{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002062","ANALYSIS_ID":"AN003361","VERSION":"1","CREATED_ON":"November 19, 2021, 12:32 pm"},

"PROJECT":{"PROJECT_TITLE":"MICROMETABOLITE","PROJECT_SUMMARY":"MICROMETABOLITE combines experts on microbial diversity assessment, plant-microbe interactions, plant and microbial genome and transcriptome analysis, plant-based production systems, natural product chemistry and technology, metabolomics, secondary metabolites identification and metabolite production and application.","INSTITUTE":"Aristotle University of Thessaloniki","DEPARTMENT":"School of Chemical Engineering","LABORATORY":"Organic Chemistry Laboratory","LAST_NAME":"Rodic","FIRST_NAME":"Nebojsa","ADDRESS":"Stepe Stepanovica 5, Conoplja, Serbia","EMAIL":"nebojsa.rodic@hotmail.com","PHONE":"+381648766400","FUNDING_SOURCE":"This research was supported by the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 721635"},

"STUDY":{"STUDY_TITLE":"Endophytic bacteria are key players in the modulation of the secondary metabolome of Lithospermum officinale L.","STUDY_SUMMARY":"Endophytic bacteria influence plant growth and development and therefore are an attractive resource for applications in agriculture. However, little is known about the impact of these microorganisms on secondary metabolite (SM) production by medicinal plants. Here we assessed, for the first time, the effects of root endophytic bacteria on the modulation of SMs in the medicinal plant Lithospermum officinale (Boraginaceae family), with a focus on the naphthoquinones alkannin/shikonin (A/S). The study was conducted using a newly developed in vitro system as well as in the greenhouse. Targeted and non-targeted metabolomics approaches were used and supported by expression analysis of the gene PGT, encoding a key enzyme in the A/S biosynthesis pathway. Three bacterial strains, Chitinophaga sp. R-73072, Xanthomonas sp. R-73098 and Pseudomonas sp. R-71838 induced a significant increase of diverse SMs, including A/S, in L. officinale in both systems, demonstrating the strength of our approach for screening A/S derivative-inducing bacteria. Our results highlight the impact of root-endophytic bacteria on secondary metabolism in plants and indicate that production of A/S derivatives in planta likely involves cross-modulation of different metabolic pathways that can be manipulated by bacterial endophytes.","INSTITUTE":"Aristotle University of Thessaloniki","LAST_NAME":"Rodic","FIRST_NAME":"Nebojsa","ADDRESS":"Stepe Stepanovica 5","EMAIL":"nebojsa.rodic@hotmail.com","PHONE":"+381648766400","NUM_GROUPS":"7"},

"SUBJECT":{"SUBJECT_TYPE":"Plant","SUBJECT_SPECIES":"Lithospermum officinale"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"71838_1",
"Factors":{"Treatment":"Pseudomonas sp. R-71838"},
"Additional sample data":{"RAW_FILE_NAME":"71838_1.raw"}
},
{
"Subject ID":"-",
"Sample ID":"71838_2",
"Factors":{"Treatment":"Pseudomonas sp. R-71838"},
"Additional sample data":{"RAW_FILE_NAME":"71838_2.raw"}
},
{
"Subject ID":"-",
"Sample ID":"71838_3",
"Factors":{"Treatment":"Pseudomonas sp. R-71838"},
"Additional sample data":{"RAW_FILE_NAME":"71838_3.raw"}
},
{
"Subject ID":"-",
"Sample ID":"71838_4",
"Factors":{"Treatment":"Pseudomonas sp. R-71838"},
"Additional sample data":{"RAW_FILE_NAME":"71838_4.raw"}
},
{
"Subject ID":"-",
"Sample ID":"71838_5",
"Factors":{"Treatment":"Pseudomonas sp. R-71838"},
"Additional sample data":{"RAW_FILE_NAME":"71838_5.raw"}
},
{
"Subject ID":"-",
"Sample ID":"71838_6",
