In the present study, the mode of action of coumarin using the germination process as a target was investigated. A dose-response curve, built using a range of concentrations from 0 to 800 µM, allowed us to identify a key concentration (400 µM) inhibiting the germination process, reducing its speed without compromising seed development. Successively, short time-course (0-48 h) experiments were carried out to evaluate the biochemical and metabolic processes involved in coumarin-induced germination delay. The results pointed out that coumarin delayed K+, Ca2+, and Mg2+ reabsorption, suggesting a late membrane reorganisation. Similarly, seed respiration was inhibited during the first 24 h but recovered after 48 h. Those results agreed with ATP levels, which followed the same trend. In addition, the untargeted metabolomic analysis allowed to identify, among the pathways significantly impacted by the treatment, amino acids metabolism, the TCA cycle, and the glyoxylate pathway. The results highlighted that coumarin was able to interact with membranes reorganisation, delaying them and reducing the production of ATP, as also supported by pathway analysis and cell respiration. The in vivo 31P-NMR analysis supported the hypothesis that the concentration chosen was able to affect plant metabolism, maintaining, on the other hand, its viability, which is extremely important for studying natural compounds' mode of action.
Keywords: 31P-NMR; phytotoxicity; plant metabolism; respiration; seed germination.