#METABOLOMICS WORKBENCH MicrometaboliteNR_20210811_060415 DATATRACK_ID:2794 STUDY_ID:ST002062 ANALYSIS_ID:AN003361 PROJECT_ID:PR001283 VERSION 1 CREATED_ON November 19, 2021, 12:32 pm #PROJECT PR:PROJECT_TITLE MICROMETABOLITE PR:PROJECT_SUMMARY MICROMETABOLITE combines experts on microbial diversity assessment, PR:PROJECT_SUMMARY plant-microbe interactions, plant and microbial genome and transcriptome PR:PROJECT_SUMMARY analysis, plant-based production systems, natural product chemistry and PR:PROJECT_SUMMARY technology, metabolomics, secondary metabolites identification and metabolite PR:PROJECT_SUMMARY production and application. PR:INSTITUTE Aristotle University of Thessaloniki PR:DEPARTMENT School of Chemical Engineering PR:LABORATORY Organic Chemistry Laboratory PR:LAST_NAME Rodic PR:FIRST_NAME Nebojsa PR:ADDRESS Stepe Stepanovica 5, Conoplja, Serbia 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 Endophytic bacteria are key players in the modulation of the secondary ST:STUDY_TITLE metabolome of Lithospermum officinale L. ST:STUDY_SUMMARY Endophytic bacteria influence plant growth and development and therefore are an ST:STUDY_SUMMARY attractive resource for applications in agriculture. However, little is known ST:STUDY_SUMMARY about the impact of these microorganisms on secondary metabolite (SM) production ST:STUDY_SUMMARY by medicinal plants. Here we assessed, for the first time, the effects of root ST:STUDY_SUMMARY endophytic bacteria on the modulation of SMs in the medicinal plant Lithospermum ST:STUDY_SUMMARY officinale (Boraginaceae family), with a focus on the naphthoquinones ST:STUDY_SUMMARY alkannin/shikonin (A/S). The study was conducted using a newly developed in ST:STUDY_SUMMARY vitro system as well as in the greenhouse. Targeted and non-targeted ST:STUDY_SUMMARY metabolomics approaches were used and supported by expression analysis of the ST:STUDY_SUMMARY gene PGT, encoding a key enzyme in the A/S biosynthesis pathway. Three bacterial ST:STUDY_SUMMARY strains, Chitinophaga sp. R-73072, Xanthomonas sp. R-73098 and Pseudomonas sp. ST:STUDY_SUMMARY R-71838 induced a significant increase of diverse SMs, including A/S, in L. ST:STUDY_SUMMARY officinale in both systems, demonstrating the strength of our approach for ST:STUDY_SUMMARY screening A/S derivative-inducing bacteria. Our results highlight the impact of ST:STUDY_SUMMARY root-endophytic bacteria on secondary metabolism in plants and indicate that ST:STUDY_SUMMARY production of A/S derivatives in planta likely involves cross-modulation of ST:STUDY_SUMMARY different metabolic pathways that can be manipulated by bacterial endophytes. ST:INSTITUTE Aristotle University of Thessaloniki ST:LAST_NAME Rodic ST:FIRST_NAME Nebojsa ST:ADDRESS Stepe Stepanovica 5 ST:EMAIL nebojsa.rodic@hotmail.com ST:PHONE +381648766400 ST:NUM_GROUPS 7 #SUBJECT SU:SUBJECT_TYPE Plant SU:SUBJECT_SPECIES Lithospermum officinale #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 - 71838_1 Treatment:Pseudomonas sp. R-71838 RAW_FILE_NAME=71838_1.raw SUBJECT_SAMPLE_FACTORS - 71838_2 Treatment:Pseudomonas sp. R-71838 