Genome-wide discovery and characterization of nitrate-responsive miRNAs in roots of maize seedlings

Abstract Nitrogen availability affects crops productivity and environment. The natural abundance of useable nitrogen is so low that the massive human alteration of the nitrogen cycle has been required to sustain the feeding of the world's population. Tons of nitrogenous fertilizers are added to the soil worldwide annually, giving rise to environmental diseases. In this scenario, the knowledge of post-transcriptional regulation of plant response to nutrients is important to improve nitrogen use efficiency of crop. With the identification of stress-responsive miRNAs, a layer of post-transcriptional gene regulation has been uncovered. We used a maize miRNAs-microarray platform to discover previously unknown nitrate-responsive miRNAs. Six mature miRNAs were identified and their expression profiles were studied by quantitative Real Time PCR (qPCR) and in situ hybridization (ISH) in maize roots grown in different nitrate availabilities. Significant differences in miRNAs’ transcripts accumulation were evidenced between nitrate-supplied and nitrate-depleted roots. Real time PCR analyses and in situ detection of miRNAs confirmed the arrays data and evidenced distinct miRNAs spatiotemporal expression patterns in maize roots. An in silico approach was used to select target genes of the miRNAs identified. Their transcripts accumulation has been investigated in both nitrate-supplied and nitrate-depleted roots by means of qPCR and ISH. Our results suggest that miRNAs play some role in modulating N-responsive gene expression by inducing post-transcriptionally the expression of target genes. In particular, the repression of the transcription of miRNA identified upon nitrate shortage could represent a crucial step integrating nitrate signals into developmental changes in maize roots.