NMR spectra of oilseed rape (Brassica napus) root exudates

Surface sterilised oilseed rape seeds were germinated 3-5 days on petri dishes containing 0.5× Murashige-Skoog medium (including vitamins) and 0.6% bacto agar. Plantlets (n = 8) were then transferred to 50 ml plastic tubes filled with autoclaved MilliQ water, so that the seedling roots were immersed into the water (see photo). Root exudates were then collected for four days after which they were freeze dried. Blank samples did not contain any seedlings but were otherwise treated the same. Lyophilised root exudate samples were dissolved in a few millilitres of MilliQ water, transferred to 15 ml plastic tubes, and dried in a vacuum centrifuge. NMR samples were prepared by adding 750 μl KH2PO4 buffer in D2O (45 mM, pD 7.0 (apparent pH 6.6)+DSS-d6 to each sample. The samples were then centrifuged for 10 min at 17000xg. For each sample, 600 µl supernatant was added to a 5 mm NMR tube. NMR experiments: - 1D-1H-NOESY, (1H,1H)-TOCSY, (1H,13C)-HSQC, CPMG, and 1D-diffusion NMR spectra were recorded of intact root exudates. - 1D-1H spectra of a sample treated with ultrafiltration and solid phase extraction (SPE), respectively. - 1D-1H spectra from a spike-in experiment where different concentrations of five metabolites were added to a pooled root exudate sample as well as a blank sample.