"Factors":{"Treatment":"Pseudomonas sp. R-71838"},
"Additional sample data":{"RAW_FILE_NAME":"71838_6.raw"}
},
{
"Subject ID":"-",
"Sample ID":"71875_1",
"Factors":{"Treatment":"Brevibacterium sp. R-71875"},
"Additional sample data":{"RAW_FILE_NAME":"71875_1.raw"}
},
{
"Subject ID":"-",
"Sample ID":"71875_2",
"Factors":{"Treatment":"Brevibacterium sp. R-71875"},
"Additional sample data":{"RAW_FILE_NAME":"71875_2.raw"}
},
{
"Subject ID":"-",
"Sample ID":"71875_3",
"Factors":{"Treatment":"Brevibacterium sp. R-71875"},
"Additional sample data":{"RAW_FILE_NAME":"71875_3.raw"}
},
{
"Subject ID":"-",
"Sample ID":"71875_4",
"Factors":{"Treatment":"Brevibacterium sp. R-71875"},
"Additional sample data":{"RAW_FILE_NAME":"71875_4.raw"}
},
{
"Subject ID":"-",
"Sample ID":"71875_5",
"Factors":{"Treatment":"Brevibacterium sp. R-71875"},
"Additional sample data":{"RAW_FILE_NAME":"71875_5.raw"}
},
{
"Subject ID":"-",
"Sample ID":"71875_6",
"Factors":{"Treatment":"Brevibacterium sp. R-71875"},
"Additional sample data":{"RAW_FILE_NAME":"71875_6.raw"}
},
{
"Subject ID":"-",
"Sample ID":"72269_1",
"Factors":{"Treatment":"Chitinophaga sp. R-72269"},
"Additional sample data":{"RAW_FILE_NAME":"72269_1.raw"}
},
{
"Subject ID":"-",
"Sample ID":"72269_2",
"Factors":{"Treatment":"Chitinophaga sp. R-72269"},
"Additional sample data":{"RAW_FILE_NAME":"72269_2.raw"}
},
{
"Subject ID":"-",
"Sample ID":"72269_3",
"Factors":{"Treatment":"Chitinophaga sp. R-72269"},
"Additional sample data":{"RAW_FILE_NAME":"72269_3.raw"}
},
{
"Subject ID":"-",
"Sample ID":"72269_4",
"Factors":{"Treatment":"Chitinophaga sp. R-72269"},
"Additional sample data":{"RAW_FILE_NAME":"72269_4.raw"}
},
{
"Subject ID":"-",
"Sample ID":"72269_5",
"Factors":{"Treatment":"Chitinophaga sp. R-72269"},
"Additional sample data":{"RAW_FILE_NAME":"72269_5.raw"}
},
{
"Subject ID":"-",
"Sample ID":"72269_6",
"Factors":{"Treatment":"Chitinophaga sp. R-72269"},
"Additional sample data":{"RAW_FILE_NAME":"72269_6.raw"}
},
{
"Subject ID":"-",
"Sample ID":"72433_1",
"Factors":{"Treatment":"Rhizobium sp. R-72433"},
"Additional sample data":{"RAW_FILE_NAME":"72433_1.raw"}
},
{
"Subject ID":"-",
"Sample ID":"72433_2",
"Factors":{"Treatment":"Rhizobium sp. R-72433"},
"Additional sample data":{"RAW_FILE_NAME":"72433_2.raw"}
},
{
"Subject ID":"-",
"Sample ID":"72433_3",
"Factors":{"Treatment":"Rhizobium sp. R-72433"},
"Additional sample data":{"RAW_FILE_NAME":"72433_3.raw"}
},
{
"Subject ID":"-",
"Sample ID":"72433_4",
"Factors":{"Treatment":"Rhizobium sp. R-72433"},
"Additional sample data":{"RAW_FILE_NAME":"72433_4.raw"}
},
{
"Subject ID":"-",
"Sample ID":"72433_5",
"Factors":{"Treatment":"Rhizobium sp. R-72433"},
"Additional sample data":{"RAW_FILE_NAME":"72433_5.raw"}
},
{
"Subject ID":"-",
"Sample ID":"72433_6",
"Factors":{"Treatment":"Rhizobium sp. R-72433"},
"Additional sample data":{"RAW_FILE_NAME":"72433_6.raw"}
},
{
"Subject ID":"-",
"Sample ID":"73072_1",
"Factors":{"Treatment":"Chitinophaga sp. R-73072"},
"Additional sample data":{"RAW_FILE_NAME":"73072_1.raw"}
},
{
"Subject ID":"-",
"Sample ID":"73072_2",
"Factors":{"Treatment":"Chitinophaga sp. R-73072"},
"Additional sample data":{"RAW_FILE_NAME":"73072_2.raw"}
},
{
"Subject ID":"-",
"Sample ID":"73072_3",
"Factors":{"Treatment":"Chitinophaga sp. R-73072"},
"Additional sample data":{"RAW_FILE_NAME":"73072_3.raw"}
},
{
"Subject ID":"-",
"Sample ID":"73072_4",
"Factors":{"Treatment":"Chitinophaga sp. R-73072"},
"Additional sample data":{"RAW_FILE_NAME":"73072_4.raw"}
},
{
"Subject ID":"-",
"Sample ID":"73072_5",
