#METABOLOMICS WORKBENCH Cristina_20220225_081936 DATATRACK_ID:3106 STUDY_ID:ST002148 ANALYSIS_ID:AN003518 PROJECT_ID:PR001361 VERSION 1 CREATED_ON March 21, 2022, 10:47 am #PROJECT PR:PROJECT_TITLE Untargeted metabolomics of Quercus ilex leaves in Drought PR:PROJECT_TYPE LC-MS analysis PR:PROJECT_SUMMARY UPLC-MS analysis of samples from Quercus ilex leaves. The objective of the study PR:PROJECT_SUMMARY is to characterize the leaf metabolome of Q. ilex in six-month-old seedlings and PR:PROJECT_SUMMARY the changes that have taken place in response to drought stress by water PR:PROJECT_SUMMARY withholding under high temperature and irradiance conditions. PR:INSTITUTE Universidad de Córdoba PR:DEPARTMENT Department Biochemistry and Molecular Biology PR:LABORATORY Agroforestry and Plant Biochemistry, Proteomics and Systems Biology PR:LAST_NAME López-Hidalgo PR:FIRST_NAME Cristina PR:ADDRESS Campus de Rabanales; Edificio C6, Planta Baja PR:EMAIL lopezhcristina@uniovi.es PR:PHONE 626894948 PR:FUNDING_SOURCE This work was supported by the University of Cordoba and financial support from PR:FUNDING_SOURCE the Spanish Ministry of Economy and Competitiveness (Project ENCINOMICS-2 PR:FUNDING_SOURCE PID2019-10908RB-100) PR:PUBLICATIONS Untargeted MS-based metabolomics analysis of the responses to drought stress in PR:PUBLICATIONS Quercus ilex leaf seedlings, and identification of putative compounds related to PR:PUBLICATIONS tolerance PR:CONTRIBUTORS Marta Tienda-Parrilla, Cristina López-Hidalgo, Víctor M. Guerrero-Sánchez, PR:CONTRIBUTORS Álvaro Infantes-González, Rocío Valderrama, Jesús V. Jorrin-Novo, PR:CONTRIBUTORS María-Dolores Rey #STUDY ST:STUDY_TITLE Untargeted MS-based metabolomics analysis of the responses to drought stress in ST:STUDY_TITLE Quercus ilex leaf seedlings, and identification of putative compounds related to ST:STUDY_TITLE tolerance ST:STUDY_TYPE Untargeted MS-based metabolomics ST:STUDY_SUMMARY The effect and responses to drought stress have been analyzed in Quercus ilex ST:STUDY_SUMMARY seedlings by using a non-targeted metabolomic approach, implementing previous ST:STUDY_SUMMARY studies pub-lished in which other -omics platforms, transcriptomics, and ST:STUDY_SUMMARY proteomics, have been em-ployed. This work is aimed at characterizing the Q. ST:STUDY_SUMMARY ilex leaf metabolome, determining possible mechanisms and molecular markers of ST:STUDY_SUMMARY drought tolerance, and identifying puta-tive bioactive compounds. Six-month-old ST:STUDY_SUMMARY seedling leaves, subjected to drought stress im-posed by water withholding under ST:STUDY_SUMMARY high temperature and irradiance conditions, were col-lected when leaf ST:STUDY_SUMMARY fluorescence decreased by 20 % (day 17) and 45 % (day 24) relative to ir-rigated ST:STUDY_SUMMARY seedlings. A total of 3934 compounds were resolved, with 616 being variable, and ST:STUDY_SUMMARY 342 identified, belonging to five chemical families. Out of the identified ST:STUDY_SUMMARY compounds 33 were variable, mostly corresponding to amino acids, carboxylic ST:STUDY_SUMMARY acids, benzenoids, flavo-noids and isoprenoids. Epigallocatechin, ellagic acid, ST:STUDY_SUMMARY pulegone, indole-3-acrylic acid and dihydrozeatin-O-glucoside were ST:STUDY_SUMMARY up-accumulated under drought conditions at both sam-pling times. An integrated ST:STUDY_SUMMARY multi-omics analysis of phenolic compounds and related en-zymes was performed, ST:STUDY_SUMMARY revealing that some enzymes involved in the flavonoid pathways (chalcone ST:STUDY_SUMMARY synthase, anthocyanidin synthase and anthocyanidin reductase) were ST:STUDY_SUMMARY up-accumulated at day 24 in non-irrigated seedlings. Some putative markers of ST:STUDY_SUMMARY drought tol-erance to drought in Q. ilex are proposed for assisting breeding ST:STUDY_SUMMARY programs based on the se-lection of elite genotypes. ST:INSTITUTE new ST:DEPARTMENT Universidad de Córdoba ST:LABORATORY Agroforestry and Plant Biochemistry, Proteomics and Systems Biology ST:LAST_NAME López Hidalgo ST:FIRST_NAME Cristina ST:ADDRESS Campus de Rabanales; Edificio C6, Planta Baja ST:EMAIL lopezhcristina@uniovi.es ST:PHONE 626894948 #SUBJECT SU:SUBJECT_TYPE Plant SU:SUBJECT_SPECIES Quercus ilex SU:TAXONOMY_ID 58334 #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 - 190218_1_C_T1_R1_neg Factor:C_T1 RAW_FILE_NAME=190218_1_C_T1_R1_neg.mzXML SUBJECT_SAMPLE_FACTORS - 190218_2_C_T1_R2_neg Factor:C_T1 RAW_FILE_NAME=190218_2_C_T1_R2_neg.mzXML SUBJECT_SAMPLE_FACTORS - 190218_3_C_T1_R3_neg Factor:C_T1 RAW_FILE_NAME=190218_3_C_T1_R3_neg.mzXML SUBJECT_SAMPLE_FACTORS - 190218_4_C_T2_R1_neg Factor:C_T2 