#METABOLOMICS WORKBENCH Simon_Miranda_20240412_055127 DATATRACK_ID:4776 STUDY_ID:ST003173 ANALYSIS_ID:AN005208 PROJECT_ID:PR001973 VERSION 1 CREATED_ON April 17, 2024, 7:41 am #PROJECT PR:PROJECT_TITLE Assessment and partial characterization of candidate genes in dihydrochalcone PR:PROJECT_TITLE and arbutin biosynthesis in an apple-pear hybrid by de novo transcriptome PR:PROJECT_TITLE assembly PR:PROJECT_SUMMARY The goal of the study was to determine the phenolic profile of young and old PR:PROJECT_SUMMARY leaves, as well as fruit of apple (Malus x domestica), pear (Pyrus communis) and PR:PROJECT_SUMMARY an intergeneric apple-pear hybrid. Three independent replicates were obtained PR:PROJECT_SUMMARY for each genotype from the germplasm collection at Fondazione Edmund Mach PR:PROJECT_SUMMARY (Italy) and analyzed by a phenolic targeted LC/MS-MS method. In addition, PR:PROJECT_SUMMARY candidate genes retrieved from a de novo transcriptome assembly were tested in PR:PROJECT_SUMMARY recombinant proteins (n = 3) to determine the conversion of hydroquinone to PR:PROJECT_SUMMARY arbutin. Combining RNA-Seq, in silico functional annotation prediction, targeted PR:PROJECT_SUMMARY gene expression analysis and expression – metabolite correlations with the PR:PROJECT_SUMMARY data submitted to Metabolomics Workbench, we identified candidate genes for PR:PROJECT_SUMMARY functional characterisation, resulting in the identification of active arbutin PR:PROJECT_SUMMARY synthases in the hybrid and parental genotypes. We found that the putative PR:PROJECT_SUMMARY arbutin synthases of pear (PcAS) and apple-pear hybrid (HybAS) were able to PR:PROJECT_SUMMARY convert hydroquinone into arbutin. Interestingly, also one out of two putative PR:PROJECT_SUMMARY arbutin synthases isolated from apple (MdAS1) could produce arbutin in vitro. PR:PROJECT_SUMMARY However, the metabolomic profiling of phenolic compounds showed that apple lacks PR:PROJECT_SUMMARY of arbutin and was found to accumulate the precursor hydroquinone in traces in PR:PROJECT_SUMMARY young and old leaves of apple. Although quercetin was accumulated in similar PR:PROJECT_SUMMARY amounts in the same tissues, a luminiscence-based assay showed that quercetin PR:PROJECT_SUMMARY was converted only 25% compared to activity towards hydroquinone in the tested PR:PROJECT_SUMMARY conditions. In summary, the metabolomic profiling submitted to Metabolomics PR:PROJECT_SUMMARY workbench also shows that: 1) arbutin is accumulated mainly in young leaves of PR:PROJECT_SUMMARY pear, followed by the apple-pear hybrid and was found in traces in apple fruit; PR:PROJECT_SUMMARY 2) rutin was found mainly in pear and apple-pear hybrid tissues; 3) phenolic PR:PROJECT_SUMMARY profile of apple is dominated by phloridzin and undetectable in all pear tissues PR:PROJECT_SUMMARY analyzed, with young leaves being the tissue showing highest accumulation. PR:INSTITUTE Fondazione Edmund Mach PR:LAST_NAME Miranda Chavez PR:FIRST_NAME Simon David PR:ADDRESS Via Mach, 1, San Michele all'Adige, Trento, 38098, Italy PR:EMAIL simondavid.mirandachavez@fmach.it PR:PHONE +390461615231 #STUDY ST:STUDY_TITLE Assessment and partial characterization of candidate genes in dihydrochalcone ST:STUDY_TITLE and arbutin biosynthesis in an apple-pear hybrid by de novo transcriptome ST:STUDY_TITLE assembly ST:STUDY_SUMMARY The goal of the study was to determine the phenolic profile of young and old ST:STUDY_SUMMARY leaves, as well as fruit of apple (Malus x domestica), pear (Pyrus