SPL12 Regulates AGL6 and AGL21 to Modulate Nodulation and Root Regeneration under Osmotic Stress and Nitrate Sufficiency Conditions in Medicago sativa

The highly conserved plant microRNA, miR156, affects root architecture, nodulation, symbiotic nitrogen fixation, and stress response. In Medicago sativa, transcripts of eleven SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE, SPLs, including SPL12, are targeted for cleavage by miR156. Our previous research revealed the role of SPL12 and its target gene, AGL6, in nodulation in alfalfa. Here, we investigated the involvement of SPL12, AGL6 and AGL21 in nodulation under osmotic stress and different nitrate availability conditions. Characterization of phenotypic and molecular parameters revealed that the SPL12/AGL6 module plays a negative role in maintaining nodulation under osmotic stress. While there was a decrease in the nodule numbers in WT plants under osmotic stress, the SPL12-RNAi and AGL6-RNAi genotypes maintained nodulation under osmotic stress. Moreover, the results showed that SPL12 regulates nodulation under a high concentration of nitrate by silencing AGL21. AGL21 transcript levels were increased under nitrate treatment in WT plants, but SPL12 was not affected throughout the treatment period. Given that AGL21 was significantly upregulated in SPL12-RNAi plants, we conclude that SPL12 may be involved in regulating nitrate inhibition of nodulation in alfalfa by targeting AGL21. Taken together, our results suggest that SPL12, AGL6, and AGL21 form a genetic module that regulates nodulation in alfalfa under osmotic stress and in response to nitrate.