Flooding tolerance is regulated through the MiR156/SPL13 module in Medicago sativa

Climate change is expected to increased the mean annual temperature and precipitation resulting in frequent flooding events. To select or generate flooding-tolerant plants, it is imperative to understand the underlying tolerance mechanisms. microRNA156 (miR156) is highly conserved across plant species and is reported to have impart effects on plant development, chemical composition and stress physiology. To investigate whether miR156 affects the response of Medicago sativa (alfalfa) to flooding stress, we used mR156 overexpressors, miR156-regulated SPL13RNAi, flooding- tolerant (CRS1001) and sensitive (AC-caribou) cultivars, exposed to two weeks of flooding. Physiological analysis, hormone profiling and global transcriptomic data illustrated the positive role of miR156 in flooding stress tolerance. During flooding, moderate miR156 over-expressers, SPL13RNAi, and CRS1001 alfalfa plants maintained Vcmax and Jmax, maximum rate of rubisco carboxylase activity and photosynthetic electron transport rate, as well high photosynthetic assimilation rate. Global transcriptomic-based pathway analysis revealed the enrichment of transcripts related to photosynthesis and secondary metabolites pathways. Moreover, hormone profiling showed an increase in the abundance of total ABA metabolites contributed mainly by increased Phaseic acid, a catabolite of ABA, in flooding tolerant alfalfa genotypes. Together, our results suggest the role of miR156/SPL13 in regulating flooding response in alfalfa is mediated in part by Phaseic acid and other secondary metabolites.