#METABOLOMICS WORKBENCH MicrometaboliteNR_20211128_084635 DATATRACK_ID:2951 STUDY_ID:ST002021 ANALYSIS_ID:AN003291 PROJECT_ID:PR001283 VERSION 1 CREATED_ON November 29, 2021, 7:54 am #PROJECT PR:PROJECT_TITLE MICROMETABOLITE PR:PROJECT_SUMMARY The overall objective of MICROMETABOLITE is to explore interactions between PR:PROJECT_SUMMARY plants and microorganisms involved in the production of secondary metabolites PR:PROJECT_SUMMARY (SM) for introducing novel ingredients in pharmaceutical and cosmeceutical PR:PROJECT_SUMMARY industry. Effects of microorganisms on the plant metabolome and the biosynthesis PR:PROJECT_SUMMARY of bioactive SM will be studied in the Boraginaceae plant family, aimed at PR:PROJECT_SUMMARY optimising plant cultivation and alkannins/shikonins (A/S) production. PR:PROJECT_SUMMARY Microorganisms will be integrated in plant production systems, and protocols PR:PROJECT_SUMMARY needed for efficient implementation in industry will be elaborated. Thereby a PR:PROJECT_SUMMARY platform will be established that will support long-term interactions between PR:PROJECT_SUMMARY academia and industry. PR:INSTITUTE Aristotle University of Thessaloniki, MICROMETABOLITE project PR:DEPARTMENT School of Chemical Engineering PR:LAST_NAME Rodic PR:FIRST_NAME Nebojsa PR:ADDRESS Stepe Stepanovica 5, Conoplja, Vojvodina, 25210, Yugoslavia PR:EMAIL nebojsa.rodic@hotmail.com PR:PHONE +381648766400 PR:FUNDING_SOURCE This research was supported by the European Union's Horizon 2020 research and PR:FUNDING_SOURCE innovation programme under the Marie Skłodowska-Curie grant agreement No 721635 #STUDY ST:STUDY_TITLE An integrated-omics approach reveals specific bacterial and fungal taxa ST:STUDY_TITLE associated with roots of Alkanna tinctoria L. Tausch correlating with ST:STUDY_TITLE medicinally relevant alkannin derivatives and other secondary metabolites ST:STUDY_SUMMARY Plants are naturally associated with diverse microbial communities, which play ST:STUDY_SUMMARY significant roles in plant performance, such as growth promotion or fending off ST:STUDY_SUMMARY pathogens. The roots of Alkanna tinctoria L. are rich in naphthoquinones, ST:STUDY_SUMMARY particularly the medicinally used chiral compounds alkannin, shikonin and their ST:STUDY_SUMMARY derivatives. Former studies already have shown that microorganisms may modulate ST:STUDY_SUMMARY plant metabolism. To further investigate the potential interaction between A. ST:STUDY_SUMMARY tinctoria and associated microorganisms we performed a greenhouse experiment, in ST:STUDY_SUMMARY which A. tinctoria plants were grown in the presence of three distinct soil ST:STUDY_SUMMARY microbiomes. At four defined plant developmental stages we made an in-depth ST:STUDY_SUMMARY assessment of bacterial and fungal root-associated microbiomes as well as all ST:STUDY_SUMMARY primary and secondary metabolites. Our results showed that the plant ST:STUDY_SUMMARY developmental stage was the most important driver influencing the plant ST:STUDY_SUMMARY metabolite content, revealing peak contents of alkannin/shikonin at