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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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