communis) and ST:STUDY_SUMMARY an intergeneric apple-pear hybrid. Three independent replicates were obtained ST:STUDY_SUMMARY for each genotype from the germplasm collection at Fondazione Edmund Mach ST:STUDY_SUMMARY (Italy) and analyzed by a targeted phenolic LC/MS-MS method. In addition, ST:STUDY_SUMMARY candidate genes from apple, pear and apple-pear hybrid retrieved from a de novo ST:STUDY_SUMMARY transcriptome assembly were expressed in E. coli and recombinant proteins were ST:STUDY_SUMMARY tested (in triplicate) to determine the conversion of hydroquinone to arbutin. ST:STUDY_SUMMARY Combining RNA-Seq, in silico functional annotation prediction, targeted gene ST:STUDY_SUMMARY expression analysis and expression – metabolite correlations with the data ST:STUDY_SUMMARY submitted to Metabolomics Workbench, we identified candidate genes for ST:STUDY_SUMMARY functional characterisation, resulting in the identification of active arbutin ST:STUDY_SUMMARY synthases in the hybrid and parental genotypes. We found that the putative ST:STUDY_SUMMARY arbutin synthases of pear (PcAS) and apple-pear hybrid (HybAS) were able to ST:STUDY_SUMMARY convert hydroquinone into arbutin. Interestingly, also one out of two putative ST:STUDY_SUMMARY arbutin synthases isolated from apple (MdAS1) could produce arbutin in vitro. ST:STUDY_SUMMARY However, the metabolomic profiling of phenolic compounds showed that apple lacks ST:STUDY_SUMMARY of arbutin and was found to accumulate the precursor hydroquinone in traces in ST:STUDY_SUMMARY young and old leaves of apple. Although quercetin was accumulated in similar ST:STUDY_SUMMARY amounts in the same tissues, a luminiscence-based assay showed that quercetin ST:STUDY_SUMMARY was converted only 25% compared to activity towards hydroquinone in the tested ST:STUDY_SUMMARY conditions. In summary, the metabolomic profiling submitted to Metabolomics ST:STUDY_SUMMARY workbench also shows that: 1) arbutin is accumulated mainly in young leaves of ST:STUDY_SUMMARY pear, followed by the apple-pear hybrid and was found in traces in apple fruit; ST:STUDY_SUMMARY 2) rutin was found mainly in pear and apple-pear hybrid tissues; 3) phenolic ST:STUDY_SUMMARY profile of apple is dominated by phloridzin and undetectable in all pear tissues ST:STUDY_SUMMARY analyzed, with young leaves being the tissue showing highest accumulation. ST:INSTITUTE Fondazione Edmund Mach ST:LAST_NAME Miranda Chavez ST:FIRST_NAME Simon David ST:ADDRESS Via Mach, 1, San Michele all'Adige, Trento, 38098, Italy ST:EMAIL simondavid.mirandachavez@fmach.it ST:PHONE +390461615231 #SUBJECT SU:SUBJECT_TYPE Plant SU:SUBJECT_SPECIES Malus domestica/Pyrus communis/Apple-pear intergeneric hybrid #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS Young Leaf_Malus domestica_YLR1 Young Leaf_Malus domestica_YLR1 Sample source:Young Leaf | Genotype:Malus domestica | Treatment:Control Batch=YLR1; RAW_FILE_NAME=Apple_YoungLeaves_R1.mzML SUBJECT_SAMPLE_FACTORS Young Leaf_Malus domestica_YLR2 Young Leaf_Malus domestica_YLR2 Sample source:Young Leaf | Genotype:Malus domestica | Treatment:Control Batch=YLR2; RAW_FILE_NAME=Apple_YoungLeaves_R2.mzML SUBJECT_SAMPLE_FACTORS Young Leaf_Malus domestica_YLR3 Young Leaf_Malus domestica_YLR3 Sample source:Young Leaf | Genotype:Malus domestica | Treatment:Control Batch=YLR3; RAW_FILE_NAME=Apple_YoungLeaves_R3.mzML SUBJECT_SAMPLE_FACTORS Old Leaf_Malus domestica_OLR1 Old Leaf_Malus domestica_OLR1 Sample source:Old Leaf | Genotype:Malus domestica | Treatment:Control