the fruiting ST:STUDY_SUMMARY stage. In contrast, the soil microbiome had the biggest impact on the plant root ST:STUDY_SUMMARY microbiome. Correlation analyses performed on the measured metabolite content ST:STUDY_SUMMARY and the abundance of individual bacterial and fungal taxa suggested a dynamic, ST:STUDY_SUMMARY at times positive or negative relationship between root-associated ST:STUDY_SUMMARY microorganisms and root metabolism. In particular, the bacterial ST:STUDY_SUMMARY Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium group and the fungal species ST:STUDY_SUMMARY Penicillium jensenii were found to be positively correlated with higher content ST:STUDY_SUMMARY of alkannins. ST:INSTITUTE Aristotle University of Thessaloniki, MICROMETABOLITE project ST:DEPARTMENT School of Chemical Engineering ST:LAST_NAME Rodic ST:FIRST_NAME Nebojsa ST:ADDRESS Stepe Stepanovica 5, Conoplja, Vojvodina, 25210, Yugoslavia ST:EMAIL nebojsa.rodic@hotmail.com ST:PHONE +381648766400 #SUBJECT SU:SUBJECT_TYPE Plant SU:SUBJECT_SPECIES Alkanna tinctoria #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 - I.1 developmental stage:growth | soil type:Greek A dilution factor=1; RAW_FILE_NAME=1-3_RA_1.raw SUBJECT_SAMPLE_FACTORS - I.2 developmental stage:growth | soil type:Greek A dilution factor=1; RAW_FILE_NAME=1-3_RA_2.raw SUBJECT_SAMPLE_FACTORS - I.3 developmental stage:growth | soil type:Greek A dilution factor=1; RAW_FILE_NAME=1-3_RA_3.raw SUBJECT_SAMPLE_FACTORS - I.4 developmental stage:growth | soil type:Greek A dilution factor=1; RAW_FILE_NAME=1-3_RA_4.raw SUBJECT_SAMPLE_FACTORS - I.5 developmental stage:growth | soil type:Greek A dilution factor=1; RAW_FILE_NAME=1-3_RA_5.raw SUBJECT_SAMPLE_FACTORS - I.6 developmental stage:growth | soil type:Greek A dilution factor=1; RAW_FILE_NAME=1-3_RA_6.raw SUBJECT_SAMPLE_FACTORS - I.7 developmental stage:growth | soil type:Greek B dilution factor=1; RAW_FILE_NAME=1-3_RA_7.raw SUBJECT_SAMPLE_FACTORS - I.8 developmental stage:growth | soil type:Greek B dilution factor=1; RAW_FILE_NAME=1-3_RA_8.raw SUBJECT_SAMPLE_FACTORS - I.9 developmental stage:growth | soil type:Greek B dilution factor=1; RAW_FILE_NAME=1-3_RA_9.raw SUBJECT_SAMPLE_FACTORS - I.10 developmental stage:growth | soil type:Greek B dilution factor=1; RAW_FILE_NAME=1-3_RA_10.raw SUBJECT_SAMPLE_FACTORS - I.11 developmental stage:growth | soil type:Greek B dilution factor=1; RAW_FILE_NAME=1-3_RA_11.raw SUBJECT_SAMPLE_FACTORS - I.12 developmental stage:growth | soil type:Greek B dilution factor=1; RAW_FILE_NAME=1-3_RA_12.raw SUBJECT_SAMPLE_FACTORS - I.13 developmental stage:growth | soil type:Austrian dilution factor=1; RAW_FILE_NAME=1-3_RA_13.raw SUBJECT_SAMPLE_FACTORS - I.14 developmental stage:growth | soil type:Austrian dilution factor=1; RAW_FILE_NAME=1-3_RA_14.raw SUBJECT_SAMPLE_FACTORS - I.15 developmental stage:growth | soil type:Austrian dilution factor=1; RAW_FILE_NAME=1-3_RA_15.raw SUBJECT_SAMPLE_FACTORS - I.16 developmental stage:growth | soil type:Austrian dilution factor=1; RAW_FILE_NAME=1-3_RA_16.raw SUBJECT_SAMPLE_FACTORS - I.17 