#METABOLOMICS WORKBENCH b72tipam_20231128_132046 DATATRACK_ID:4480 STUDY_ID:ST002995 ANALYSIS_ID:AN004919 PROJECT_ID:PR001865 VERSION 1 CREATED_ON November 29, 2023, 3:32 pm #PROJECT PR:PROJECT_TITLE Metabolomic study of the Quercus ilex response to Phytophthora cinnamomi, the PR:PROJECT_TITLE main causal agent of the decline syndrome, and the identification of putative PR:PROJECT_TITLE markers of resistance PR:PROJECT_TYPE LC-MS analysis PR:PROJECT_SUMMARY Holm oak (Quercus ilex) is considered one of the major structural elements of PR:PROJECT_SUMMARY the Mediterranean forests and the agrosilvopastoral Spanish “dehesa”, PR:PROJECT_SUMMARY representing an outstanding example of ecological and socio-economic PR:PROJECT_SUMMARY sustainability of forest ecosystems. The exotic pathogen Phytophthora cinnamomi PR:PROJECT_SUMMARY is one of the most aggressive of woody species, and together drought is PR:PROJECT_SUMMARY considered one of the main drivers of holm oak decline. The effect and responses PR:PROJECT_SUMMARY of P. cinnamomi inoculation has been studied on the offspring of mother trees PR:PROJECT_SUMMARY growing in declined and non-declined areas of two Andalusian populations PR:PROJECT_SUMMARY (Cordoba and Huelva). Damage symptoms, mortality, and chlorophyll fluorescence PR:PROJECT_SUMMARY have been evaluated in seedlings inoculated under humid and drought conditions. PR:PROJECT_SUMMARY The effect and responses depended on the population, being more accused in PR:PROJECT_SUMMARY Huelva than in Cordoba population. An integrative proteomic and metabolomic PR:PROJECT_SUMMARY analysis revealed the involvement of different metabolic pathways in response to PR:PROJECT_SUMMARY the pathogen in both populations, such as amino acid metabolism pathways in PR:PROJECT_SUMMARY Huelva, and terpenoids and flavonoids biosynthesis in Cordoba. However, a PR:PROJECT_SUMMARY differential response was not observed between seedlings inoculated under humid PR:PROJECT_SUMMARY and drought conditions. A protective mechanism of the photosynthetic apparatus PR:PROJECT_SUMMARY is launched in response to defective photosynthetic activity in inoculated PR:PROJECT_SUMMARY plants, which seems to be more efficient in the Cordoba population. In addition, PR:PROJECT_SUMMARY enzymes and metabolites of the phenylpropanoid and flavonoid biosynthesis PR:PROJECT_SUMMARY pathways may confer higher resistance to Cordoba population. Some of these PR:PROJECT_SUMMARY enzymes are proposed as markers of resilience, among which glyoxalase I, PR:PROJECT_SUMMARY glutathione reductase, thioredoxin reductase, and cinnamyl alcohol dehydrogenase PR:PROJECT_SUMMARY are candidates. PR:INSTITUTE University of Cordoba PR:DEPARTMENT Department of Biochemistry and Molecular Biology PR:LABORATORY Agroforestry and Plant Biochemistry, Proteomics and Systems Biology PR:LAST_NAME Tienda Parrilla PR:FIRST_NAME Marta PR:ADDRESS Campus de Rabanales, Córdoba, Córdoba, 14014, Spain PR:EMAIL b72tipam@uco.es PR:PHONE 634925272 PR:FUNDING_SOURCE This research was funded by the Spanish Ministry of Economy and Competitiveness PR:FUNDING_SOURCE in the framework of Projects PID2019-109038RB-I00 and PID2022-141599OB-I00, and PR:FUNDING_SOURCE PROYEXCEL_00881 grants from the Junta de Andalucía #STUDY ST:STUDY_TITLE Untargeted metabolomics of