Batch=OLR1; RAW_FILE_NAME=Apple_OldLeaves_R1.mzML SUBJECT_SAMPLE_FACTORS Old Leaf_Malus domestica_OLR2 Old Leaf_Malus domestica_OLR2 Sample source:Old Leaf | Genotype:Malus domestica | Treatment:Control Batch=OLR2; RAW_FILE_NAME=Apple_OldLeaves_R2.mzML SUBJECT_SAMPLE_FACTORS Old Leaf_Malus domestica_OLR3 Old Leaf_Malus domestica_OLR3 Sample source:Old Leaf | Genotype:Malus domestica | Treatment:Control Batch=OLR3; RAW_FILE_NAME=Apple_OldLeaves_R3.mzML SUBJECT_SAMPLE_FACTORS Fruit_Malus domestica_FR1 Fruit_Malus domestica_FR1 Sample source:Fruit | Genotype:Malus domestica | Treatment:Control Batch=FR1; RAW_FILE_NAME=Apple_Fruit_R1.mzML SUBJECT_SAMPLE_FACTORS Fruit_Malus domestica_FR2 Fruit_Malus domestica_FR2 Sample source:Fruit | Genotype:Malus domestica | Treatment:Control Batch=FR2; RAW_FILE_NAME=Apple_Fruit_R2.mzML SUBJECT_SAMPLE_FACTORS Fruit_Malus domestica_FR3 Fruit_Malus domestica_FR3 Sample source:Fruit | Genotype:Malus domestica | Treatment:Control Batch=FR3; RAW_FILE_NAME=Apple_Fruit_R3.mzML SUBJECT_SAMPLE_FACTORS Young Leaf_Pyrus communis_YLR1 Young Leaf_Pyrus communis_YLR1 Sample source:Young Leaf | Genotype:Pyrus communis | Treatment:Control Batch=YLR1; RAW_FILE_NAME=Pear_YoungLeaves_R1.mzML SUBJECT_SAMPLE_FACTORS Young Leaf_Pyrus communis_YLR2 Young Leaf_Pyrus communis_YLR2 Sample source:Young Leaf | Genotype:Pyrus communis | Treatment:Control Batch=YLR2; RAW_FILE_NAME=Pear_YoungLeaves_R2.mzML SUBJECT_SAMPLE_FACTORS Young Leaf_Pyrus communis_YLR3 Young Leaf_Pyrus communis_YLR3 Sample source:Young Leaf | Genotype:Pyrus communis | Treatment:Control Batch=YLR3; RAW_FILE_NAME=Pear_YoungLeaves_R3.mzML SUBJECT_SAMPLE_FACTORS Old Leaf_Pyrus communis_OLR1 Old Leaf_Pyrus communis_OLR1 Sample source:Old Leaf | Genotype:Pyrus communis | Treatment:Control Batch=OLR1; RAW_FILE_NAME=Pear_OldLeaves_R1.mzML SUBJECT_SAMPLE_FACTORS Old Leaf_Pyrus communis_OLR2 Old Leaf_Pyrus communis_OLR2 Sample source:Old Leaf | Genotype:Pyrus communis | Treatment:Control Batch=OLR2; RAW_FILE_NAME=Pear_OldLeaves_R2.mzML SUBJECT_SAMPLE_FACTORS Old Leaf_Pyrus communis_OLR3 Old Leaf_Pyrus communis_OLR3 Sample source:Old Leaf | Genotype:Pyrus communis | Treatment:Control Batch=OLR3; RAW_FILE_NAME=Pear_OldLeaves_R3.mzML SUBJECT_SAMPLE_FACTORS Fruit_Pyrus communis_FR1 Fruit_Pyrus communis_FR1 Sample source:Fruit | Genotype:Pyrus communis | Treatment:Control Batch=FR1; RAW_FILE_NAME=Pear_Fruit_R1.mzML SUBJECT_SAMPLE_FACTORS Fruit_Pyrus communis_FR2 Fruit_Pyrus communis_FR2 Sample source:Fruit | Genotype:Pyrus communis | Treatment:Control Batch=FR2; RAW_FILE_NAME=Pear_Fruit_R2.mzML SUBJECT_SAMPLE_FACTORS Fruit_Pyrus communis_FR3 Fruit_Pyrus communis_FR3 Sample source:Fruit | Genotype:Pyrus communis | Treatment:Control Batch=FR3; RAW_FILE_NAME=Pear_Fruit_R3.mzML SUBJECT_SAMPLE_FACTORS Young Leaf_Apple pear hybrid_YLR1 Young Leaf_Apple pear hybrid_YLR1 Sample source:Young Leaf | Genotype:Apple pear hybrid | Treatment:Control Batch=YLR1; RAW_FILE_NAME=Hybrid_YoungLeaves_R1.mzML SUBJECT_SAMPLE_FACTORS Young Leaf_Apple pear hybrid_YLR2 Young Leaf_Apple pear hybrid_YLR2 Sample source:Young Leaf | Genotype:Apple pear hybrid | Treatment:Control Batch=YLR2; RAW_FILE_NAME=Hybrid_YoungLeaves_R2.mzML SUBJECT_SAMPLE_FACTORS Young Leaf_Apple pear hybrid_YLR3 Young Leaf_Apple pear hybrid_YLR3 Sample source:Young Leaf | Genotype:Apple pear hybrid | Treatment:Control Batch=YLR3; RAW_FILE_NAME=Hybrid_YoungLeaves_R3.mzML SUBJECT_SAMPLE_FACTORS Old Leaf_Apple pear hybrid_OLR1 Old Leaf_Apple pear hybrid_OLR1 Sample