developmental stage:growth | soil type:Austrian dilution factor=1; RAW_FILE_NAME=1-3_RA_17.raw SUBJECT_SAMPLE_FACTORS - I.18 developmental stage:growth | soil type:Austrian dilution factor=1; RAW_FILE_NAME=1-3_RA_18.raw SUBJECT_SAMPLE_FACTORS - II.1 developmental stage:blooming | soil type:Greek A dilution factor=1; RAW_FILE_NAME=15-3_RA_1.raw SUBJECT_SAMPLE_FACTORS - II.2 developmental stage:blooming | soil type:Greek A dilution factor=1; RAW_FILE_NAME=15-3_RA_2.raw SUBJECT_SAMPLE_FACTORS - II.3 developmental stage:blooming | soil type:Greek A dilution factor=1; RAW_FILE_NAME=15-3_RA_3.raw SUBJECT_SAMPLE_FACTORS - II.4 developmental stage:blooming | soil type:Greek A dilution factor=1; RAW_FILE_NAME=15-3_RA_4.raw SUBJECT_SAMPLE_FACTORS - II.5 developmental stage:blooming | soil type:Greek A dilution factor=1; RAW_FILE_NAME=15-3_RA_5.raw SUBJECT_SAMPLE_FACTORS - II.6 developmental stage:blooming | soil type:Greek A dilution factor=1; RAW_FILE_NAME=15-3_RA_6.raw SUBJECT_SAMPLE_FACTORS - II.7 developmental stage:blooming | soil type:Greek B dilution factor=1; RAW_FILE_NAME=15-3_RA_7.raw SUBJECT_SAMPLE_FACTORS - II.8 developmental stage:blooming | soil type:Greek B dilution factor=1; RAW_FILE_NAME=15-3_RA_8.raw SUBJECT_SAMPLE_FACTORS - II.9 developmental stage:blooming | soil type:Greek B dilution factor=1; RAW_FILE_NAME=15-3_RA_9.raw SUBJECT_SAMPLE_FACTORS - II.10 developmental stage:blooming | soil type:Greek B dilution factor=1; RAW_FILE_NAME=15-3_RA_10.raw SUBJECT_SAMPLE_FACTORS - II.11 developmental stage:blooming | soil type:Greek B dilution factor=1; RAW_FILE_NAME=15-3_RA_11.raw SUBJECT_SAMPLE_FACTORS - II.12 developmental stage:blooming | soil type:Greek B dilution factor=1; RAW_FILE_NAME=15-3_RA_12.raw SUBJECT_SAMPLE_FACTORS - II.13 developmental stage:blooming | soil type:Austrian dilution factor=1; RAW_FILE_NAME=15-3_RA_13.raw SUBJECT_SAMPLE_FACTORS - II.14 developmental stage:blooming | soil type:Austrian dilution factor=1; RAW_FILE_NAME=15-3_RA_14.raw SUBJECT_SAMPLE_FACTORS - II.15 developmental stage:blooming | soil type:Austrian dilution factor=1; RAW_FILE_NAME=15-3_RA_15.raw SUBJECT_SAMPLE_FACTORS - II.16 developmental stage:blooming | soil type:Austrian dilution factor=1; RAW_FILE_NAME=15-3_RA_16.raw SUBJECT_SAMPLE_FACTORS - II.17 developmental stage:blooming | soil type:Austrian dilution factor=1; RAW_FILE_NAME=15-3_RA_17.raw SUBJECT_SAMPLE_FACTORS - II.18 developmental stage:blooming | soil type:Austrian dilution factor=1; RAW_FILE_NAME=15-3_RA_18.raw SUBJECT_SAMPLE_FACTORS - III.1 developmental stage:fruiting | soil type:Greek A dilution factor=1; RAW_FILE_NAME=12-4_RA_1.raw SUBJECT_SAMPLE_FACTORS - III.2 developmental stage:fruiting | soil type:Greek A dilution factor=1; RAW_FILE_NAME=12-4_RA_2.raw SUBJECT_SAMPLE_FACTORS - III.3 developmental stage:fruiting | soil type:Greek A dilution factor=1; RAW_FILE_NAME=12-4_RA_3.raw SUBJECT_SAMPLE_FACTORS - III.4 developmental stage:fruiting | soil type:Greek A dilution factor=3; RAW_FILE_NAME=12-4_RA_4D.raw SUBJECT_SAMPLE_FACTORS - III.5 developmental stage:fruiting | soil type:Greek