Expression and tissue-specific localization of nitrate-responsive miRNAs in roots of maize seedlings.

Nitrogen availability seriously affects crop productivity and environment. The knowledge of post-transcriptional regulation of plant response to nutrients is important to improve nitrogen use efficiency of crop. This research was aimed at understanding the role of miRNAs in the molecular control of plant response to nitrate. The expression profiles of six mature miRNAs were deeply studied by quantitative real time polymerase chain reaction and in situ hybridization (ISH). To this aim, a novel optimized protocol was set up for the use of digoxygenin-labelled Zip Nucleic Acid-modified oligonucleotides as probes for ISH. Significant differences in miRNAs' transcripts accumulation were evidenced between nitrate-supplied and nitrate-depleted roots. Real-time PCR analyses and in situ detection of miRNA confirmed the array data and allowed us to evidence distinct miRNAs spatio-temporal expression patterns in maize roots. Our results suggest that a prolonged nitrate depletion may induce post-transcriptionally the expression of target genes by repressing the transcription of specific miRNAs. In particular, the repression of the transcription of miR528a/b, miR528a*/b*, miR169i/j/k, miR169i*/j*/k*, miR166j/k/n and miR408/b upon nitrate shortage could represent a crucial step integrating nitrate signals into developmental changes in maize roots.