source:Old Leaf | Genotype:Apple pear hybrid | Treatment:Control Batch=OLR1; RAW_FILE_NAME=Hybrid_OldLeaves_R1.mzML SUBJECT_SAMPLE_FACTORS Old Leaf_Apple pear hybrid_OLR2 Old Leaf_Apple pear hybrid_OLR2 Sample source:Old Leaf | Genotype:Apple pear hybrid | Treatment:Control Batch=OLR2; RAW_FILE_NAME=Hybrid_OldLeaves_R2.mzML SUBJECT_SAMPLE_FACTORS Old Leaf_Apple pear hybrid_OLR3 Old Leaf_Apple pear hybrid_OLR3 Sample source:Old Leaf | Genotype:Apple pear hybrid | Treatment:Control Batch=OLR3; RAW_FILE_NAME=Hybrid_OldLeaves_R3.mzML SUBJECT_SAMPLE_FACTORS Fruit_Apple pear hybrid_FR1 Fruit_Apple pear hybrid_FR1 Sample source:Fruit | Genotype:Apple pear hybrid | Treatment:Control Batch=FR1; RAW_FILE_NAME=Hybrid_Fruit_R1.mzML SUBJECT_SAMPLE_FACTORS Fruit_Apple pear hybrid_FR2 Fruit_Apple pear hybrid_FR2 Sample source:Fruit | Genotype:Apple pear hybrid | Treatment:Control Batch=FR2; RAW_FILE_NAME=Hybrid_Fruit_R2.mzML SUBJECT_SAMPLE_FACTORS Fruit_Apple pear hybrid_FR3 Fruit_Apple pear hybrid_FR3 Sample source:Fruit | Genotype:Apple pear hybrid | Treatment:Control Batch=FR3; RAW_FILE_NAME=Hybrid_Fruit_R3.mzML SUBJECT_SAMPLE_FACTORS Enzyme assay_Malus domestica_MdAS1R1 Enzyme assay_Malus domestica_MdAS1R1 Sample source:Enzyme assay | Genotype:Malus domestica | Treatment:5ug Batch=MdAS1R1; RAW_FILE_NAME=MdAS1_R1.mzML SUBJECT_SAMPLE_FACTORS Enzyme assay_Malus domestica_MdAS1R2 Enzyme assay_Malus domestica_MdAS1R2 Sample source:Enzyme assay | Genotype:Malus domestica | Treatment:5ug Batch=MdAS1R2; RAW_FILE_NAME=MdAS1_R2.mzML SUBJECT_SAMPLE_FACTORS Enzyme assay_Malus domestica_MdAS1R3 Enzyme assay_Malus domestica_MdAS1R3 Sample source:Enzyme assay | Genotype:Malus domestica | Treatment:5ug Batch=MdAS1R3; RAW_FILE_NAME=MdAS1_R3.mzML SUBJECT_SAMPLE_FACTORS Enzyme assay_Malus domestica_MdAS2R1 Enzyme assay_Malus domestica_MdAS2R1 Sample source:Enzyme assay | Genotype:Malus domestica | Treatment:5ug Batch=MdAS2R1; RAW_FILE_NAME=MdAS2_R1.mzML SUBJECT_SAMPLE_FACTORS Enzyme assay_Malus domestica_MdAS2R2 Enzyme assay_Malus domestica_MdAS2R2 Sample source:Enzyme assay | Genotype:Malus domestica | Treatment:5ug Batch=MdAS2R2; RAW_FILE_NAME=MdAS2_R2.mzML SUBJECT_SAMPLE_FACTORS Enzyme assay_Malus domestica_MdAS2R3 Enzyme assay_Malus domestica_MdAS2R3 Sample source:Enzyme assay | Genotype:Malus domestica | Treatment:5ug Batch=MdAS2R3; RAW_FILE_NAME=MdAS2_R3.mzML SUBJECT_SAMPLE_FACTORS Enzyme assay_Pyrus communis_PcASR1 Enzyme assay_Pyrus communis_PcASR1 Sample source:Enzyme assay | Genotype:Pyrus communis | Treatment:5ug Batch=PcASR1; RAW_FILE_NAME=PcAS_R1.mzML SUBJECT_SAMPLE_FACTORS Enzyme assay_Pyrus communis_PcASR2 Enzyme assay_Pyrus communis_PcASR2 Sample source:Enzyme assay | Genotype:Pyrus communis | Treatment:5ug Batch=PcASR2; RAW_FILE_NAME=PcAS_R2.mzML SUBJECT_SAMPLE_FACTORS Enzyme assay_Pyrus communis_PcASR3 Enzyme assay_Pyrus communis_PcASR3 Sample source:Enzyme assay | Genotype:Pyrus communis | Treatment:5ug Batch=PcASR3; RAW_FILE_NAME=PcAS_R3.mzML SUBJECT_SAMPLE_FACTORS Enzyme assay_Apple pear hybrid_HybASR1 Enzyme assay_Apple pear hybrid_HybASR1 Sample source:Enzyme assay | Genotype:Apple pear hybrid | Treatment:5ug Batch=HybASR1; RAW_FILE_NAME=HybAS_R1.mzML SUBJECT_SAMPLE_FACTORS Enzyme assay_Apple pear hybrid_HybASR2 Enzyme assay_Apple pear hybrid_HybASR2 Sample source:Enzyme assay | Genotype:Apple pear hybrid | Treatment:5ug Batch=HybASR2; RAW_FILE_NAME=HybAS_R2.mzML SUBJECT_SAMPLE_FACTORS Enzyme assay_Apple pear hybrid_HybASR3 Enzyme assay_Apple pear hybrid_HybASR3 Sample