A dilution factor=1; RAW_FILE_NAME=12-4_RA_5.raw SUBJECT_SAMPLE_FACTORS - III.6 developmental stage:fruiting | soil type:Greek A dilution factor=3; RAW_FILE_NAME=12-4_RA_6D.raw SUBJECT_SAMPLE_FACTORS - III.7 developmental stage:fruiting | soil type:Greek B dilution factor=1; RAW_FILE_NAME=12-4_RA_7.raw SUBJECT_SAMPLE_FACTORS - III.8 developmental stage:fruiting | soil type:Greek B dilution factor=3; RAW_FILE_NAME=12-4_RA_8D.raw SUBJECT_SAMPLE_FACTORS - III.9 developmental stage:fruiting | soil type:Greek B dilution factor=3; RAW_FILE_NAME=12-4_RA_9D.raw SUBJECT_SAMPLE_FACTORS - III.10 developmental stage:fruiting | soil type:Greek B dilution factor=1; RAW_FILE_NAME=12-4_RA_10.raw SUBJECT_SAMPLE_FACTORS - III.11 developmental stage:fruiting | soil type:Greek B dilution factor=1; RAW_FILE_NAME=12-4_RA_11.raw SUBJECT_SAMPLE_FACTORS - III.12 developmental stage:fruiting | soil type:Greek B dilution factor=3; RAW_FILE_NAME=12-4_RA_12D.raw SUBJECT_SAMPLE_FACTORS - III.13 developmental stage:fruiting | soil type:Austrian dilution factor=1; RAW_FILE_NAME=12-4_RA_13.raw SUBJECT_SAMPLE_FACTORS - III.14 developmental stage:fruiting | soil type:Austrian dilution factor=1; RAW_FILE_NAME=12-4_RA_14.raw SUBJECT_SAMPLE_FACTORS - III.15 developmental stage:fruiting | soil type:Austrian dilution factor=3; RAW_FILE_NAME=12-4_RA_15D.raw SUBJECT_SAMPLE_FACTORS - III.16 developmental stage:fruiting | soil type:Austrian dilution factor=3; RAW_FILE_NAME=12-4_RA_16D.raw SUBJECT_SAMPLE_FACTORS - III.17 developmental stage:fruiting | soil type:Austrian dilution factor=1; RAW_FILE_NAME=12-4_RA_17.raw SUBJECT_SAMPLE_FACTORS - III.18 developmental stage:fruiting | soil type:Austrian dilution factor=3; RAW_FILE_NAME=12-4_RA_18D.raw SUBJECT_SAMPLE_FACTORS - QC_UCL_AT_01 developmental stage:QC | soil type:NA dilution factor=1; RAW_FILE_NAME=QC_UCL_AT.raw SUBJECT_SAMPLE_FACTORS - QC_UCL_AT_02 developmental stage:QC | soil type:NA dilution factor=1; RAW_FILE_NAME=QC_UCL_AT_180730210457.raw SUBJECT_SAMPLE_FACTORS - QC_UCL_AT_03 developmental stage:QC | soil type:NA dilution factor=1; RAW_FILE_NAME=QC_UCL_AT_180731013653.raw SUBJECT_SAMPLE_FACTORS - QC_UCL_AT_04 developmental stage:QC | soil type:NA dilution factor=1; RAW_FILE_NAME=QC_UCL_AT_04.raw SUBJECT_SAMPLE_FACTORS - QC_UCL_AT_05 developmental stage:QC | soil type:NA dilution factor=1; RAW_FILE_NAME=QC_UCL_AT_05.raw SUBJECT_SAMPLE_FACTORS - QC_UCL_AT_06 developmental stage:QC | soil type:NA dilution factor=1; RAW_FILE_NAME=QC_UCL_AT_06.raw SUBJECT_SAMPLE_FACTORS - QC_UCL_AT_07 developmental stage:QC | soil type:NA dilution factor=1; RAW_FILE_NAME=QC_UCL_AT_07.raw SUBJECT_SAMPLE_FACTORS - QC_UCL_AT_08 developmental stage:QC | soil type:NA dilution factor=1; RAW_FILE_NAME=QC_UCL_AT_08.raw SUBJECT_SAMPLE_FACTORS - QC_UCL_AT_09 developmental stage:QC | soil type:NA dilution factor=1; RAW_FILE_NAME=QC_UCL_AT_09.raw #COLLECTION CO:COLLECTION_SUMMARY Alkanna tinctoria plants were provided as rooted acclimatized individuals, CO:COLLECTION_SUMMARY originally collected and identified from natural populations CO:SAMPLE_TYPE