Quercus ilex seedlings under drought and Phytophthora ST:STUDY_TITLE cinnamomi inoculation stresses ST:STUDY_SUMMARY Holm oak (Quercus ilex) is considered one of the major structural elements of ST:STUDY_SUMMARY the Mediterranean forests and the agrosilvopastoral Spanish “dehesa”, ST:STUDY_SUMMARY representing an outstanding example of ecological and socio-economic ST:STUDY_SUMMARY sustainability of forest ecosystems. The exotic pathogen Phytophthora cinnamomi ST:STUDY_SUMMARY is one of the most aggressive of woody species, and together drought is ST:STUDY_SUMMARY considered one of the main drivers of holm oak decline. The effect and responses ST:STUDY_SUMMARY of P. cinnamomi inoculation has been studied on the offspring of mother trees ST:STUDY_SUMMARY growing in declined and non-declined areas of two Andalusian populations ST:STUDY_SUMMARY (Cordoba and Huelva). Damage symptoms, mortality, and chlorophyll fluorescence ST:STUDY_SUMMARY have been evaluated in seedlings inoculated under humid and drought conditions. ST:STUDY_SUMMARY The effect and responses depended on the population, being more accused in ST:STUDY_SUMMARY Huelva than in Cordoba population. An integrative proteomic and metabolomic ST:STUDY_SUMMARY analysis revealed the involvement of different metabolic pathways in response to ST:STUDY_SUMMARY the pathogen in both populations, such as amino acid metabolism pathways in ST:STUDY_SUMMARY Huelva, and terpenoids and flavonoids biosynthesis in Cordoba. However, a ST:STUDY_SUMMARY differential response was not observed between seedlings inoculated under humid ST:STUDY_SUMMARY and drought conditions. A protective mechanism of the photosynthetic apparatus ST:STUDY_SUMMARY is launched in response to defective photosynthetic activity in inoculated ST:STUDY_SUMMARY plants, which seems to be more efficient in the Cordoba population. In addition, ST:STUDY_SUMMARY enzymes and metabolites of the phenylpropanoid and flavonoid biosynthesis ST:STUDY_SUMMARY pathways may confer higher resistance to Cordoba population. Some of these ST:STUDY_SUMMARY enzymes are proposed as markers of resilience, among which glyoxalase I, ST:STUDY_SUMMARY glutathione reductase, thioredoxin reductase, and cinnamyl alcohol dehydrogenase ST:STUDY_SUMMARY are candidates. ST:INSTITUTE University of Cordoba ST:DEPARTMENT Department of Biochemistry and Molecular Biology ST:LABORATORY Agroforestry and Plant Biochemistry, Proteomics and Systems Biology ST:LAST_NAME Tienda Parrilla ST:FIRST_NAME Marta ST:ADDRESS Campus de Rabanales, Córdoba, Córdoba, 14014, Spain ST:EMAIL b72tipam@uco.es ST:STUDY_TYPE Untargeted MS-based metabolomics ST:PHONE 634925272 #SUBJECT SU:SUBJECT_TYPE Plant SU:SUBJECT_SPECIES Quercus ilex SU:TAXONOMY_ID 1753 SU:AGE_OR_AGE_RANGE Six-month-old seedlings #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 - CO_Control_1_pos Factor:CO_Control RAW_FILE_NAME=220505_2_CO_Control_11_pos.mzXML SUBJECT_SAMPLE_FACTORS - CO_Control_2_pos Factor:CO_Control RAW_FILE_NAME=220505_3_CO_Control_12_pos.mzXML SUBJECT_SAMPLE_FACTORS - CO_Control_3_pos Factor:CO_Control RAW_FILE_NAME=220505_1_CO_Control_10_pos.mzXML SUBJECT_SAMPLE_FACTORS - CO_Combined_1_pos Factor:CO_Combined RAW_FILE_NAME=220505_5_CO_Combinado_10_pos.mzXML SUBJECT_SAMPLE_FACTORS - CO_Combined_2_pos Factor:CO_Combined