source:Enzyme assay | Genotype:Apple pear hybrid | Treatment:5ug Batch=HybASR3; RAW_FILE_NAME=HybAS_R3.mzML #COLLECTION CO:COLLECTION_SUMMARY For metabolite profiling, ripe fruit and young and old leaves of apple, pear and CO:COLLECTION_SUMMARY hybrid were collected from each individual maintained in the germplasm CO:COLLECTION_SUMMARY collection of Fondazione Edmund Mach. 100 mg of fresh tissue (FW) was extracted CO:COLLECTION_SUMMARY in 4 mL 80% v·v-1 methanol, sonicated for 20 min at 60 Hz in a water bath at CO:COLLECTION_SUMMARY 25ºC, agitated for further 20 min and kept in dark for 48 h, filtered through a CO:COLLECTION_SUMMARY 0.22 µm PTFE filter and stored at 4 ºC. For enzyme assays, E. coli strains CO:COLLECTION_SUMMARY harbouring pGEX-4T-1 with putative AS were grown in Terrific Broth (12 g·L-1 CO:COLLECTION_SUMMARY tryptone, 24 g·L-1 yeast extract, 9.4 g·L-1 K2HPO4, 2.2 g·L-1 KH2PO4, 4 CO:COLLECTION_SUMMARY mL·L-1 glycerol) at 37 °C and recombinant proteins were induced by CO:COLLECTION_SUMMARY supplementation of 0.5 mM IPTG at optical density OD600 of 0.5 – 0.6 and CO:COLLECTION_SUMMARY incubation at 20 ºC with agitation at 200 rpm for 16 h. Protein extraction was CO:COLLECTION_SUMMARY carried out by resuspending cells with B-PER™ Complete reagent supplemented CO:COLLECTION_SUMMARY with cOmplete™ protease inhibitor cocktail (Roche) followed by protein CO:COLLECTION_SUMMARY purification by Pierce™ GST spin purification kit, according to manufacter’s CO:COLLECTION_SUMMARY instructions. Quantitation of proteins was carried out by Pierce™ BCA protein CO:COLLECTION_SUMMARY assay kit and Bradford reagent (Sigma) after crude extraction and Glutathione CO:COLLECTION_SUMMARY S-Transferase (GST) - fusion protein purification, respectively. Enzyme activity CO:COLLECTION_SUMMARY was assayed in 200 µL reactions using 1 mM hydroquinone, 2 mM UDP-glucose, 5 CO:COLLECTION_SUMMARY µg purified protein in 200 mM Tris HCl, pH 7.5 buffer, incubated at 50 °C for CO:COLLECTION_SUMMARY 1 h and terminated by adding 300 µL methanol, as previously described35. CO:SAMPLE_TYPE Plant tissue/Enzyme assay CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY All samples were wildtype genotypes from Malus domestica, Pyrus communis and TR:TREATMENT_SUMMARY apple-pear hybrid, each in three replicates grown in the germplasm collection at TR:TREATMENT_SUMMARY Fondazione Edmund Mach, Italy. For recombinant protein assay, each protein was TR:TREATMENT_SUMMARY supplemented with the putative hydroquinone substrate. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY For phenolic targeted profiling, 100 mg of fresh tissue (FW) was extracted in 4 SP:SAMPLEPREP_SUMMARY mL 80% v·v-1 methanol, sonicated for 20 min at 60 Hz in a water bath at 25ºC, SP:SAMPLEPREP_SUMMARY agitated for further 20 min and kept in dark for 48 h, filtered through a 0.22 SP:SAMPLEPREP_SUMMARY µm PTFE filter and stored at 4 ºC. For recombinant protein assays, each 200 SP:SAMPLEPREP_SUMMARY µL reaction was extracted with 300 µL methanol for injection. