Journal: Frontiers in Plant Science

Article Title: Systematic characterization of extracellular vesicles from potato ( Solanum tuberosum cv. Laura) roots and peels: biophysical properties and proteomic profiling

doi: 10.3389/fpls.2024.1477614

Figure Lengend Snippet: The experimental design used for the collection, isolation, and characterization of extracellular vesicles released by potato (cv. Laura) roots and peels. Independent experiments were carried out to collect initial sampling solutions containing root exudates of potato ( Solanum tuberosum , cultivar: Laura) plants grown hydroponically and apoplastic washes collected from potato peels of the same cultivar by a vacuum infiltration method using a vesicle isolation buffer. Three replicates were conducted for each experiment. The purification process was a combination of differential centrifugation steps and size exclusion chromatography (SEC), where 20 purified fractions (500 µL each) of the samples were collected for further examination. Biophysical and chemical analyses were conducted on these extracellular vesicles (EVs) using the Bradford protein assay, nanoparticle tracking analysis (NTA), and transmission electron microscopy (TEM). The EV proteome of the samples was evaluated using mass spectrometry where purified EV samples (n = 3) and crude samples (n = 3) from each potato peel and root sample were used for comparison.

Article Snippet: EVs were isolated from concentrated sample solutions using size exclusion chromatography (SEC) (Econo-pac ® Disposable chromatography column, Bio-Rad, Berkeley, CA, USA) packed with a 10-cm column of cross-linked 4% agarose matrix with 90-μm beads (Sepharose 4 fast flowTM, GE HealthCare Bio-Sciences AB, Uppsala, Sweden) and using Dulbecco’s phosphate-buffered saline (DPBS; Sigma-Aldrich, St. Louis, MO, USA) as the equilibration buffer.

Techniques: Isolation, Sampling, Purification, Centrifugation, Size-exclusion Chromatography, Bradford Protein Assay, Transmission Assay, Electron Microscopy, Mass Spectrometry, Comparison

Journal: Frontiers in Plant Science

Article Title: Systematic characterization of extracellular vesicles from potato ( Solanum tuberosum cv. Laura) roots and peels: biophysical properties and proteomic profiling

doi: 10.3389/fpls.2024.1477614

Figure Lengend Snippet: The particle and protein distribution for fractions collected from Solanum tuberosum (cv. Laura) peel apoplastic fluid (A) and root growth medium (B) using size exclusion chromatography (SEC). Fractions 4–8 and 5–8 contain the highest number of particles (vesicles) with a minimal amount of protein contamination for peel and root samples, respectively. n = 3; error bars represent the standard error of the mean ( ± SEM). Particle number was measured using the scatter mode of Nanoparticle Tracking Analysis (NTA), and the total protein concentration was measured using the Bradford protein assay.

Article Snippet: EVs were isolated from concentrated sample solutions using size exclusion chromatography (SEC) (Econo-pac ® Disposable chromatography column, Bio-Rad, Berkeley, CA, USA) packed with a 10-cm column of cross-linked 4% agarose matrix with 90-μm beads (Sepharose 4 fast flowTM, GE HealthCare Bio-Sciences AB, Uppsala, Sweden) and using Dulbecco’s phosphate-buffered saline (DPBS; Sigma-Aldrich, St. Louis, MO, USA) as the equilibration buffer.

Techniques: Size-exclusion Chromatography, Protein Concentration, Bradford Protein Assay