Notes S1: Identification and quantification of compounds
The compounds present in exudates was determined using replicate natural abundance and 15N
samples collected after 7 hours since these samples contained the largest number of compounds
at the greatest concentration. Identification of compounds was an iterative multi-step process
that took several months. The first step involved integrating all peaks with a signal to noise ratio
greater than 5. Next, peaks originating from inorganic ions (e.g. salt-formate clusters) and
contaminating exogenous organic compounds were deleted. Contaminating exogenous
compounds were identified by comparing the exudate composition of 15N plants with natural
abundance plants. Exogenous compounds occurred in both natural abundance and 15N samples
without the corresponding mass shift, whereas endogenous compounds appeared in the natural
abundance and 15N samples with their mass spectra shifted according to the number of N atoms
in the compound (Giavalisco et al. 2008). The third step involved identification of “known”
molecules based on comparison of migration times and [M+H]+ with 75 authentic standards run
under the same conditions on the same instrument. Only about one-half of compounds were
identified in this step. For compounds that were not identified via standards the fourth step
involved building a short list of candidate compounds by de novo interpretation of mass spectra
(Warren 2014) or searching a CE-MS database (Baran et al. 2006) or the MS/MS databases
Metlin (Smith et al. 2005), HMDB (Wishart et al. 2007) and MassBank (Horai et al. 2010). The
list of candidates was further refined by seeing if the number of N atoms in the candidate
compound was the same as the number of N atoms that were calculated by subtracting [M+H]+
of the 14N isotopologue from [M+H]+ of the 15N isotopologue. In the final step, additional
standards were purchased and analysed under the same conditions as samples so as to confirm
putative identifications. For compounds where authentic standards were available,
concentrations were determined based on standard curves. For some compounds authentic
standards were not available, but it was possible to estimate approximate concentrations based on
the standard curve measured for a closely related compound.
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