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Ultraperformance liquid chromatography was performed on a Waters Acquity UPLC CH:CHROMATOGRAPHY_SUMMARY system (Milford, MA) consisting of a binary pump, an online vacuum degasser, an CH:CHROMATOGRAPHY_SUMMARY autosampler, and a column compartment. Separation of the phenolic compounds was CH:CHROMATOGRAPHY_SUMMARY achieved on a Waters Acquity HSS T3 column 1.8 μm, 100 mm × 2.1 mm (Milford, CH:CHROMATOGRAPHY_SUMMARY MA, USA), kept at 40 °C. Mobile phase A was water containing 0.1% formic acid; CH:CHROMATOGRAPHY_SUMMARY mobile phase B was acetonitrile containing 0.1% formic acid. The flow was 0.4 CH:CHROMATOGRAPHY_SUMMARY mL/min, and the gradient profile was 0-0.1 min, 5% B; from 0 to 3 min, linear CH:CHROMATOGRAPHY_SUMMARY gradient to 20% B; from 3 to 4.3 min, isocratic 20% B; from 4.3 to 9 min, linear CH:CHROMATOGRAPHY_SUMMARY gradient to 45% B; from 9 to 11 min, linear gradient to 100% B; from 11 to 13 CH:CHROMATOGRAPHY_SUMMARY min, wash at 100% B; from 13.01 to 15 min, back to the initial conditions of 5% CH:CHROMATOGRAPHY_SUMMARY B. The injection volume of both the standard solutions and the samples was 2 CH:CHROMATOGRAPHY_SUMMARY μL. After each injection, the needle was rinsed with 600 μL of weak wash CH:CHROMATOGRAPHY_SUMMARY solution (water/methanol, 90:10) and 200 μL of strong wash solution CH:CHROMATOGRAPHY_SUMMARY (methanol/water, 90:10). Samples were kept at 6 °C during the analysis. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters Acquity CH:COLUMN_NAME Waters ACQUITY UPLC HSS T3 (100 x 2.1mm,1.8um) CH:SOLVENT_A 99.9% water/0.1% formic acid CH:SOLVENT_B 99.9% acetonitrile/0.1% formic acid CH: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 CH:FLOW_GRADIENT min: linear 45% B, 9.0-11.0 min: linear 100% B, 11.0-13.0 min: wash 100% B, CH:FLOW_GRADIENT 13.01 – 15.0 min: back to initial 5% B CH:FLOW_RATE 0.4 ml/min CH:COLUMN_TEMPERATURE 40 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Waters Xevo TQ-XS MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Capillary voltage was 3.5 kV in positive mode and −2.5 kV in negative mode; MS:MS_COMMENTS the source was kept at 150 °C; desolvation temperature was 500 °C; cone gas MS:MS_COMMENTS flow, 50 L/h; and desolvation gas flow, 800 L/h. Unit resolution was applied to MS:MS_COMMENTS each quadrupole. Flow injections of each individual metabolite were used to MS:MS_COMMENTS optimize the MRM conditions. For the majority of the metabolites, this was done MS:MS_COMMENTS automatically by the Waters Intellistart software, whereas for some compounds MS:MS_COMMENTS the optimal cone voltages and collision energies were identified during MS:MS_COMMENTS collision-induced dissociation (CID) experiments and manually set. A dwell time MS:MS_COMMENTS of at least 25 ms was applied to each MRM transition. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS mg/L MS_METABOLITE_DATA_START Samples Young Leaf_Malus domestica_YLR1 Young Leaf_Malus domestica_YLR2 Young Leaf_Malus domestica_YLR3 Old Leaf_Malus domestica_OLR1 Old Leaf_Malus domestica_OLR2 Old Leaf_Malus domestica_OLR3 Fruit_Malus domestica_FR1 Fruit_Malus domestica_FR2 Fruit_Malus domestica_FR3 Young Leaf_Pyrus communis_YLR1 Young Leaf_Pyrus communis_YLR2 Young Leaf_Pyrus communis_YLR3 Old Leaf_Pyrus communis_OLR1 Old Leaf_Pyrus communis_OLR2 Old Leaf_Pyrus communis_OLR3 Fruit_Pyrus communis_FR1 Fruit_Pyrus communis_FR2 Fruit_Pyrus communis_FR3 Young Leaf_Apple pear hybrid_YLR1 Young Leaf_Apple pear hybrid_YLR2 Young Leaf_Apple pear hybrid_YLR3 Old Leaf_Apple pear hybrid_OLR1 Old Leaf_Apple pear hybrid_OLR2 Old Leaf_Apple pear hybrid_OLR3 Fruit_Apple pear hybrid_FR1 Fruit_Apple pear hybrid_FR2 Fruit_Apple pear hybrid_FR3 Enzyme assay_Malus domestica_MdAS1R1 Enzyme assay_Malus domestica_MdAS1R2 Enzyme assay_Malus domestica_MdAS1R3 Enzyme assay_Malus domestica_MdAS2R1 Enzyme assay_Malus domestica_MdAS2R2 Enzyme assay_Malus domestica_MdAS2R3 Enzyme assay_Pyrus communis_PcASR1 Enzyme