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In agricultural soils, nitrate (NO₃⁻) is the major nitrogen (N) nutrient for plants, but few studies have analyzed molecular and biochemical responses involved in its acquisition by grapevine roots. In viticulture, considering grafting, NO₃⁻ acquisition is strictly dependent on rootstock. To improve the knowledge about N nutrition in grapevine, this study analyzed biochemical and proteomic changes induced by, NO₃⁻ availability, in a hydroponic system, in the roots of M4, a recently selected grapevine rootstock. The evaluation of biochemical parameters, such as NO₃⁻, sugar and amino acid contents in roots, and the abundance of nitrate reductase, allowed us to define the time course of the metabolic adaptations to NO₃⁻ supply. On the basis of these results, the proteomic analysis was conducted by comparing the root profiles in N-starved plants and after 30 h of NO₃⁻ resupply. The analysis quantified 461 proteins, 26% of which differed in abundance between conditions. Overall, this approach highlighted, together with an increased N assimilatory metabolism, a concomitant rise in the oxidative pentose phosphate pathway and glycolysis, needed to fulfill the redox power and carbon skeleton demands, respectively. Moreover, a wide modulation of protein and amino acid metabolisms and changes of proteins involved in root development were observed. Finally, some results open new questions about the importance of redox-related post-translational modifications and of NO₃⁻ availability in modulating the dialog between root and rhizosphere.