Plant #TREATMENT TR:TREATMENT_SUMMARY Plants were produced by micropropagation from several mother plants by the TR:TREATMENT_SUMMARY Hellenic Agricultural Organization (HAO, Thessaloniki, Greece). The plants were TR:TREATMENT_SUMMARY transferred to 5 L pots containing 4.5 L of sterilized (121° C for 15 min) peat TR:TREATMENT_SUMMARY moss and perlite (volume ratio 2:1), mixed with 200 g field soil collected TR:TREATMENT_SUMMARY either in Austria or in Greece. Thus, all plants were grown in a substrate with TR:TREATMENT_SUMMARY highly similar chemical and physical characteristics, but hosting those TR:TREATMENT_SUMMARY microbial communities prevailing in these three distinct soils. Plants were TR:TREATMENT_SUMMARY grown in the greenhouse at 16 h light / 8 h dark photoperiod, 25°C with 50% TR:TREATMENT_SUMMARY relative humidity (RH) and a photosynthetic photon flux density (PPFD) of 96 TR:TREATMENT_SUMMARY μmolm^-2 s^-1. Plants were watered twice per week with deionized water and TR:TREATMENT_SUMMARY moved randomly once per week. Plants were harvested at four different defined TR:TREATMENT_SUMMARY developmental stages, the first stage (“vegetative growth”) was defined when TR:TREATMENT_SUMMARY more than 50% of the individuals started to produce new leaves, “blooming” TR:TREATMENT_SUMMARY was the stage when more than 50% of the individuals had flowers, “fruiting” TR:TREATMENT_SUMMARY when more than 50% of the individual plants began to produce fruits. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Plant roots were ground to a fine powder using a ball mill (Fritsch Pulverisette SP:SAMPLEPREP_SUMMARY 0, Germany). Each powdered sample was weighed (70 mg) into microcentrifuge SP:SAMPLEPREP_SUMMARY tubes, followed by extraction with 3 mL of methanol by ultrasound at 10% power SP:SAMPLEPREP_SUMMARY for 3 h (Bandelin Sonorex Digital 10P, Berlin, Germany) and centrifugation for SP:SAMPLEPREP_SUMMARY 10 minutes at 12.500 rpm (Hermle Z 216 MK, Wehingen, Germany). The supernatants SP:SAMPLEPREP_SUMMARY were collected and subjected to UHPLC-HRMS analysis after filtering with 0.22 SP:SAMPLEPREP_SUMMARY μm syringe filters. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Accela AS CH:COLUMN_NAME Waters Acquity UPLC HSS C18 SB 1.8 μm 2.1 x 100 mm CH:FLOW_RATE 0.3mL/min CH:COLUMN_TEMPERATURE 50 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo LTQ Discovery Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS The MS/MS data were obtained for the six most intense m/z peaks in each full MS:MS_COMMENTS scan, with the normalized collision energy set to 35 eV. The acquisition and MS:MS_COMMENTS initial processing of the data were by means of XcaliburTM (Thermo Scientific, MS:MS_COMMENTS USA) software, while data alignment and feature extraction were performed MS:MS_COMMENTS utilizing the XCMS Online platform (The Scripps Research Institute, USA). The MS:MS_COMMENTS batch error correction was done with the help of MetaboAnalyst 5.0. MS:MS_RESULTS_FILE ST002021_AN003291_Results.txt UNITS:intensity units Has m/z:Yes Has RT:Yes RT units:Minutes #END