assay_Pyrus communis_PcASR2 Enzyme assay_Pyrus communis_PcASR3 Enzyme assay_Apple pear hybrid_HybASR1 Enzyme assay_Apple pear hybrid_HybASR2 Enzyme assay_Apple pear hybrid_HybASR3 Factors Sample source:Young Leaf | Genotype:Malus domestica | Treatment:Control Sample source:Young Leaf | Genotype:Malus domestica | Treatment:Control Sample source:Young Leaf | Genotype:Malus domestica | Treatment:Control Sample source:Old Leaf | Genotype:Malus domestica | Treatment:Control Sample source:Old Leaf | Genotype:Malus domestica | Treatment:Control Sample source:Old Leaf | Genotype:Malus domestica | Treatment:Control Sample source:Fruit | Genotype:Malus domestica | Treatment:Control Sample source:Fruit | Genotype:Malus domestica | Treatment:Control Sample source:Fruit | Genotype:Malus domestica | Treatment:Control Sample source:Young Leaf | Genotype:Pyrus communis | Treatment:Control Sample source:Young Leaf | Genotype:Pyrus communis | Treatment:Control Sample source:Young Leaf | Genotype:Pyrus communis | Treatment:Control Sample source:Old Leaf | Genotype:Pyrus communis | Treatment:Control Sample source:Old Leaf | Genotype:Pyrus communis | Treatment:Control Sample source:Old Leaf | Genotype:Pyrus communis | Treatment:Control Sample source:Fruit | Genotype:Pyrus communis | Treatment:Control Sample source:Fruit | Genotype:Pyrus communis | Treatment:Control Sample source:Fruit | Genotype:Pyrus communis | Treatment:Control Sample source:Young Leaf | Genotype:Apple pear hybrid | Treatment:Control Sample source:Young Leaf | Genotype:Apple pear hybrid | Treatment:Control Sample source:Young Leaf | Genotype:Apple pear hybrid | Treatment:Control Sample source:Old Leaf | Genotype:Apple pear hybrid | Treatment:Control Sample source:Old Leaf | Genotype:Apple pear hybrid | Treatment:Control Sample source:Old Leaf | Genotype:Apple pear hybrid | Treatment:Control Sample source:Fruit | Genotype:Apple pear hybrid | Treatment:Control Sample source:Fruit | Genotype:Apple pear hybrid | Treatment:Control Sample source:Fruit | Genotype:Apple pear hybrid | Treatment:Control Sample source:Enzyme assay | Genotype:Malus domestica | Treatment:5ug Sample source:Enzyme assay | Genotype:Malus domestica | Treatment:5ug Sample source:Enzyme assay | Genotype:Malus domestica | Treatment:5ug Sample source:Enzyme assay | Genotype:Malus domestica | Treatment:5ug Sample source:Enzyme assay | Genotype:Malus domestica | Treatment:5ug Sample source:Enzyme assay | Genotype:Malus domestica | Treatment:5ug Sample source:Enzyme assay | Genotype:Pyrus communis | Treatment:5ug Sample source:Enzyme assay | Genotype:Pyrus communis | Treatment:5ug Sample source:Enzyme assay | Genotype:Pyrus communis | Treatment:5ug Sample source:Enzyme assay | Genotype:Apple pear hybrid | Treatment:5ug Sample source:Enzyme assay | Genotype:Apple pear hybrid | Treatment:5ug Sample source:Enzyme assay | Genotype:Apple pear hybrid | Treatment:5ug Phloretin 0.04 0.02 0.02 0.27 0.29 0.27 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.91 0.71 0.82 0.33 0.52 0.57 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Phloridzin 67.70 60.16 61.57 54.08 56.11 49.38 0.12 0.10 0.10 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 39.88 46.32 45.40 24.46 25.18 25.44 0.02 0.02 0.02 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Ellagic acid 0.46 0.43 0.35 0.19 0.43 0.31 0.00 0.00 0.00 0.24 0.29 0.31 0.00 0.18 0.02 0.01 0.00 0.00 0.33 0.24 0.05 0.21 0.00 0.17 0.01 0.01 0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Neochlorogenic acid 