Mar 19, 2025
Protocol for Roots and Rhizodeposition Metabolite extraction from Plant-Agar Plates for LC-MS/MS analysis
- 1Oak Ridge National Laboratory
Protocol Citation: Paul E. Abraham 2025. Protocol for Roots and Rhizodeposition Metabolite extraction from Plant-Agar Plates for LC-MS/MS analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj9rpnlk5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 02, 2024
Last Modified: March 19, 2025
Protocol Integer ID: 111439
Keywords: Metabolomics, Extraction, Plant, Roots, Rhizosphere, Exudate, Rhizodeposition
Funders Acknowledgements:
U.S. Department of Energy’s Office of Biological and Environmental Research
Grant ID: Plant–Microbe Interfaces Scientific Focus Area
Abstract
Our research has demonstrated that Populus root-associated microbiomes assembled in a plant-agar system formed a nearly identical composition to that observed in a clay soil matrix. This finding suggests that a plant agar matrix can replicate a soil-like environment while providing a more controlled and reproducible workflow for studying rhizodeposition. Building on this, we developed a novel biphasic extraction method specifically tailored for rhizodeposition studies using the plant agar-plate system. Rhizodeposition encompasses a diverse mixture of metabolites with varying polarities, from polar organic acids and amino acids to nonpolar phenolics and lipophilic compounds. To capture this full range, we employed a 50/50 water and ethyl acetate biphasic extraction mixture to enable a comprehensive profiling of root-derived metabolites involved in rhizodeposition
Preparing Plant-agar Plate for Spatial Metabolite Extraction
Preparing Plant-agar Plate for Spatial Metabolite Extraction
Prepare Agar Media: Mix agar with the appropriate growth media according to the specifications of the experiment. Adjust pH as needed, then autoclave to sterilize. Pour the agar media into sterile Petri dishes (typically 90 mm or 100 mm diameter) and allow it to cool and solidify under sterile conditions.
Note
Note that while this metabolite extraction protocol is compatible with both agar and agarose, using agar may introduce a higher background of matrix compounds in comparison to agarose. Although one can manipulate the amount, we have tested and can only recommend 2% agar and 0.3% agarose.
Transplant Plant onto Agar Plate: In a sterile laminar flow hood, using sterile forceps, carefully place roots gently on the agar surface, avoiding damage to the root tissue. To place the cuttings on the plates, lay the root system down on the surface of the media and use sterile forceps to spread the roots out so that one root traverses the middle of the plate and the other roots are pushed away from each other. Use the sterile forceps to gently push the roots into the media so they are fully embedded.
Note
Example of container used when growing plants with agar inoculated with microbes.
Close container: Place each plate with a cutting on an inverted lid of a sterile plastic container and position an inverted container over the cutting and use the sidewall to hold the cutting upright, then seal the container by pressing it down and snapping it in place with the inverted lid. You now have an open plate with a cutting embedded in the media and sitting inside a sterile inverted plastic container. Label the container and place the cuttings in a light room or plant incubation chamber.
Metabolite Extraction from Root Tissue
Metabolite Extraction from Root Tissue
Excise Root Tissue: Excise roots from each plant with a sterile scalpel.
Freeze and Store: Transfer the root tissue from each plant into a separate vial. Freeze the sample vials immediately in liquid nitrogen and store at -80°C until further processing.
Freeze-dry and Record Weights: Freeze-dry the root tissue (e.g., Labconco, USA) and record the dry weights for each sample.
Grind Root Tissue: Using a cryogrinder (e.g., SPEX Sample Prep 2010 GENO/GRINDER) or mortar and pestle, grind the freeze-dried root tissue in liquid nitrogen into a fine powder and store samples on ice.
Preparation of Solvents for Metabolite Extraction: Mix HPLC-grade ethyl acetate with HPLC grade water in a 1:1 ratio to create the hydrated ethyl acetate solvent. Transfer the hydrated ethyl acetate and HPLC grade water to separate containers, and chill both on ice prior to extraction.