"Factors":{"Treatment":"Chitinophaga sp. R-73072"},
"Additional sample data":{"RAW_FILE_NAME":"73072_5.raw"}
},
{
"Subject ID":"-",
"Sample ID":"73072_6",
"Factors":{"Treatment":"Chitinophaga sp. R-73072"},
"Additional sample data":{"RAW_FILE_NAME":"73072_6.raw"}
},
{
"Subject ID":"-",
"Sample ID":"73098_1",
"Factors":{"Treatment":"Xanthomonas sp. R-73098"},
"Additional sample data":{"RAW_FILE_NAME":"73098_1.raw"}
},
{
"Subject ID":"-",
"Sample ID":"73098_2",
"Factors":{"Treatment":"Xanthomonas sp. R-73098"},
"Additional sample data":{"RAW_FILE_NAME":"73098_2.raw"}
},
{
"Subject ID":"-",
"Sample ID":"73098_3",
"Factors":{"Treatment":"Xanthomonas sp. R-73098"},
"Additional sample data":{"RAW_FILE_NAME":"73098_3.raw"}
},
{
"Subject ID":"-",
"Sample ID":"73098_4",
"Factors":{"Treatment":"Xanthomonas sp. R-73098"},
"Additional sample data":{"RAW_FILE_NAME":"73098_4.raw"}
},
{
"Subject ID":"-",
"Sample ID":"73098_5",
"Factors":{"Treatment":"Xanthomonas sp. R-73098"},
"Additional sample data":{"RAW_FILE_NAME":"73098_5.raw"}
},
{
"Subject ID":"-",
"Sample ID":"73098_6",
"Factors":{"Treatment":"Xanthomonas sp. R-73098"},
"Additional sample data":{"RAW_FILE_NAME":"73098_6.raw"}
},
{
"Subject ID":"-",
"Sample ID":"Cminus_1",
"Factors":{"Treatment":"Control"},
"Additional sample data":{"RAW_FILE_NAME":"Cminus_1.raw"}
},
{
"Subject ID":"-",
"Sample ID":"Cminus_2",
"Factors":{"Treatment":"Control"},
"Additional sample data":{"RAW_FILE_NAME":"Cminus_2.raw"}
},
{
"Subject ID":"-",
"Sample ID":"Cminus_3",
"Factors":{"Treatment":"Control"},
"Additional sample data":{"RAW_FILE_NAME":"Cminus_3.raw"}
},
{
"Subject ID":"-",
"Sample ID":"Cminus_4",
"Factors":{"Treatment":"Control"},
"Additional sample data":{"RAW_FILE_NAME":"Cminus_4.raw"}
},
{
"Subject ID":"-",
"Sample ID":"Cminus_5",
"Factors":{"Treatment":"Control"},
"Additional sample data":{"RAW_FILE_NAME":"Cminus_5.raw"}
},
{
"Subject ID":"-",
"Sample ID":"Cminus_6",
"Factors":{"Treatment":"Control"},
"Additional sample data":{"RAW_FILE_NAME":"Cminus_6.raw"}
},
{
"Subject ID":"-",
"Sample ID":"QC_6_BACTERIA_LE_24_01",
"Factors":{"Treatment":"QC"},
"Additional sample data":{"RAW_FILE_NAME":"QC_6_BACTERIA_LE_24_01.raw"}
},
{
"Subject ID":"-",
"Sample ID":"QC_6_BACTERIA_LE_24_02",
"Factors":{"Treatment":"QC"},
"Additional sample data":{"RAW_FILE_NAME":"QC_6_BACTERIA_LE_24_02.raw"}
},
{
"Subject ID":"-",
"Sample ID":"QC_6_BACTERIA_LE_24_03",
"Factors":{"Treatment":"QC"},
"Additional sample data":{"RAW_FILE_NAME":"QC_6_BACTERIA_LE_24_03.raw"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Six glass jars with three plants each were used per treatment. Each jar containing three plants was thus considered as a biological replicate; six biological replicates per treatment were used. The treatments consisted of inoculating bacteria. PBS was used as a negative control treatment. Plants were grown for seven weeks. The root system of each plant was harvested separately and fresh weight was recorded before lyophilisation. The dry weight was then measured and the samples stored at -80C for analysis of the content of A/Sd by targeted and non-targeted metabolomics","SAMPLE_TYPE":"Plant"},

