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Introduction: Nitrogen (N) and iron (Fe) are involved in several biochemical processes in living organisms, and their limited bioavailability is a strong constraint for plant growth and yield. This work investigated the interplay between Fe and N nutritional pathways in tomato plants kept under N and Fe deficiency and then resupplied with Fe and N (as nitrate, ammonium, or urea) through a physiological, metabolomics and gene expression study. Results: After 24 hours of Fe resupply, the Fe concentration in Fe-deficient roots was dependent on the applied N form (following the pattern: nitrate > urea > ammonium > Fe-deficient control), and whereas in leaves of urea treated plants the Fe concentration was lower in comparison to the other N forms. Untargeted metabolomics pointed out distinctive modulations of plant metabolism in a treatment-dependent manner. Overall, N-containing metabolites were affected by the treatments in both leaves and roots, while N form significantly shaped the phytohormone profile. Moreover, the simultaneous application of Fe with N to Fe-deficient plants elicited secondary metabolites’ accumulation, such as phenylpropanoids, depending on the applied N form (mainly by urea, followed by nitrate and ammonium). After 4 hours of treatment, ammonium- and urea-treated roots showed a reduction of enzymatic activity of Fe(III)-chelate reductase (FCR), compared to nitrate or N-depleted plants (maintained in Fe deficiency, where FCR was maintained at high levels). The response of nitrate-treated plants leads to the improvement of Fe concentration in tomato roots and the increase of Fe(II) transporter (IRT1) gene expression in tomato roots. Conclusions: Our results strengthen and improve the understanding about the interaction between N and Fe nutritional pathways, thinning the current knowledge gap. [ABSTRACT FROM AUTHOR]