Peanut Root Exudate Metabolite Profiling and Mimicry

NPRE were collected, lyophilized, and the dry material (50 mg) was extracted using 1.2 ml of methanol by vortexing 1 minute and then sonicated at room temperature for 20 min. After centrifugation, 500 µL of the supernatant was transferred into two centrifuge tubes for measurement of sugars and acids. For sugars, 20 µL of 3 mg/ml phenyl-β-D-glucopyranoside and 20 µL of 0.1 mg/ml 3-(4-hydroxyphenyl)-propionic acid were added as internal standards. The derivatization of sugars was performed by adding 120 μL methoxyamine-hydrochloride (20 mg/ml in pyridine) for 60 min at 30°C and with 120 μL N,O-bis (trimethylsilyl) trifluoro-acetamide (BSTFA) for 120 min at 45°C, 700 rpm. For amino acids and organic acids, 20 µL of 0.1 mg/ml 3-(4-hydroxyphenyl)-propionic acid was added to the sample as the internal standard and the derivatization was performed by adding 120 µL of methoxylamin-hydrochloride (20 mg/ml in pyridine) for 90 min at 37°C, 700 rpm, followed by addition of 120µl of dimethyl-tert-butylsilyl (TBDMS) and incubated for 30 min at 60°C, 700 rpm. After centrifuging, 100 µL of the supernatant was transferred to vials for detection using TSQ Duo GC-MS/MS (Thermo Fisher Scientific).
Based on NPRE composition, medium mimicking peanut root exudates (MMPRE) was recomposed as follows: Maltose 1.09 g/l; Glucose 0.88 g/l; Arabinose 0.67 g/l; Fructose 0.28 g/l; Trehalose 0.14 g/l; Myo-inositol 0.07 g/l; Lactic acid 2.73 g/l; Succinic acid 0.13 g/l; Malonic acid 0.11 g/l; Fumaric acid 0.07 g/l; Pyruvic acid 0.03 g/l; Pyroglutamic acid 0.02 g/l; Malic acid 0.02 g/l; Citric acid 0.02 g/l; Valine 1.05 g/l; Leucine 0.57 g/l; Threonine 0.57 g/l; Isoleucine 0.42 g/l; Alanine 0.32 g/l; Phenylalanine 0.32 g/l; Tyrosine 0.15 g/l; Serine 0.14 g/l; Glycine 0.11 g/l; Asparagine 0.08 g/l; Glutamic acid 0.03 g/l. Salt and micronutrients were added with these proportions: 0.25 g/l MgSO₄·7H₂O; 0.34 g/l; K₂HPO₄; 3-morpholino-1-propanesulfonic acid MOPS 10.5 g/l; 0.25 g/l KCl, 1 g/l (NH₄)₂SO₄; 1.2 mg/l Fe₂(SO₄)₃; 0.4 mg/l MnSO₄; 1.6 mg/l CuSO₄, 4 mg/l Na₂ MoO₄, pH adjusted to 7. In case of assay on solid MMPRE, 20 g/l of agar was used for this preparation.