"TREATMENT":{"TREATMENT_SUMMARY":"A set of 50 bacteria previously isolated from another A/Sd-producing plant, Alkanna tinctoria L. (Tausch), was selected based on diversity and on phenotypic traits tested in vitro (Rat et al., 2021). The 50 strains were first grown in 35 ml Reasoner’s 2A broth (R2B, Acumedia) at 28°C, 100 rpm for 72 h. Before preparing the inocula, 5 ml of each bacterial culture was sampled and used to estimate bacterial concentrations via counting of colony-forming units (CFU), while the remaining culture was used as inoculum. The inoculum was centrifuged at 4°C, 14000 rpm for 10 min, the supernatant was discarded, and the pellet was preserved in 2 ml of R2B medium supplemented with 10% glycerol. Bacterial pellets were then stored at -20°C until inoculation. To prepare the inoculum, a pellet was suspended in 28 ml of sterile phosphate-buffered saline (PBS) at pH 7.4. The resuspended inoculum was then adjusted to a concentration of 104-106 CFU/ml, and ten µl were used to inoculate each plant. The bacterial suspension was first injected in the MSRmod medium with a micropipette. Then, a shoot tip of L. officinale of 3.5 cm length was selected and the top two-three leaves were removed by cutting. The plant was finally transferred in sterile conditions and inserted at the positions where the bacteria had been injected in the medium. Plants treated with only PBS were used as non-inoculated controls. To avoid light in the root compartment, which can inhibit the production of shikonin (Yazaki et al., 1999), the surface of the medium was covered with sterilized (in an oven at 145°C during 10 h) quartz sand and the lower half of the jar was wrapped with aluminium foil (Figure 1). The jars were then incubated in a growth chamber at 20ºC, 16:8 h light:dark, with a light intensity of 50 µmol m-2s-1. Plants were harvested for analysis after seven weeks of incubation"},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"The lyophilised roots were ground to a fine powder using a ball mill (Fritsch Pulverisette 0, Germany). For each powdered sample, a subsample of 35 mg was placed into microcentrifuge tubes for SMs extraction with 1.5 ml of methanol (LC-MS grade, Honeywell Riedel de Haën, USA) in an ultrasound bath at 10% power for three hours (Bandelin Sonorex Digital 10P, Berlin, Germany) followed by centrifugation for 10 minutes at 12500 rpm (Hermle Z 216 MK, Wehingen, Germany). The supernatants were collected and subjected to Ultra-High-Performance Liquid Chromatography-High Resolution Mass Spectrometry (UHPLC-HRMS) analysis, after filtering with 0.22 μm polytetrafluorethylene (PTFE) filters."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Thermo Vanquish","COLUMN_NAME":"Waters Acquity UPLC HSS C18 SB 1.8 μm 2.1 x 100 mm"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"INSTRUMENT_NAME":"Thermo Q Exactive Focus","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"POSITIVE","MS_COMMENTS":"The solvents used were ultrapure water (A) and methanol (B), both with 0.1% (v/v) formic acid. The gradient elution program was as follows: 0 min 95A/5B, 1 min 50A/50B, 8 min 0A/100B, 13 min 0A/100B, 13.01 min 95A/5B, 16 min 95A/5B. Data were acquired in positive ionisation mode, with the capillary temperature set to 320 oC using the instrument's MS/MS discovery feature. The normalised collision energy was set to 35 eV. The instrument control, acquisition and initial processing of the data were conducted by the Xcalibur software (version 4.1, Thermo Scientific, USA). Furthermore, data alignment and feature extraction were performed on the XCMS online platform (Huan et al., 2017). Identification of some detected compounds was performed using the software Compound Discoverer (version 3.2, Thermo Scientific, USA).","MS_RESULTS_FILE":"ST002062_AN003361_Results.txt UNITS:intensity units Has m/z:Yes Has RT:Yes RT units:Minutes"}

}