Gene expression in response to signaling molecules has been well studied in the leaves of the model plant species Arabidopsis thaliana. However, knowledge of gene expression and metabolic regulation at the root level is limited. Here, the signaling compounds salicylic acid (SA), methyl jasmonate (MeJA) and nitric oxide (NO) were applied exogenously to induce various defense responses in roots, and their effect was studied using a combination of genomic, molecular and biochemical approaches. Genes involved in defense signaling/activation, cellular redox state, metabolism, transcription factors and membrane transport were altered in expression following treatment with SA, MeJA and NO. In addition, it was found that SA-, MeJA- and NO-elicited roots increased the root exudation of phytochemicals compared with the roots of nontreated control plants. Transport systems likely to be involved in the root exudation of phytochemicals, including the MATE, ABC, MFS, amino acid, sugar and inorganic solute transporters, showed altered expression profiles in response to treatments. Overall, significant differences were found in the signaling compound-elicited expression profiles of genes in roots vs those in leaves. These differences could be correlated to the underground nature of roots and their exposure to higher microbial inoculum rates under natural conditions.