The MsNAC73 – MsMPK3 Complex Modulates Salt Tolerance and Shoot Branching of Alfalfa via Activating MsPG2 and MsPAE12 Expressions

Balancing plant growth and survival is an important strategy for plants to adapt to different environments. However, the molecular mechanisms of the balance strategy are poorly understood. Our previous study demonstrated that MsNAC73 interacts with the promoter of MsPAE12, which positively regulates alfalfa shoot branching. In the present study, MsNAC73 was further found to interact with the promoter of MsPG2 and negatively regulate MsPG2 expression through MODMS database analysis and experimental verification (Y1H, EMSA and Dual-LUC assays). The transgenic alfalfa plants with overexpression or knockdown of MsNAC73 and MsPG2 were obtained, and their responses to abiotic stress were analysed. Overexpressing MsNAC73 negatively and overexpressing MsPG2 positively affected the salt tolerance of alfalfa. MsPG2 increased salt tolerance via hydrolysing pectins, increasing cell wall extensibility and reducing Na⁺/K⁺ ratio, stomatal aperture and vessel diameter. Co-IP, Y2H, split-LUC and BiFC assays demonstrated that MsMPK3 interacted with MsNAC73 and phosphorylated MsNAC73 at the Thr-123 site. Furthermore, low abundances of MsMPK3 and MsNAC73 under normal conditions diminished the MsNAC73 phosphorylation, thereby promoting MsPAE12 expression and increasing alfalfa shoot branching. Under salt stress, however, MsNAC73 and MsMPK3 were upregulated at transcript and protein levels. The increased phosphorylation of MsNAC73 (MsNAC73^T123D) promoted MsPG2 expression. Additionally, overexpression of MsNAC73^T123D and MsNAC73^T123A (dephosphorylation of MsNAC73) in alfalfa hairy roots increased root elongation under salt conditions and lateral root amounts under normal conditions respectively. In brief, these results revealed that the MsNAC73-MsMPK3-MsPG2/MsPAE12 module plays a key role in the trade-off between shoot branching and plant survival in response to different environments.