Nitrate regulates maize root transcriptome through nitric oxide dependent and independent mechanisms

Maize root responds to nitrate by modulating its development thorough the coordinated action of many players, interacting one each other. Among them, nitric oxide is produced in primary root of previously deprived seedlings early after the nitrate provision, thus inducing root elongation. In this study, a molecular untargeted approach was applied to discriminate the signaling and physiological pathways regulated by nitrate through nitric oxide from those regulated by nitrate itself of by further downstream factors. To this aim an RNA-sequencing approach was applied to discover the main molecular signatures distinguishing the response of maize root to nitrate according to their dependency, or independency on nitric oxide. A set of subsequent detailed functional annotation tools (Gene Ontology enrichment, MapMan, KEGG reconstruction pathway, transcription factors detection) were used to try to gain further information on the importance of this molecule in the regulation of the maize transcriptional response to nitrate. Besides, the lateral root density was measured both in the presence of nitrate and in the presence of nitrate plus cPTIO, a specific NO scavenger, and compared to that observed for N-depleted roots. Our results led to identify six clusters of transcripts according to their responsiveness to nitric oxide and to their regulation by nitrate provision. In general, common and specific features for the six clusters were identified allowing to discompose the overall root response to nitrate according to its dependency on nitric oxide. Illumina RNA-Seq analysis of maize root apexes samples collected from seedlings grown for 24 h in nutrient solution supplied with 1mM NO3- or 1 mM NO3- plus 1 mM cPTIO, compared to negative controls grown in a N-deprived nutrient solution. 3 days old maize B73 inbreed seedlings were treated for 24 h prior to roots collection and RNA extraction. 3 biological replicates per condition were produced, each composed by 10-15 plants.