Perform Metabolite Extraction: Add equal volumes of ice-cold, hydrated LC-MS grade ethyl acetate and LC-MS grade water (total volume = 2 mL) to each powdered root sample. Vortex the samples at maximum speed for 1 minute to mix thoroughly. Ensure samples remain cold throughout this process by keeping them on ice, then transfer them to a fridge set at 4°C and incubate on ice overnight.
Note
Total solvent volume can be adjusted depending on the sample amount.
Centrifuge Samples: Centrifuge the samples at maximum speed for 10 minutes at 4°C.
Separate Fractions: Using aspiration, carefully separate the organic (ethyl acetate) and aqueous supernatant fractions. Ensure samples remain cold throughout this process by keeping them on ice.
Prepare Organic Fraction: Dry the ethyl acetate extracts completely in a chemical fume hood. Resuspend each extract in 50 μL of organic solvent (70% acetonitrile with 0.1% formic acid) prior to LC-MS/MS measurement.
Prepare the Aqueous Fraction: Pre-wet 10 kDa centrifugal filters (e.g., Sartorius VivaSpin). Filter the aqueous extracts by centrifuging at 4,500 x g at 4°C to remove residual media and particles. Lyophilize the aqueous extracts (e.g., Labconco, USA), then resuspend in 50 μL of aqueous solvent (98% LC-MS water, 2% acetonitrile with 0.1% formic acid) prior to LC-MS/MS measurement.
Store Extracts: Store both organic and aqueous extractions at 4°C for short-term storage until LC-MS/MS analysis.
Metabolite Extraction from Media (Rhizodeposits)
Metabolite Extraction from Media (Rhizodeposits)
Collect Spatially Resolved Rhizodeposits: Use an inverted 1 mL pipette tip to cut and collect agar plugs of equal volume from distinct spatial regions surrounding the plant roots (e.g., root base, root tips, and regions distant from the root base and tip).
Freeze and Store Agar Plugs: Immediately freeze the plugs in liquid nitrogen and store them at -80°C until further processing.
Freeze-dry: Freeze-dry the agar plugs (e.g., Labconco, USA).
Perform Metabolite Extraction: Add equal volumes of ice-cold, hydrated LC-MS grade ethyl acetate and LC-MS grade water (total volume = 2 mL) to each agar plug sample. Vortex the samples at maximum speed for 1 minute to mix thoroughly. Ensure samples remain cold throughout this process by keeping them on ice, then transfer them to a fridge set at 4°C and incubate on ice overnight.
Note
Total solvent volume can be adjusted depending on the sample amount.
Centrifuge Samples: Centrifuge the samples at maximum speed for 10 minutes at 4°C.
Separate Fractions: Using aspiration, carefully separate the organic (ethyl acetate) and aqueous supernatant fractions. Ensure samples remain cold throughout this process by keeping them on ice.
Prepare Organic Fraction: Dry the ethyl acetate extracts completely in a chemical fume hood. Resuspend each extract in 50 μL of organic solvent (70% acetonitrile with 0.1% formic acid) prior to LC-MS/MS measurement.
Prepare the Aqueous Fraction: Pre-wet 10 kDa centrifugal filters (e.g., Sartorius VivaSpin). Filter the aqueous extracts by centrifuging at 4,500 x g at 4°C to remove residual media and particles. Lyophilize the aqueous extracts (e.g., Labconco, USA), then resuspend in 50 μL of aqueous solvent (98% LC-MS water, 2% acetonitrile with 0.1% formic acid) prior to LC-MS/MS measurement.
Store Extracts: Store both organic and aqueous extractions at 4°C for short-term storage until LC-MS/MS analysis.
Acknowledgements
This research is supported by the Plant–Microbe Interfaces (https://pmiweb.ornl.gov/) Scientific Focus Areas funded by the Genomic Science Program of the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research (BER)