybrid Control
SA343175Old Leaf_Apple pear hybrid_OLR1Old Leaf Apple pear hybrid Control
SA343176Old Leaf_Malus domestica_OLR2Old Leaf Malus domestica Control
SA343177Old Leaf_Malus domestica_OLR1Old Leaf Malus domestica Control
SA343178Old Leaf_Malus domestica_OLR3Old Leaf Malus domestica Control
SA343179Old Leaf_Pyrus communis_OLR3Old Leaf Pyrus communis Control
SA343180Old Leaf_Pyrus communis_OLR2Old Leaf Pyrus communis Control
SA343181Old Leaf_Pyrus communis_OLR1Old Leaf Pyrus communis Control
SA343182Young Leaf_Apple pear hybrid_YLR2Young Leaf Apple pear hybrid Control
SA343183Young Leaf_Apple pear hybrid_YLR3Young Leaf Apple pear hybrid Control
SA343184Young Leaf_Apple pear hybrid_YLR1Young Leaf Apple pear hybrid Control
SA343185Young Leaf_Malus domestica_YLR2Young Leaf Malus domestica Control
SA343186Young Leaf_Malus domestica_YLR1Young Leaf Malus domestica Control
SA343187Young Leaf_Malus domestica_YLR3Young Leaf Malus domestica Control
SA343188Young Leaf_Pyrus communis_YLR3Young Leaf Pyrus communis Control
SA343189Young Leaf_Pyrus communis_YLR2Young Leaf Pyrus communis Control
SA343190Young Leaf_Pyrus communis_YLR1Young 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
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