miR156-SPL12-AGL6 module regulates root architecture and nodulation in Medicago sativa (alfalfa)

Developing alfalfa cultivars that can withstand drought is critical for the sustainable production of this forage crop. miR156 is highly conserved in plants, where it functions by silencing a group of SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors. We previously showed that miR156 overexpression in alfalfa improves drought tolerance. In this study, we used three alfalfa genotypes with different levels of miR156 overexpression and three genotypes with reduced SPL13 expression to study drought tolerance strategies by miR156 at the phenotypic, physiological, metabolic and molecular levels. These analyses on stems, roots and leaves of the used genotypes revealed a coordinated response to drought mediated by SPL13 silencing. Low to moderate levels of miR156 improved drought tolerance in alfalfa by silencing SPL13, increasing accumulation of stress mitigating metabolites such as proline, gamma-aminobutyric-acid, anthocyanins and other flavonoids, as well as enhancing photosynthetic assimilation rate, Fv/Fm ratio and root development. Moreover, transcription of genes involved in secondary metabolite synthesis and photosynthesis were increased significantly in the moderate miR156 over-expressors. We also demonstrate that the SPL13 protein binds to the promoter region of DIHYDROFLAVONOL-4-REDUCTASE gene in vivo to regulate its expression level. We conclude that a moderate increase in miR156 levels (0.5 to 1.5) is sufficient to enhance drought resilience in alfalfa, but higher miR156 overexpression may result in drought susceptibility.