RAW_FILE_NAME=190218_4_C_T2_R1_neg.mzXML SUBJECT_SAMPLE_FACTORS - 190218_5_C_T2_R2_neg Factor:C_T2 RAW_FILE_NAME=190218_5_C_T2_R2_neg.mzXML SUBJECT_SAMPLE_FACTORS - 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190218_QCMIX_pos_TOP5_1 Factor:Quality Control mixture RAW_FILE_NAME=190218_QCMIX_pos_TOP5_1.mzXML SUBJECT_SAMPLE_FACTORS - 190218_QCMIX_pos_TOP5_2 Factor:Quality Control mixture RAW_FILE_NAME=190218_QCMIX_pos_TOP5_2.mzXML SUBJECT_SAMPLE_FACTORS - 190218_QCMIX_pos_TOP5_3 Factor:Quality Control mixture RAW_FILE_NAME=190218_QCMIX_pos_TOP5_3.mzXML #COLLECTION CO:COLLECTION_SUMMARY Out of the biological replicates, three asymptomatic (non-damaged) non-irrigated CO:COLLECTION_SUMMARY seedlings were randomly selected for metabolomics. All leaves were collected CO:COLLECTION_SUMMARY when the chlorophyll fluorescence dropped by 20 and 45 % in the non-irrigated CO:COLLECTION_SUMMARY seedlings compared to irrigated ones (at days 17 and 24, respectively) [7]. CO:COLLECTION_SUMMARY After collection, leaves from the three biological replicates per treatment and CO:COLLECTION_SUMMARY sampling time were washed with tap water, blot dried with filter paper, CO:COLLECTION_SUMMARY shock-frozen in liquid nitrogen, and stored at -80 °C until metabolite CO:COLLECTION_SUMMARY extraction. CO:SAMPLE_TYPE new CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY An experiment based on a completely randomized design was developed by using ten TR:TREATMENT_SUMMARY biological replicates per treatment [7]. Out of the biological replicates, three TR:TREATMENT_SUMMARY asymptomatic (non-damaged) non-irrigated seedlings were randomly selected for TR:TREATMENT_SUMMARY metabolomics. All leaves were collected when the chlorophyll fluorescence TR:TREATMENT_SUMMARY dropped by 20 and 45 % in the non-irrigated seedlings compared to irrigated ones TR:TREATMENT_SUMMARY (at days 17 and 24, respectively) TR:TREATMENT Drought TR:PLANT_PLOT_DESIGN Randomized design TR:PLANT_LIGHT_PERIOD 28 W m-2 solar irradiance TR:PLANT_HUMIDITY 41% humidity TR:PLANT_TEMP 37 °C temperature TR:PLANT_GROWTH_STAGE Six-month-old seedlings TR:PLANT_METAB_QUENCH_METHOD Liquid N2 TR:PLANT_STORAGE -80ºC #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Metabolites were extracted from leaves as described by Valledor et al., [41], SP:SAMPLEPREP_SUMMARY with minor modifications. An extraction solution containing 600 µL of ice-cold SP:SAMPLEPREP_SUMMARY methanol: chloroform: water (5:2:2) was added to 50 mg dry weight leaf tissue SP:SAMPLEPREP_SUMMARY and vortexed for 10 sec. The mixture was sonicated (ultrasonic bath, 40 kHz for SP:SAMPLEPREP_SUMMARY 10 min) and after centrifugation at 20,000 × g at 4°C for 4 mins, the SP:SAMPLEPREP_SUMMARY supernatant was transferred to a new tube. Then, 200 µL of cold methanol: SP:SAMPLEPREP_SUMMARY chloroform: water (5:2:2) was added to the pellets and the process was repeated SP:SAMPLEPREP_SUMMARY once. After combining both supernatants, they were vacuum dried at 30°C SP:SAMPLEPREP_SUMMARY (Speedvac, Eppendorf Vacuum Concentrator Plus/5301, Eppendorf, Leicestershire, SP:SAMPLEPREP_SUMMARY UK). Dried extracts were reconstituted in methanol, centrifuged at 20,000×g for SP:SAMPLEPREP_SUMMARY 10 min, and filtered through 0.22 μm PTPE membranes (Thermo Fisher Scientific, SP:SAMPLEPREP_SUMMARY Courtaboeuf, France), and the filtrate was collected in 1.5 mL LC/MS certified SP:SAMPLEPREP_SUMMARY sample vials. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 RS CH:COLUMN_NAME Waters Acquity BEH C8 (100 x 2.1mm, 1.7um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive HF hybrid Orbitrap MS:INSTRUMENT_TYPE QTRAP MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS The operating parameters, in positive ion mode, were sheath gas flow rate at 60 MS:MS_COMMENTS kV, auxiliary gas flow rate at 25 kV, sweep gas flow rate at 2 kV, spray voltage MS:MS_COMMENTS at 3.5 kV, capillary temperature at 320 °C, S-lens RF level at 50 kV, and MS:MS_COMMENTS auxiliary gas heater temperature at 400 °C. The Xcalibur v3.1 software was used MS:MS_COMMENTS for instrument control and data acquisition. Spectra data were acquired in a MS:MS_COMMENTS full scan (FS) mode at a resolution of 70,000 (full width half maximum, FWHM at MS:MS_COMMENTS m/z 200) for MS1, and a data dependent (dd-MS2/dd-SIM) manner for MS2, MS:MS_COMMENTS fragmenting the five most abundant precursor ions per MS1 scan (TopN, 5), MS:MS_COMMENTS acquiring MS/MS data between 200 and 2000 m/z at a resolution of 17.500. MS:MS_RESULTS_FILE ST002148_AN003518_Results.txt UNITS:peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END