RAW_FILE_NAME=220505_4_CO_Combinado_8_pos.mzXML SUBJECT_SAMPLE_FACTORS - CO_Combined_3_pos Factor:CO_Combined RAW_FILE_NAME=220505_6_CO_Combinado_asterisco_pos.mzXML SUBJECT_SAMPLE_FACTORS - HU_Control_1_pos Factor:HU_Control RAW_FILE_NAME=220505_7_HU_Control_9_pos.mzXML SUBJECT_SAMPLE_FACTORS - HU_Control_2_pos Factor:HU_Control RAW_FILE_NAME=220505_9_HU_Control_11_pos.mzXML SUBJECT_SAMPLE_FACTORS - HU_Control_3_pos Factor:HU_Control RAW_FILE_NAME=220505_8_HU_Control_10_pos.mzXML SUBJECT_SAMPLE_FACTORS - HU_Combined_1_pos Factor:HU_Combined RAW_FILE_NAME=220505_11_HU_Combinado_7_pos.mzXML SUBJECT_SAMPLE_FACTORS - HU_Combined_2_pos Factor:HU_Combined RAW_FILE_NAME=220505_10_HU_Combinado_1_pos.mzXML SUBJECT_SAMPLE_FACTORS - HU_Combined_3_pos Factor:HU_Combined RAW_FILE_NAME=220505_12_HU_Combinado_11_pos.mzXML SUBJECT_SAMPLE_FACTORS - CO_Control_1_neg Factor:CO_Control RAW_FILE_NAME=220510_2_CO_Control_11_neg.mzXML SUBJECT_SAMPLE_FACTORS - CO_Control_2_neg Factor:CO_Control RAW_FILE_NAME=220510_3_CO_Control_12_neg.mzXML SUBJECT_SAMPLE_FACTORS - CO_Control_3_neg Factor:CO_Control RAW_FILE_NAME=220510_1_CO_Control_10_neg.mzXML SUBJECT_SAMPLE_FACTORS - CO_Combined_1_neg Factor:CO_Combined RAW_FILE_NAME=220510_5_CO_Combinado_10_neg.mzXML SUBJECT_SAMPLE_FACTORS - CO_Combined_2_neg Factor:CO_Combined RAW_FILE_NAME=220510_4_CO_Combinado_8_neg.mzXML SUBJECT_SAMPLE_FACTORS - CO_Combined_3_neg Factor:CO_Combined RAW_FILE_NAME=220510_6_CO_Combinado_asterisco_neg.mzXML SUBJECT_SAMPLE_FACTORS - HU_Control_1_neg Factor:HU_Control RAW_FILE_NAME=220510_7_HU_Control_9_neg.mzXML SUBJECT_SAMPLE_FACTORS - HU_Control_2_neg Factor:HU_Control RAW_FILE_NAME=220510_9_HU_Control_11_neg.mzXML SUBJECT_SAMPLE_FACTORS - HU_Control_3_neg Factor:HU_Control RAW_FILE_NAME=220510_8_HU_Control_10_neg.mzXML SUBJECT_SAMPLE_FACTORS - HU_Combined_1_neg Factor:HU_Combined RAW_FILE_NAME=220510_11_HU_Combinado_7_neg.mzXML SUBJECT_SAMPLE_FACTORS - HU_Combined_2_neg Factor:HU_Combined RAW_FILE_NAME=220510_10_HU_Combinado_1_neg.mzXML SUBJECT_SAMPLE_FACTORS - HU_Combined_3_neg Factor:HU_Combined RAW_FILE_NAME=220510_12_HU_Combinado_11_neg.mzXML #COLLECTION CO:COLLECTION_SUMMARY Metabolomics analyses were performed when leaf chlorophyll fluorescence CO:COLLECTION_SUMMARY decreased 35% (day 12 of the experiment) on plant subjected to combined CO:COLLECTION_SUMMARY stresses, relative to the control plants. For that, all healthy leaves from CO:COLLECTION_SUMMARY three biological replicates per treatment (control and combined stresses) and CO:COLLECTION_SUMMARY population (Córdoba and Huelva) of seedlings from the non-infected adult trees CO:COLLECTION_SUMMARY were collected, washed with distilled water, dried with paper, frozen in liquid CO:COLLECTION_SUMMARY nitrogen and stored in the dark and cold (-80 ºC) until use. Six-month-old CO:COLLECTION_SUMMARY seedlings were subjected to two different treatments: (1) control (irrigation to CO:COLLECTION_SUMMARY field capacity, non-inoculated with P. cinnamomi) and (2) combined stress CO:COLLECTION_SUMMARY (inoculated with P. cinnamomi and non-irrigated) CO:SAMPLE_TYPE Plant CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY The treatments were performed as described by Ruiz-Gómez et al. 2018 and TR:TREATMENT_SUMMARY San-Eufrasio et al. 2021. Briefly, inoculation with