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.66 1.56 1.71 11.77 12.03 11.93 0.00 0.00 0.00 0.36 0.40 0.40 1.32 1.59 1.71 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Chlorogenic acid 0.12 0.14 0.11 0.72 0.69 0.80 0.38 0.34 0.39 66.36 66.27 69.76 52.64 50.93 45.82 0.44 0.40 0.43 30.15 32.63 33.07 35.25 38.85 40.58 0.55 0.65 0.58 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Cryptochlorogenic acid 0.07 0.12 0.11 0.06 0.06 0.03 0.02 0.01 0.01 0.07 0.06 0.07 1.64 1.65 1.71 0.01 0.01 0.00 0.32 0.31 0.34 0.35 0.42 0.44 0.07 0.01 0.06 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Naringenin 0.01 0.02 0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.02 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Eriodictyol 0.48 0.24 0.16 0.14 0.15 0.24 0.03 0.03 0.03 0.14 0.14 0.15 0.17 0.14 0.12 0.03 0.03 0.03 0.00 0.16 0.20 0.14 0.14 0.28 0.03 0.03 0.03 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Catechin 0.22 0.18 0.15 0.04 0.03 0.00 0.08 0.05 0.02 4.85 4.96 4.94 3.15 2.95 2.81 0.07 0.07 0.07 7.31 7.98 8.45 7.75 8.20 8.06 0.29 0.31 0.30 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Epicatechin 0.63 0.69 0.74 1.97 1.58 1.74 0.90 0.80 0.79 6.16 5.95 6.16 10.63 11.12 10.12 0.24 0.25 0.24 1.48 1.70 2.44 4.32 4.86 5.09 0.88 0.80 0.84 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Gallocatechin 0.16 0.19 0.17 0.00 0.00 0.00 0.00 0.00 0.00 0.28 0.26 0.19 0.00 0.00 0.00 0.00 0.00 0.00 0.20 0.23 0.13 0.00 0.00 0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Epigallocatechin 0.00 0.00 0.00 0.06 0.00 0.07 0.01 0.01 0.01 0.00 0.00 0.05 0.04 0.04 0.03 0.01 0.01 0.01 0.05 0.06 0.00 0.03 0.04 0.03 0.01 0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Epicatechin gallate 0.03 0.02 0.02 0.00 0.00 0.00 0.00 0.01 0.00 0.00 0.00 0.00 0.03 0.00 0.00 0.01 0.01 0.00 0.00 0.03 0.03 0.00 0.03 0.03 0.01 0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Gallocatechin gallate 0.00 0.03 0.03 0.03 0.03 0.03 0.01 0.01 0.01 0.03 0.04 0.04 0.00 0.00 0.00 0.01 0.01 0.00 0.03 0.04 0.00 0.03 0.03 0.00 0.00 0.00 0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Epigallocatechin gallate 0.00 0.17 0.17 0.19 0.19 0.16 0.04 0.00 0.04 0.19 0.19 0.20 0.00 0.00 0.00 0.04 0.04 0.00 0.18 0.20 0.00 0.19 0.18 0.00 0.04 0.00 0.04 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Procyanidin B1 1.33 0.33 0.31 0.35 2.23 1.63 0.00 0.00 0.00 0.78 0.69 0.27 0.18 1.01 0.90 0.05 0.10 0.13 1.05 1.24 1.35 8.71 0.00 0.11 2.63 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Procyanidin B2+B4 0.31 0.21 0.20 0.58 0.55 0.64 0.97 0.73 0.90 1.59 1.51 1.62 4.46 4.93 4.19 0.12 0.14 0.17 0.46 0.56 0.44 0.68 1.07 0.95 1.51 1.48 1.52 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Procyanidin B3 0.08 0.07 0.07 0.10 0.18 0.08 0.17 0.13 0.13 0.69 0.57 0.49 0.19 1.81 2.30 0.08 0.07 0.07 0.28 0.20 0.25 1.57 0.25 0.22 0.85 0.83 0.05 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Arbutin 0.00 0.13 0.06 0.13 0.12 0.19 0.00 0.00 0.00 145.50 145.50 150.22 129.56 124.02 108.78 0.53 0.52 0.46 45.78 48.36 53.14 28.99 31.21 30.75 0.15 0.21 0.17 7.13 7.06 7.19 0.13 0.07 0.44 4.47 5.25 6.11 7.57 6.67 8.39 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Phloretin Phloridzin Ellagic acid Neochlorogenic acid Chlorogenic acid Cryptochlorogenic acid Naringenin Eriodictyol Catechin Epicatechin Gallocatechin Epigallocatechin Epicatechin gallate Gallocatechin gallate Epigallocatechin gallate Procyanidin B1 Procyanidin B2+B4 Procyanidin B3 Arbutin METABOLITES_END #END