Posttranscriptional regulation of a variety of mRNAs by small 21-to 24-nucleotide RNAs, notably the microRNAs (miRNAs), is emerging as a novel developmental mechanism. In legumes like the model Medicago truncatula, roots are able to develop a de novo meristem through the symbiotic interaction with nitrogen-fixing rhizobia. We used deep sequencing of small RNAs from root apexes and nodules of M. truncatula to identify 100 novel candidate miRNAs encoded by 265 hairpin precursors. New atypical precursor classes producing only specific 21-and 24-nucleotide small RNAs were found. Statistical analysis on sequencing reads abundance revealed specific miRNA isoforms in a same family showing contrasting expression patterns between nodules and root apexes. The differentially expressed conserved and nonconserved miRNAs may target a large variety of mRNAs. In root nodules, which show diverse cell types ranging from a persistent meristem to a fully differentiated central region, we discovered miRNAs spatially enriched in nodule meristematic tissues, vascular bundles, and bacterial infection zones using in situ hybridization. Spatial regulation of miRNAs may determine specialization of regulatory RNA networks in plant differentiation processes, such as root nodule formation.

Summary In plants, roots are essential for water and nutrient acquisition. MicroRNAs (miRNAs) regulate their target mRNAs by transcript cleavage and/or inhibition of protein translation and are known as major post‐transcriptional regulators of various developmental pathways and stress responses. In Arabidopsis thaliana, four isoforms of miR169 are encoded by 14 different genes and target diverse mRNAs, encoding subunits A of the NF‐Y transcription factor complex. These miRNA isoforms and their targets have previously been linked to nutrient signalling in plants. By using mimicry constructs against different isoforms of miR169 and miR‐resistant versions of NF‐YA genes we analysed the role of specific miR169 isoforms in root growth and branching. We identified a regulatory node involving the particular miR169defg isoform and NF‐YA2 and NF‐YA10 genes that acts in the control of primary root growth. The specific expression of MIM169defg constructs altered specific cell type numbers and dimensions in the root meristem. Preventing miR169defg‐regulation of NF‐YA2 indirectly affected laterial root initiation. We also showed that the miR169defg isoform affects NF‐YA2 transcripts both at mRNA stability and translation levels. We propose that a specific miR169 isoform and the NF‐YA2 target control root architecture in Arabidopsis.