P. cinnamomi was performed TR:TREATMENT_SUMMARY by root immersion during 10 min in Carrot-Agar (CA) liquid medium at a TR:TREATMENT_SUMMARY concentration of 43 chlamydospores/μl. Roots of control seedlings were also TR:TREATMENT_SUMMARY immersed in CA liquid without P. cinnamomi. After inoculation, seedlings were TR:TREATMENT_SUMMARY transplanted into pots with fresh perlite and flooded for 48 h. Then, excess TR:TREATMENT_SUMMARY water was removed before drought treatment. Drought was imposed by withholding TR:TREATMENT_SUMMARY water for 28 days [36]. The presence of P. cinnamomi in the inoculated plants TR:TREATMENT_SUMMARY was confirmed on day 12 of the experiment by using fine roots (<2 mm thick, ~1 TR:TREATMENT_SUMMARY cm long) placed in Petri dishes of selective medium (PARPBH) TR:TREATMENT Abiotic (drought) and biotic (inoculation with P. cinnamomi) stresses TR:PLANT_GROWTH_LOCATION Córdoba, Andalucía TR:PLANT_PLOT_DESIGN Randomized design TR:PLANT_GROWTH_STAGE Six-month-old seedlings TR:PLANT_METAB_QUENCH_METHOD Liquid N2 TR:PLANT_STORAGE Fresh #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Metabolites were extracted from freeze-dried leaf powder. Briefly, a buffer SP:SAMPLEPREP_SUMMARY containing 1200 μL of cold ethanol: water (80:20) was added to 30 mg of leaf SP:SAMPLEPREP_SUMMARY powder, tissue disruption was driven by maceration with pistil, vortexed (10 s) SP:SAMPLEPREP_SUMMARY and sonicated (ultrasonic bath, 40 kHZ for 10 min). After centrifugation SP:SAMPLEPREP_SUMMARY (16,000×g, 4°C, 6 min) the supernatant was vacuum dried at 30 °C (Speedvac, SP:SAMPLEPREP_SUMMARY Eppendorf Vacuum Concentrator Plus/5301, Eppendorf, Leicestershire, UK). SP:PROCESSING_METHOD Ethanol:Water SP:PROCESSING_STORAGE_CONDITIONS On ice SP:EXTRACTION_METHOD Ethanol:Water SP:EXTRACT_CLEANUP Centrifugation SP:EXTRACT_STORAGE -80℃ SP:SAMPLE_RESUSPENSION Methanol SP:SAMPLE_DERIVATIZATION NO SP:SAMPLE_SPIKING NO #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Chromatographic separations were performed using an Acquity UPLC BEH C18 column CH:CHROMATOGRAPHY_SUMMARY (2.1 x 100 mm, 1.7 µm) (Waters). The column was maintained at 40 °C and eluted CH:CHROMATOGRAPHY_SUMMARY under the following conditions: 5% B for 1 min, linear gradient from 5% to 100% CH:CHROMATOGRAPHY_SUMMARY in solvent B for 9 min, isocratic at 100% B for 2 min, and return to initial CH:CHROMATOGRAPHY_SUMMARY conditions, 5% B for 3 min. A flow rate of 0.5 mL/min was used. Eluent A was CH:CHROMATOGRAPHY_SUMMARY 0.1% formic acid in water and eluent B was 0.1% formic acid in methanol. CH:CHROMATOGRAPHY_SUMMARY Injection volume was 5 µl. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters Acquity CH:COLUMN_NAME Waters ACQUITY UPLC BEH C18 (100 x 2.1mm,1.7um) CH:SOLVENT_A 0.1% formic acid in water CH:SOLVENT_B 0.1% formic acid in methanol CH:FLOW_GRADIENT 5% B for 1 min, linear gradient from 5% to 100% in solvent B for 9 min, CH:FLOW_GRADIENT isocratic at 100% B for 2 min, and return to initial conditions, 5% B for 3 min CH:FLOW_RATE 0.5 mL/min CH:COLUMN_TEMPERATURE 40 °C #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE QTRAP MS:MS_TYPE Other MS:ION_MODE POSITIVE MS:MS_COMMENTS POS MS:MS_RESULTS_FILE ST002995_AN004919_Results.txt UNITS:Peak Area Has m/z:Neutral masses Has RT:Yes RT units:Minutes #END