Summary of Study ST002062

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001283. The data can be accessed directly via it's Project DOI: 10.21228/M8VX1B This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study ID	ST002062
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, Conoplja, Serbia
Email	nebojsa.rodic@hotmail.com
Phone	+381648766400
Submit Date	2021-08-11
Num Groups	7
Raw Data Available	Yes
Raw Data File Type(s)	raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2022-01-31
Release Version	1

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Project:

Project ID:	PR001283
Project DOI:	doi: 10.21228/M8VX1B
Project Title:	MICROMETABOLITE
Project Summary:	The overall objective of MICROMETABOLITE is to explore interactions between plants and microorganisms involved in the production of secondary metabolites (SM) for introducing novel ingredients in pharmaceutical and cosmeceutical industry. Effects of microorganisms on the plant metabolome and the biosynthesis of bioactive SM will be studied in the Boraginaceae plant family, aimed at optimising plant cultivation and alkannins/shikonins (A/S) production. Microorganisms will be integrated in plant production systems, and protocols needed for efficient implementation in industry will be elaborated. Thereby a platform will be established that will support long-term interactions between academia and industry.
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, Vojvodina, 25210, 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

Subject:

Subject ID:	SU002144
Subject Type:	Plant
Subject Species:	Lithospermum officinale
Taxonomy ID:	475907
Species Group:	Plants

Factors:

Subject type: Plant; Subject species: Lithospermum officinale (Factor headings shown in green)

mb_sample_id	local_sample_id	Treatment
SA194033	71875_3	Brevibacterium sp. R-71875
SA194034	71875_1	Brevibacterium sp. R-71875
SA194035	71875_4	Brevibacterium sp. R-71875
SA194036	71875_5	Brevibacterium sp. R-71875
SA194037	71875_6	Brevibacterium sp. R-71875
SA194038	71875_2	Brevibacterium sp. R-71875
SA194039	72269_2	Chitinophaga sp. R-72269
SA194040	72269_6	Chitinophaga sp. R-72269
SA194041	72269_4	Chitinophaga sp. R-72269
SA194042	72269_3	Chitinophaga sp. R-72269
SA194043	72269_1	Chitinophaga sp. R-72269
SA194044	72269_5	Chitinophaga sp. R-72269
SA194045	73072_1	Chitinophaga sp. R-73072
SA194046	73072_6	Chitinophaga sp. R-73072
SA194047	73072_4	Chitinophaga sp. R-73072
SA194048	73072_3	Chitinophaga sp. R-73072
SA194049	73072_2	Chitinophaga sp. R-73072
SA194050	73072_5	Chitinophaga sp. R-73072
SA194051	Cminus_5	Control
SA194052	Cminus_2	Control
SA194053	Cminus_3	Control
SA194054	Cminus_4	Control
SA194055	Cminus_6	Control
SA194056	Cminus_1	Control
SA194057	71838_2	Pseudomonas sp. R-71838
SA194058	71838_3	Pseudomonas sp. R-71838
SA194059	71838_6	Pseudomonas sp. R-71838
SA194060	71838_4	Pseudomonas sp. R-71838
SA194061	71838_1	Pseudomonas sp. R-71838
SA194062	71838_5	Pseudomonas sp. R-71838
SA194063	QC_6_BACTERIA_LE_24_03	QC
SA194064	QC_6_BACTERIA_LE_24_01	QC
SA194065	QC_6_BACTERIA_LE_24_02	QC
SA194066	72433_5	Rhizobium sp. R-72433
SA194067	72433_4	Rhizobium sp. R-72433
SA194068	72433_3	Rhizobium sp. R-72433
SA194069	72433_2	Rhizobium sp. R-72433
SA194070	72433_1	Rhizobium sp. R-72433
SA194071	72433_6	Rhizobium sp. R-72433
SA194072	73098_1	Xanthomonas sp. R-73098
SA194073	73098_5	Xanthomonas sp. R-73098
SA194074	73098_4	Xanthomonas sp. R-73098
SA194075	73098_3	Xanthomonas sp. R-73098
SA194076	73098_2	Xanthomonas sp. R-73098
SA194077	73098_6	Xanthomonas sp. R-73098

Showing results 1 to 45 of 45

Showing results 1 to 45 of 45

Collection:

Collection ID:	CO002137
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 -80 C for analysis of the content of A/Sd by targeted and non-targeted metabolomics
Sample Type:	Plant

Treatment:

Treatment ID:	TR002156
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

Treatment ID:

TR002156

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

Sample Preparation:

Sampleprep ID:	SP002150
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.

Sampleprep ID:

SP002150

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.

Combined analysis:

Analysis ID	AN003361
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Thermo Vanquish
Column	Waters Acquity UPLC HSS C18 SB (100 x 2.1mm, 1.8um)
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Thermo Q Exactive Focus
Ion Mode	POSITIVE
Units	intensity units

Chromatography:

Chromatography ID:	CH002487
Instrument Name:	Thermo Vanquish
Column Name:	Waters Acquity UPLC HSS C18 SB (100 x 2.1mm, 1.8um)
Flow Gradient:	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.
Solvent A:	100% water; 0.1% formic acid;
Solvent B:	100% methanol; 0.1% formic acid
Chromatography Type:	Reversed phase

MS:

MS ID:	MS003130
Analysis ID:	AN003361
Instrument Name:	Thermo Q Exactive Focus
Instrument Type:	Orbitrap
MS Type:	ESI
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).
Ion Mode:	POSITIVE