Suspect screening of ¹³C-pulse-labeled root exudates collected with EcoRoot by GC-HRMS under climate stress

Understanding root exudation in natural soils remains a methodological challenge because of the complexity of the rhizosphere and rapid assimilation of exuded compounds by soil microbiota. To overcome this challenge, this study aims to develop a novel analytical strategy combining sampling via the EcoRoot methodology with ¹³CO₂ pulse labeling (PL) and gas chromatography coupled with Orbitrap high resolution mass spectrometry (GCHRMS). Applied to Linum usitatissimum (flax) under both current and simulated 2050 climate conditions, this approach enabled to unambiguously trap and analyze primary metabolites liberated through root exudation, notably monosaccharides. This study demonstrates that the high resolving power of the Orbitrap (60,000 resolution) is mandatory to accurately separate isobaric isotopologues involving ¹³C and derivatization-induced ²⁹Si and allowed to revealed the incorporation of ¹³C into specific sugar fragments. Despite identified space-charge effect at low ion intensities (threshold defined at I(M0/TIC)>0.1), isotopic enrichment was successfully quantified, reaching up to 27% of labeled molecules (%LM) for galactose 5TMS, with a mean isotopic enrichment (MIE) of 0.45 for the m/z 217.10746 fragment. Moreover, climate change simulation led to increased exudation of labeled sugars, with M1 fraction, rising from 5% (control) to 8% (climate change) (p < 0.05). This suggests altered carbon allocation strategies in response to environmental stress. GCHRMS enrichment results were compared with the ones measured by GC-combustion-IRMS and were found to be of the same order of magnitude. This finding validates the relevance of this approach while offering key advantages such as compatibility with untargeted analysis and improved sensitivity for low-abundance compounds.