The proteome of Medicago truncatula in response to ammonium and urea nutrition reveals the role of membrane proteins and enzymes of root lignification

Plants differ widely in their growth and tolerance responses to ammonium and urea nutrition, while derived phenotypes seem markedly different from plants grown under nitrate supply. Plant responses to N sources are complex, and the traits involved remain unknown. This work reports a comprehensive and quantitative root proteomic study on the NH4+-tolerant legume Medicago truncatula grown under axenic conditions with either nitrate, NH4+ or urea supply as sole N source by using the iTRAQ method. Sixty-one different proteins among the three N sources were identified. Interestingly, among the proteomic responses, urea nutrition displayed greater similarity to nitrate than to ammonium nutrition. We found remarkable differences in membrane proteins that play roles in sensing the N form, and regulate the intracellular pH and the uptake of N. Also, several groups of proteins were differentially expressed in the C metabolism pathway involved in reorganizing N assimilation. In addition, enzymes related to phenylpropanoid metabolism, including the peroxidases POD2, POD6, POD7 and POD11, which were up-regulated under ammonium nutrition, contributed to the reinforcement of cell walls, as confirmed by specific staining of lignin. Thus, we identified cell wall lignification as an important tolerance mechanism of root cells associated with the stunted phenotype typical of plants grown under ammonium nutrition. This work was supported by the grants AGL2014-52396-P and AGL2017-86293-P from the Spanish Ministry of Economy and Competitiveness-MINECO. RE received a Juan de la Cierva-incorporación grant IJCI-2014-21452. JB is a holder of a PhD fellowship from the Public University of Navarre. The Proteomics Unit of Navarrabiomed is a member of ProteoRed, PRB3-ISCIII-Spain, and is supported by grant PT17/0019/009, of the PE I + D+I 2018-2020 funded by ISCIII and FEDER.