MsmiR156 affects global gene expression and promotes root regenerative capacity and nitrogen fixation activity in alfalfa

MicroRNA156 (miR156) regulates a network of downstream genes to affect plant growth and development. We previously generated alfalfa (Medicago sativa) plants that overexpress homologous miR156 (MsmiR156OE), and identified three of its SPL target genes. These plants exhibited increased vegetative yield, delayed flowering and longer roots. In this study, we aimed to elucidate the effect of miR156 on the root system, including effect on nodulation and nitrogen fixation. We found that MsmiR156 overexpression increases root regeneration capacity in alfalfa, but with little effect on root biomass at the early stages of root development. MsmiR156 also promotes nitrogen fixation activity by upregulating expression of nitrogenase-related genes FixK, NifA and RpoH in roots inoculated with Sinorrhizobium meliloti. Furthermore, we conducted transcriptomics analysis of MsmiR156OE alfalfa roots and identified differentially expressed genes belonging to 132 different functional categories, including plant cell wall organization, peptidyl-hypusine synthesis, and response to water stress. Expression analysis also revealed miR156 effects on genes involved in nodulation, root development and phytohormone biosynthesis. The present findings suggest that miR156 regulates root development and nitrogen fixation activity. Taken together, these findings highlight the important role that miR156 may play as a tool in the biotechnological improvement of alfalfa, and potentially other crops.