#METABOLOMICS WORKBENCH Simon_Miranda_20240412_055127 DATATRACK_ID:4776 STUDY_ID:ST003173 ANALYSIS_ID:AN005207 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                      	POSITIVE
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
Quercetin	0.60	0.00	0.44	0.00	0.00	0.01	0.00	0.00	0.00	0.00	0.01	0.03	0.00	0.00	0.00	0.00	0.00	0.00	0.03	0.03	0.03	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
Quercetin-3'O-rhamnoside	2.35	2.31	2.22	2.34	2.48	2.73	0.01	0.01	0.01	0.04	0.03	0.05	0.02	0.04	0.04	0.00	0.00	0.00	0.24	0.24	0.28	0.44	0.41	0.47	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
Quercetin-3'O-glucoside	1.69	1.72	1.62	1.38	1.54	1.52	0.01	0.00	0.00	0.09	0.09	0.08	0.27	0.26	0.22	0.00	0.00	0.00	0.13	0.13	0.17	0.61	0.68	0.61	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
Quercetin-3'O-galactoside	1.69	1.72	1.62	1.38	1.54	1.52	0.01	0.00	0.00	0.65	0.60	0.67	0.61	0.54	0.54	0.00	0.00	0.00	0.13	0.13	0.17	0.43	0.31	0.61	0.00	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
Rutin	0.00	0.00	0.00	0.28	0.26	0.35	0.00	0.00	0.00	13.89	14.12	15.52	11.20	11.25	11.50	0.00	0.00	0.00	10.95	11.87	12.42	5.86	6.67	6.73	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
Kaempferol-3'O-glucoside	0.13	0.14	0.12	0.05	0.04	0.05	0.00	0.00	0.00	0.16	0.16	0.16	0.06	0.07	0.08	0.00	0.00	0.00	0.26	0.26	0.28	0.15	0.16	0.17	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
Kaempferol-3'O-rutinoside	0.37	0.44	0.39	0.00	0.00	0.00	0.00	0.00	0.00	1.45	1.45	1.79	0.69	0.61	0.74	0.00	0.00	0.00	2.14	2.25	2.39	0.58	0.74	0.67	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
Laricitrin	0.05	0.00	0.04	0.05	0.05	0.04	0.01	0.00	0.00	0.05	0.00	0.05	0.05	0.00	0.04	0.00	0.01	0.00	0.04	0.05	0.00	0.05	0.04	0.05	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
Isorhamnetin-3'O-glucoside	0.01	0.02	0.01	0.00	0.00	0.00	0.00	0.00	0.00	0.03	0.03	0.05	0.06	0.09	0.09	0.01	0.00	0.00	0.00	0.01	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	0.00	0.00	0.00
Isorhamnetin-3'O-rutinoside	0.04	0.05	0.05	0.01	0.01	0.01	0.00	0.00	0.00	0.94	0.87	1.04	0.65	0.57	0.60	0.00	0.00	0.00	0.54	0.55	0.63	0.15	0.19	0.20	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
Hydroquinone	0.2	0.0	0.5	0.0	0.6	6.3	0.0	0.0	0.0	30.2	29.2	27.2	7.8	6.2	5.9	0.0	0.0	0.0	14.7	14.3	12.5	10.3	11.3	10.9	0.0	0.0	0.1	19.76	20.04	18.3	26.09	24.12	26.66	24.21	18.79	22.03	19.69	21.24	12.72
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name
Quercetin
Quercetin-3'O-rhamnoside
Quercetin-3'O-glucoside
Quercetin-3'O-galactoside
Rutin
Kaempferol-3'O-glucoside
Kaempferol-3'O-rutinoside
Laricitrin
Isorhamnetin-3'O-glucoside
Isorhamnetin-3'O-rutinoside
Hydroquinone
METABOLITES_END
#END