RAW_FILE_NAME=71838_2.raw SUBJECT_SAMPLE_FACTORS - 71838_3 Treatment:Pseudomonas sp. R-71838 RAW_FILE_NAME=71838_3.raw SUBJECT_SAMPLE_FACTORS - 71838_4 Treatment:Pseudomonas sp. R-71838 RAW_FILE_NAME=71838_4.raw SUBJECT_SAMPLE_FACTORS - 71838_5 Treatment:Pseudomonas sp. R-71838 RAW_FILE_NAME=71838_5.raw SUBJECT_SAMPLE_FACTORS - 71838_6 Treatment:Pseudomonas sp. R-71838 RAW_FILE_NAME=71838_6.raw SUBJECT_SAMPLE_FACTORS - 71875_1 Treatment:Brevibacterium sp. R-71875 RAW_FILE_NAME=71875_1.raw SUBJECT_SAMPLE_FACTORS - 71875_2 Treatment:Brevibacterium sp. R-71875 RAW_FILE_NAME=71875_2.raw SUBJECT_SAMPLE_FACTORS - 71875_3 Treatment:Brevibacterium sp. R-71875 RAW_FILE_NAME=71875_3.raw SUBJECT_SAMPLE_FACTORS - 71875_4 Treatment:Brevibacterium sp. R-71875 RAW_FILE_NAME=71875_4.raw SUBJECT_SAMPLE_FACTORS - 71875_5 Treatment:Brevibacterium sp. R-71875 RAW_FILE_NAME=71875_5.raw SUBJECT_SAMPLE_FACTORS - 71875_6 Treatment:Brevibacterium sp. R-71875 RAW_FILE_NAME=71875_6.raw SUBJECT_SAMPLE_FACTORS - 72269_1 Treatment:Chitinophaga sp. R-72269 RAW_FILE_NAME=72269_1.raw SUBJECT_SAMPLE_FACTORS - 72269_2 Treatment:Chitinophaga sp. R-72269 RAW_FILE_NAME=72269_2.raw SUBJECT_SAMPLE_FACTORS - 72269_3 Treatment:Chitinophaga sp. R-72269 RAW_FILE_NAME=72269_3.raw SUBJECT_SAMPLE_FACTORS - 72269_4 Treatment:Chitinophaga sp. R-72269 RAW_FILE_NAME=72269_4.raw SUBJECT_SAMPLE_FACTORS - 72269_5 Treatment:Chitinophaga sp. R-72269 RAW_FILE_NAME=72269_5.raw SUBJECT_SAMPLE_FACTORS - 72269_6 Treatment:Chitinophaga sp. R-72269 RAW_FILE_NAME=72269_6.raw SUBJECT_SAMPLE_FACTORS - 72433_1 Treatment:Rhizobium sp. R-72433 RAW_FILE_NAME=72433_1.raw SUBJECT_SAMPLE_FACTORS - 72433_2 Treatment:Rhizobium sp. R-72433 RAW_FILE_NAME=72433_2.raw SUBJECT_SAMPLE_FACTORS - 72433_3 Treatment:Rhizobium sp. R-72433 RAW_FILE_NAME=72433_3.raw SUBJECT_SAMPLE_FACTORS - 72433_4 Treatment:Rhizobium sp. R-72433 RAW_FILE_NAME=72433_4.raw SUBJECT_SAMPLE_FACTORS - 72433_5 Treatment:Rhizobium sp. R-72433 RAW_FILE_NAME=72433_5.raw SUBJECT_SAMPLE_FACTORS - 72433_6 Treatment:Rhizobium sp. R-72433 RAW_FILE_NAME=72433_6.raw SUBJECT_SAMPLE_FACTORS - 73072_1 Treatment:Chitinophaga sp. R-73072 RAW_FILE_NAME=73072_1.raw SUBJECT_SAMPLE_FACTORS - 73072_2 Treatment:Chitinophaga sp. R-73072 RAW_FILE_NAME=73072_2.raw SUBJECT_SAMPLE_FACTORS - 73072_3 Treatment:Chitinophaga sp. R-73072 RAW_FILE_NAME=73072_3.raw SUBJECT_SAMPLE_FACTORS - 73072_4 Treatment:Chitinophaga sp. R-73072 RAW_FILE_NAME=73072_4.raw SUBJECT_SAMPLE_FACTORS - 73072_5 Treatment:Chitinophaga sp. R-73072 RAW_FILE_NAME=73072_5.raw SUBJECT_SAMPLE_FACTORS - 73072_6 Treatment:Chitinophaga sp. R-73072 RAW_FILE_NAME=73072_6.raw SUBJECT_SAMPLE_FACTORS - 73098_1 Treatment:Xanthomonas sp. R-73098 RAW_FILE_NAME=73098_1.raw SUBJECT_SAMPLE_FACTORS - 73098_2 Treatment:Xanthomonas sp. R-73098 RAW_FILE_NAME=73098_2.raw SUBJECT_SAMPLE_FACTORS - 73098_3 Treatment:Xanthomonas sp. R-73098 RAW_FILE_NAME=73098_3.raw SUBJECT_SAMPLE_FACTORS - 73098_4 Treatment:Xanthomonas sp. R-73098 RAW_FILE_NAME=73098_4.raw SUBJECT_SAMPLE_FACTORS - 73098_5 Treatment:Xanthomonas sp. R-73098 RAW_FILE_NAME=73098_5.raw SUBJECT_SAMPLE_FACTORS - 73098_6 Treatment:Xanthomonas sp. R-73098 RAW_FILE_NAME=73098_6.raw SUBJECT_SAMPLE_FACTORS - Cminus_1 Treatment:Control RAW_FILE_NAME=Cminus_1.raw SUBJECT_SAMPLE_FACTORS - Cminus_2 Treatment:Control RAW_FILE_NAME=Cminus_2.raw SUBJECT_SAMPLE_FACTORS - Cminus_3 Treatment:Control RAW_FILE_NAME=Cminus_3.raw SUBJECT_SAMPLE_FACTORS - Cminus_4 Treatment:Control RAW_FILE_NAME=Cminus_4.raw SUBJECT_SAMPLE_FACTORS - Cminus_5 Treatment:Control RAW_FILE_NAME=Cminus_5.raw SUBJECT_SAMPLE_FACTORS - Cminus_6 Treatment:Control RAW_FILE_NAME=Cminus_6.raw SUBJECT_SAMPLE_FACTORS - QC_6_BACTERIA_LE_24_01 Treatment:QC RAW_FILE_NAME=QC_6_BACTERIA_LE_24_01.raw SUBJECT_SAMPLE_FACTORS - QC_6_BACTERIA_LE_24_02 Treatment:QC RAW_FILE_NAME=QC_6_BACTERIA_LE_24_02.raw SUBJECT_SAMPLE_FACTORS - QC_6_BACTERIA_LE_24_03 Treatment:QC RAW_FILE_NAME=QC_6_BACTERIA_LE_24_03.raw #COLLECTION CO:COLLECTION_SUMMARY Six glass jars with three plants each were used per treatment. Each jar CO:COLLECTION_SUMMARY containing three plants was thus considered as a biological replicate; six CO:COLLECTION_SUMMARY biological replicates per treatment were used. The treatments consisted of CO:COLLECTION_SUMMARY inoculating bacteria. PBS was used as a negative control treatment. Plants were CO:COLLECTION_SUMMARY grown for seven weeks. The root system of each plant was harvested separately CO:COLLECTION_SUMMARY and fresh weight was recorded before lyophilisation. The dry weight was then CO:COLLECTION_SUMMARY measured and the samples stored at -80C for analysis of the content of A/Sd CO:COLLECTION_SUMMARY by targeted and non-targeted metabolomics CO:SAMPLE_TYPE Plant #TREATMENT TR:TREATMENT_SUMMARY A set of 50 bacteria previously isolated from another A/Sd-producing plant, TR:TREATMENT_SUMMARY Alkanna tinctoria L. (Tausch), was selected based on diversity and on phenotypic TR:TREATMENT_SUMMARY traits tested in vitro (Rat et al., 2021). The 50 strains were first grown in 35 TR:TREATMENT_SUMMARY ml Reasoner’s 2A broth (R2B, Acumedia) at 28°C, 100 rpm for 72 h. Before TR:TREATMENT_SUMMARY preparing the inocula, 5 ml of each bacterial culture was sampled and used to TR:TREATMENT_SUMMARY estimate bacterial concentrations via counting of colony-forming units (CFU), TR:TREATMENT_SUMMARY while the remaining culture was used as inoculum. The inoculum was centrifuged TR:TREATMENT_SUMMARY at 4°C, 14000 rpm for 10 min, the supernatant was discarded, and the pellet was TR:TREATMENT_SUMMARY preserved in 2 ml of R2B medium supplemented with 10% glycerol. Bacterial TR:TREATMENT_SUMMARY pellets were then stored at -20°C until inoculation. To prepare the inoculum, a TR:TREATMENT_SUMMARY pellet was suspended in 28 ml of sterile phosphate-buffered saline (PBS) at pH TR:TREATMENT_SUMMARY 7.4. The resuspended inoculum was then adjusted to a concentration of 104-106 TR:TREATMENT_SUMMARY CFU/ml, and ten µl were used to inoculate each plant. The bacterial suspension TR:TREATMENT_SUMMARY was first injected in the MSRmod medium with a micropipette. Then, a shoot tip TR:TREATMENT_SUMMARY of L. officinale of 3.5 cm length was selected and the top two-three leaves were TR:TREATMENT_SUMMARY removed by cutting. The plant was finally transferred in sterile conditions and TR:TREATMENT_SUMMARY inserted at the positions where the bacteria had been injected in the medium. TR:TREATMENT_SUMMARY Plants treated with only PBS were used as non-inoculated controls. To avoid TR:TREATMENT_SUMMARY light in the root compartment, which can inhibit the production of shikonin TR:TREATMENT_SUMMARY (Yazaki et al., 1999), the surface of the medium was covered with sterilized (in TR:TREATMENT_SUMMARY an oven at 145°C during 10 h) quartz sand and the lower half of the jar was TR:TREATMENT_SUMMARY wrapped with aluminium foil (Figure 1). The jars were then incubated in a growth TR:TREATMENT_SUMMARY chamber at 20ºC, 16:8 h light:dark, with a light intensity of 50 µmol m-2s-1. TR:TREATMENT_SUMMARY Plants were harvested for analysis after seven weeks of incubation #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The lyophilised roots were ground to a fine powder using a ball mill (Fritsch SP:SAMPLEPREP_SUMMARY Pulverisette 0, Germany). For each powdered sample, a subsample of 35 mg was SP:SAMPLEPREP_SUMMARY placed into microcentrifuge tubes for SMs extraction with 1.5 ml of methanol SP:SAMPLEPREP_SUMMARY (LC-MS grade, Honeywell Riedel de Haën, USA) in an ultrasound bath at 10% power SP:SAMPLEPREP_SUMMARY for three hours (Bandelin Sonorex Digital 10P, Berlin, Germany) followed by SP:SAMPLEPREP_SUMMARY centrifugation for 10 minutes at 12500 rpm (Hermle Z 216 MK, Wehingen, Germany). SP:SAMPLEPREP_SUMMARY The supernatants were collected and subjected to Ultra-High-Performance Liquid SP:SAMPLEPREP_SUMMARY Chromatography-High Resolution Mass Spectrometry (UHPLC-HRMS) analysis, after SP:SAMPLEPREP_SUMMARY filtering with 0.22 μm polytetrafluorethylene (PTFE) filters. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Waters Acquity UPLC HSS C18 SB 1.8 μm 2.1 x 100 mm #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Focus MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS The solvents used were ultrapure water (A) and methanol (B), both with 0.1% MS:MS_COMMENTS (v/v) formic acid. The gradient elution program was as follows: 0 min 95A/5B, 1 MS:MS_COMMENTS min 50A/50B, 8 min 0A/100B, 13 min 0A/100B, 13.01 min 95A/5B, 16 min 95A/5B. MS:MS_COMMENTS Data were acquired in positive ionisation mode, with the capillary temperature MS:MS_COMMENTS set to 320 oC using the instrument's MS/MS discovery feature. The normalised MS:MS_COMMENTS collision energy was set to 35 eV. The instrument control, acquisition and MS:MS_COMMENTS initial processing of the data were conducted by the Xcalibur software (version MS:MS_COMMENTS 4.1, Thermo Scientific, USA). Furthermore, data alignment and feature extraction MS:MS_COMMENTS were performed on the XCMS online platform (Huan et al., 2017). Identification MS:MS_COMMENTS of some detected compounds was performed using the software Compound Discoverer MS:MS_COMMENTS (version 3.2, Thermo Scientific, USA). MS:MS_RESULTS_FILE ST002062_AN003361_Results.txt UNITS:intensity units Has m/z:Yes Has RT:Yes RT units:Minutes #END